1. travel
2. tourist destinations
3. italy

October 12 - 15, 2014
Hotel La Palma, Capri, Italy
FEBS WORKSHOP
Decoding Non-coding RNAs in
Development and Cancer
Scientific Organizers
Carlo M. Croce
Human Cancer Genetics, Columbus, OH, USA
Pier Paolo Pandolfi
Harvard Medical School, Boston, MA, USA
Reuven Agami
The Netherlands Cancer Institute, Amsterdam, The Netherlands
Amelia Cimmino
Institute of Genetics and Biophysics "A. Buzzati-Traverso", CNR, Naples, Italy
Workshop Coordinators
Sandro De Falco & Maria R. Matarazzo
Institute of Genetics and Biophysics “A. Buzzati-Traverso”, CNR, Naples, Italy
Workshop Organizing Secretariat
Anna Maria Aliperti & Federica Staempfli
Institute of Genetics and Biophysics “A. Buzzati-Traverso”, CNR, Naples, Italy
FEBS Workshop:
Decoding Non-coding RNAs in Development and Cancer
Edited by IGB Press/2014
Editorial Secretariat:
Anna Maria Aliperti
Donatella Jesu
Federica Staempfli
Front cover:
Design of a long non-coding RNA 2D structure and assembling
of the corresponding RNA 3D structure obtained by using Assemble2 and
UCSF Chimera software.
IGB Press 2014
c/o Institute of Genetics and Biophysics “A. Buzzati-Traverso”, CNR
Via P. Castellino, 111
80131 Naples, Italy
THIS WORKSHOP HAS BEEN ORGANIZED AND SUPPORTED BY:
FEBS – Federation of Biochemical Societies
Institute of Genetics and Biophysics “ABT” – CNR
AND SPONSORED BY:
Progetto Bandiera Epigenomica
Department of Biomedical Sciences - CNR
Nikon
M&M Biotech S.c.a.r.l.
BIORAD Laboratories
3
Previous and forthcoming Meetings organized in Capri by the
Institute of Genetics and Biophysics “ABT”, CNR
1988
Workshop on Molecular Biology of Mammalian Sex Chromosome
Organizers: H. Cooke, M.G. Persico and D. Toniolo
1989
Workshop on Molecular Biology of Development
Organizers: P. Bazzicalupo, E. Boncinelli and F. Graziani
1990
Workshop on the Molecular Biology of MHC Genes
Organizers: R. Accolla and J. Guardiola
1991
Workshop on Cellular and Molecular Cues in Neural Development
Organizer: U. di Porzio
1992
Workshop on Growth Factors and Development
Organizers: I.B. Dawid and M.G. Persico
1993
Workshop on Control Mechanisms in Oogenesis and Morphogenesis
Organizer: C. Malva
1994
Workshop on Signal Transduction Mechanisms
Organizers: M.R. Rosner and M.P. Stoppelli
1995
Workshop on Evolution and Development
Organizers: P. Bazzicalupo, U. di Porzio, J. McGhee and A. Simeone
1996
Workshop on Transcription Factors and Nuclear Oncogenes
Organizers: L. Lania, D.M. Livingston and P. Verde
1997
Workshop on Genome-Based Analysis of Gene Regulation and Its
Evolution - Organizers: M. D'Urso and D. Schlessinger
1998
Workshop on Cell Fate and the Generation of Cell Diversity
Organizers: G. Barsacchi, G. Camerino and C. Malva
1999
Workshop on Vasculogenesis and Angiogenesis
Organizers: P. Carmeliet and M. G. Persico
2000
Workshop on Plant Development: From Cell Fate to Organ Formation
Organizers: C. Bowler and R. Defez
2001
Workshop on Generating Cell Diversity in the Nervous System
Organizers: U. di Porzio and A. Simeone
2002
Workshop on From Genome Sequence to Functional Analysis and
Medical Applications - Organizers: A. Ballabio and J. Guardiola
2003
Workshop on The Molecular Biology, Genetics and Pathology of AP-1
Transcription Factors – Organizers: D. Bohmann, A.M. Musti, M. Yaniv
and P. Verde
2004
Workshop on The Biology and Development of the Eye in Health and
Disease - Organizers: S. Banfi, A. Wright and A. Ciccodicola
4
2005
Workshop on Epigenetic Bases of Genome Reprogramming
Organizers: V. Orlando, R. Feil and M. D’Esposito
2006
EMBO Workshop on Cell Migration, Tissue Invasion and Disease
Organizers: S. De Falco, G. Minchiotti and M.P. Stoppelli
2007
FEBS Workshop on Generating Neural Diversity in the Brain
Organizers: M. Studer and U. di Porzio
2008
EMBO Workshop on THE NF-KappaB Network in Development and
Disease - Organizers: G. Courtois and M.V. Ursini
2009
Workshop on Stem Cells: from Molecular Physiology to Therapeutic
Applications - Organizers: C. Mummery, S. Filosa, G. Minchiotti
2010
FEBS Workshop on Therapeutic Targets in Cancer Cell Metabolism &
Death - Organizers: I. I. Iaccarino, G. Melino, T. Mak
2012
FEBS Workshop on Molecular and Cellular Mechanisms in
Angiogenesis - Organizers: J. Ambati, S. De Falco, M. De Palma
2013
FEBS Workshop on Translating Epigenomes into Function: a NextGeneration Challenge for Human Disease Organizers: Stephan Beck and Maurizio D'Esposito
EMBO Workshop on Stem Cell Mechanobiology in Developemnt and
Disease - Organizers: Viola Vogel, Paolo Netti, Gabriella Minchiotti
2015
IGB Meeting Coordinators
1988-1989
1990-2001
2002
2003-2005
2006-2012
2013-2014
M. Graziella Persico and Edoardo Boncinelli
M. Graziella Persico and Umberto di Porzio
M. Graziella Persico
M. Graziella Persico, Ingram I. Iaccarino and M. Patrizia Stoppelli
Ingram I. Iaccarino and M. Patrizia Stoppelli
Sandro De Falco and Maria R. Matarazzo
IGB Meeting Secretariat
1989-1990
1991
1992-1996
1997-2000
2001-2007
2008-2014
Susan Hafkin
Patricia Reynolds
Patricia Reynolds and Antonietta Secondulfo
Antonietta Secondulfo
Anna Maria Aliperti and Antonietta Secondulfo
Anna Maria Aliperti and Federica Staempfli
5
Scientific Organizers
IGB Meeting Coordinators
Carlo M. Croce
Sandro De Falco
Mol Vir, Imm, Med Genetics
Human Cancer Genetics
1082 Biomedical Research Tower 460
West 12th Avenue
Columbus, OH 43210, USA
carlo.croce@osumc.edu
Abstract:
32, 62, 64, 78, 80, 87
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
sandro.defalco@igb.cnr.it
Maria Matarazzo
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
maria.matarazzo@igb.cnr.it
Abstract:
61
Pier Paolo Pandolfi
Cancer Center
Beth Israel Deaconess Medical Center
Harvard Medical School
77 Avenue Louis Pasteur
Boston, MA 02115, USA
ppandolf@bidmc.harvard.edu
Abstract:
22
Reuven Agami
IGB Meeting Secretariat
The Netherlands Cancer Institute
Plesmanlaan 121 - 1066 CX Amsterdam,
The Netherlands
r.agami@nki.nl
Abstract:
25
Anna Maria Aliperti
Federica Staempfli
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
workshop@igb.cnr.it
Amelia Cimmino
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
amelia.cimmino@igb.cnr.it
Abstract:
52, 53, 63, 76, 100
IGB Director
Antonio Simeone
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
antonio.simeone@igb.cnr.it
Abstract:
68
6
FEBS WORKSHOP PROGRAMME
Decoding Non-coding RNAs in Development and Cancer
October 12-15, 2014 - Capri, Italy
SUNDAY, OCTOBER 12TH
14.30
Registration and poster set up
16.30
Welcome address: Antonio Simeone (IGB Director)
16.45
Graziella Persico Lecture - Pier Paolo Pandolfi (USA):
The Non-Coding RNA revolution in cancer research
17.30
Coffee break
NON-CODING RNAS REGULATION AND CERNA CROSS-TALK
Chair: Pier Paolo Pandolfi
18.00
Brian Brown (USA): Understanding the relationship between microRNA
concentration and activity
18.45
Judy Lieberman (USA): Cancer crosstalk: miRNA transfer by exosomes
between cancer cells confers metastatic capability
20.30
Dinner
MONDAY, OCTOBER 13TH
NON-CODING RNAS REGULATION AND CERNA CROSS-TALK
Chair: Brian Brown
9.15
Nikolaus Rajewsky (Germany): Regulatory RNAs
7
9.45
Teresa M. Creanza (Italy), short talk: Differential miRNA-mRNA coexpression networks in colorectal cancer
10.00
Coffee break, group photo and Poster Session I (odd numbers)
NON-CODING RNAS IN TRANSCRIPTIONAL CONTROL AND CHROMATIN
Chair: Judy Lieberman
12.00
Irene Bozzoni (Italy): Regulatory circuitries controlled by long non-coding
RNAs
12.30
Massimiliano Pagani (Italy), short talk: LincRNAs landscape in human
lymphocytes highlights regulation of T cell differentiation by linc-MAF-4
13.30
Lunch
15.45
John Thomson (UK): The Dlk1-Dio3 imprinted gene cluster noncoding
RNAs are novel candidate biomarkers for liver tumour promotion
16.15
Edith Schneider (Germany), short talk: Transcriptional regulation of
microRNAs through Meis1 in the pathogenesis of acute myeloid leukemia
16.30
Maite Huarte (Spain): The lncRNA components of the p53 network
17.00
Coffee break
NON-CODING RNAS IN CANCER
Chair: Carlo Maria Croce
17.30
Muller Fabbri (USA): miRceptors and exosomic microRNAs dictate the
biology of the tumor microenvironment
18.00
Enrica Calura (Italy), short talk: miRNA and gene regulatory pathway of
stage I epithelial ovarian cancer: reconstructing cancer circuits
18.15
Danilo Fiore (Italy), short talk: Survival in glioblastoma cancer patients is
predicted by miR-340, that regulates key cancer hallmarks by inhibiting
NRAS
18.30
Mario Leonardo Squadrito (Switzerland), short talk: Endogenous RNAs
modulate microRNA sorting to exosomes and transfer to acceptor cells
8
18.45
Reuven Agami (The Netherlands): Enhancer RNAs at the service of p53
20.30
Dinner: Meet the expert I (Speakers of day 1 and 2)
TUESDAY, OCTOBER 14TH
NON-CODING RNAS IN CANCER
Chair: Reuven Agami
9.00
Carlo Maria Croce (USA): Causes and consequences of microRNA
dysregulation in cancer
9.30
Francesca Garibaldi (Italy), short
processing by mutp53 in colon cancer
9.45
Sven Diederichs (Germany): Long non-coding RNAs in cancer
10.15
talk:
Regulation
of
microRNA
Coffee break and Poster Session II (even numbers)
NON-CODING RNAS IN DEVELOPMENT
Chair: Mauro Giacca
12.15
Stefanie Dimmeler (Germany): Non-coding RNAs in angiogenesis
signaling
12.45
Harold Cremer (France): MicroRNA function in postnatal forebrain
neurogenesis
13.30
Lunch
15.45
Gabriella De Vita (Italy), short talk: A long non-coding RNA regulated in
development and cancer
16.00
Diana A. Alexieva (UK), short talk: Post transcriptional regulation of
microRNAs in embryonic stem cells
16.15
Stefano Volinia (Italy): Do pluripotent stem cell miRNAs and long noncoding RNAs have a role in cancer?
16.45
Coffee break
9
THERAPEUTIC APPLICATIONS OF NON-CODING RNAS
Chair: Roberto Corradini
17.15
Raymond Schiffelers (The Netherlands): Identification and delivery of
anti-angiogenic miRNAs
17.45
Margherita Iaboni (Italy), short talk: Aptamer-miRNA-212 conjugate
sensitizes NSCLC cells to TRAIL
18.00
Sakari Kauppinen
malignancies
18.30
Gianluigi Condorelli (Italy): Non coding RNAs in cardiac hypertrophy and
failure
19.00
Mauro Giacca (Italy): Functional high throughput screenings to identify
miRNAs inducing cardiac regeneration and overcoming cellular
senescence
20.30
Dinner: Meet the expert II (Speakers of day 3 and 4)
(Denmark):
Non-coding
RNAs
in
hematologic
WEDNESDAY, OCTOBER 15TH
THERAPEUTIC APPLICATIONS OF NON-CODING RNAS
Chair: Gianluigi Condorelli
9.30
David Carter (UK): Star roles for miRNAs in cancer drug resistance
10.00
Roberto Corradini (Italy): Modified PNA as anti-miR
10.30
Ross Conlon (Ireland), short talk: Extra-vesicular miRNA profiling of in
vitro & in vivo models of therapy resistant neuroblastoma
10.45
Susanna Obad (Denmark): Targeting of microRNAs for therapeutics
11.15
Open Discussion (R. Agami, M. Fabbri, P.P. Pandolfi):
"The revolutionary world of ncRNAs"
12.15
Concluding remarks
12.30
Lunch
10
ADDRESS LIST
OF
INVITED SPEAKERS
AND
ALL APPLICANTS
INVITED SPEAKERS
Irene Bozzoni
Dip. di Biologia e Biotecnologie
Charles Darwin
University La Sapienza
P.le Aldo Moro 5
00185 Rome, Italy
irene.bozzoni@uniroma1.it
Abstract:
26, 93
Harold Cremer
Developmental Biology
Institute of Marseille (IBDM)
Campus de Luminy
13288 Marseille, France
harold.cremer@univ-amu.fr
Abstract:
31
Sven Diederichs
German Cancer Research Center
(DKFZ) & Institute of Pathology
University Hospital Heidelberg
Im Neuenheimer Feld 280
D-69120 Heidelberg, Germany
s.diederichs@dkfz.de
Abstract:
33
Brian Brown
Department of Genetics and
Genomic Sciences
Mount Sinai School of Medicine
1425 Madison Avenue
New York, New York 10029, USA
brian.brown@mssm.edu
Abstract:
27
David Carter
Department of Biological and
Medical Sciences
Oxford Brookes University
Headington Campus, Gipsy Lane
Oxford OX3 0BP, UK
dcarter@brookes.ac.uk
Abstract:
28
Stefanie Dimmeler
Institute for Cardiovascular Regeneration,
Centre of Molecular Medicine
Goethe-University Frankfurt
Theodor-Stern-Kai 7
60590 Frankfurt, Germany
dimmeler@em.uni-frankfurt.de
Abstract:
34
Gianluigi Condorelli
Humanitas Research Hospital
Institute of Genetics and Biomedical
Research
National Research Council of Italy (CNR)
University of Milan
Via Manzoni 56,
Rozzano (MI), Italy
gianluigi.condorelli@unimi.it
Abstract:
29
Muller Fabbri
Keck School of Medicine
Norris Comprehensive Cancer Center
Children’s Center for Cancer
and Blood Diseases
Children's Hospital
Los Angeles CA, USA
mfabbri@chla.usc.edu
Abstract:
35, 53, 62, 100
Roberto Corradini
Department of Chemistry
University of Parma
Parco Area delle Scienze 17/A
43124 Parma, Italy
roberto.corradini@unipr.it
Abstract:
30
Mauro Giacca
ICGEB Trieste
Molecular Medicine Laboratory
Padriciano 99
34012 Trieste, Italy
giacca@icgeb.org
Abstract:
36, 98
12
Maite Huarte
Department of Oncology
CIMA Avd/Pio XII, 55
CIMA Building
31008 Pamplona (Navarra), Spain
maitehuarte@unav.es
Abstract:
37
Raymond Schiffelers
Department of Clinical Chemistry
and Haematology
Room G 03.647 UMC
Heidelberglaan 100
3584 CX, Utrecht The Netherlands
r.schiffelers@umcutrecht.nl
Abstract:
42
Sakari Kauppinen
Department of Clinical Medicine
Aalborg University
Department of Haematology
Aalborg University Hospital
A.C. Meyers Vænge 15
bldg. FK10B.215, DK-2450
Cph SV, Denmark
mskauppinen@gmail.com
Abstract:
38
John Thomson
MRC Human Genetics Unit
MRC IGMM,
University of Edinburgh
Western General Hospital
Crewe Road,
Edinburgh EH4 2XU, UK
jthoms24@staffmail.ed.ac.uk
Abstract:
43
Judy Lieberman
Program in Cellular and Molecular
Medicine, Boston Children's Hospital
Professor of Pediatrics
Harvard Medical School
200 Longwood Avenue, WAB 250
Boston MA 02115, USA
lieberman@idi.harvard.edu
Abstract:
39
Stefano Volinia
Department of Morphology,
Surgery and Experimental Medicine
University of Ferrara
Via Fossato di Mortara 64/b
44121 Ferrara, Italy
stefano.volinia@gmail.com
Abstract:
44, 103
Susanna Obad
Roche Innovation Center
Copenhagen A/S
Copenhagen Area,
Hørsholm, Denmark
SUO@Santaris.com
Abstract:
40
Nikolaus Rajewsky
Max Delbruck Center for
Molecular Medicine
Berlin-Buch
Robert-Rössle-Str. 10
13092 Berlin, Germany
rajewsky@mdc-berlin.de
Abstract:
41
13
APPLICANTS
Diana Alexieva
Imperial College London
Institute for Reproductive and
Developmental Biology
Du Cane Road,
Hammersmith Campus
W12 0NN, London, UK
diana.a.alexieva@gmail.com
Abstract:
46
Enrica Calura
Department of Biology
University of Padova
Via U.Bassi 58/B
Padova, Italy
enrica.calura@unipd.it
Abstract:
51
Gerolama Condorelli
Department of Molecular Medicine and
Medical Biotechnology
University of Naples "Federico II"
Via Pansini, 5
80121 Naples, Italy
gecondor@unina.it
Abstract:
64, 71, 87, 89
Lucia Altucci
Seconda Università di Napoli
Vico L. De Crecchio 7
80138 Naples, Italy
lucia.altucci@unina2.it
Abstract:
47
Anita Annese
Department of Biosciences,
Biotechnology and Biopharmaceutics
University of Bari
via Orabona 4
70126 Bari, Italy
anita.annese@gmail.com
Abstract:
48, 83
Ross Conlon
Royal College of Surgeons in Ireland
Cancer Genetics Group, 2nd Floor
York House, York St
D2 Dublin, Ireland
rossconlon@rcsi.ie
Abstract:
54
Giancarlo Bellenchi
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
giancarlo.bellenchi@igb.cnr.it
Abstract:
49, 68
Claudia Coronnello
Fondazione Ri.MED @ IBIM-CNR
via Ugo La Malfa 153
90146 Palermo, Italy
corocla@gmail.com
Abstract:
55
Teresa Maria Creanza
Institute of Intelligent Systems for
Automation
National Research Council
Via Amendola 122/D
70126 Bari, Italy
creanza@ba.issia.cnr.it
Abstract:
56
Agnieszka Belter
Institute of Bioorganic Chemistry,
Polish Academy of Sciences
Noskowskiego 12/14
61-704 Poznan, Poland
abelter@man.poznan.pl
Abstract:
50
14
Laura Crisafulli
Institute of Genetic and Biomedical
Research (IRGB)
Milan Unit
via Manzoni 113
20089 Rozzano (MI), Italy
laura.crisafulli@humanitasresearch.it
Abstract:
57
Floriana Della Ragione
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
floriana.dellaragione@igb.cnr.it
Abstract:
61
Ylenia D'Agostino
Cellular and Developmental Biology
Stazione Zoologica "Anton Dohrn"
Villa Comunale
80121 Naples, Italy
ylenia.dagostino@szn.it
Abstract:
58
Francesca Fanini
IRST Srl IRCCS
Via Piero Maroncelli 40
47014 Meldola FC, Italy
francesca.fanini@irst.emr.it
Abstract:
62, 100
Annalisa Fico
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
annalisa.fico@igb.cnr.it
Abstract:
63, 65
Roberto De Gregorio
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
roberto.degregorio@igb.cnr.it
Abstract:
49
Danilo Fiore
University of Naples "Federico II"
Department of Molecular Medicine
and Medical Biotechnology
Via Pansini 5
80131 Naples, Italy
danilo.fiore@unina.it
Abstract:
64, 71, 87, 89
Gabriella De Vita
Dip. di Medicina Molecolare e
Biotecnologie Mediche
Università degli Studi di Napoli Fed. II
Via S. Pansini 5
80131 Naples, Italy
gdevita@unina.it
Abstract:
59, 92
Alessandro Fiorenzano
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
alessandro.fiorenzano@igb.cnr.it
Abstract:
65, 63
Valentina Del Monaco
Ceinge - Biotecnologie Avanzate
Via Comunale Margherita, 484
80122 Naples, Italy
delmonaco@ceinge.unina.it
Abstract:
60
Francesca Garibaldi
IFO-Istituti Fisioterapici Ospitalieri
Istituto Nazionale dei Tumori
"Regina Elena"
Via delle Messi d'oro 156
00158 Rome, Italy
garibaldi@ifo.it
Abstract:
66, 69
Carmela Dell'Aversana
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
dellaversanalara@gmail.com
Abstract:
47
15
Giuliano Giuliani
University of Utrecht
Universiteitsweg 100
Utrecht, The Netherlands
ggiuliani@umcutrecht.nl
Abstract:
67
Florian Kuchenbauer
University Hospital of Ulm
Department of Internal Medicine III,
Albert-Einstein-Allee 23
89081 Ulm, Germany
florian.kuchenbauer@uni-ulm.de
Abstract:
73
Giovanna Grimaldi
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
giovanna.grimaldi@igb.cnr.it
Abstract:
68
Alireza Labani Motlagh
Umea University
Historiegrand 1 V, 1001
90734 Umea, Sweden
alireza.labani@climi.umu.se
Abstract:
74
Aymone Gurtner
Regina Elena National Cancer Institute
via delle Messi d'Oro 156
00158 Rome, Italy
gurner@ifo.it
Abstract:
69, 66
Shraddha Lad
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
shraddha.lad@igb.cnr.it
Abstract:
68
Adnan Hashim
University of Salerno
Campus di Medicina e Chirurgia
Via. S. Allende, 1
84081 Baronissi (SA)
margheritaiabo@libero.it
Abstract:
70
Anna Li Santi
University of Salerno
Giovanni Paolo II
84084 Fisciano (SA), Italy
alisanti@unisa.it
Abstract:
75
Margherita Iaboni
University of Naples "Federico II"
Via Pansini, 5
80121 Naples, Italy
ahashim@unisa.it
Abstract:
71, 64, 87, 89
Ada Marino
Dipartimento di Scienze Mediche
Traslazionali (Dismet)
University of Naples Federico II
Via Pansini, 5
80131, Naples, Italy
Abstract:
76
Mariia Inomistova
National Cancer Institute of the
Ministry of Public Health of Ukraine
33/43, Lomonosova
03022 Kyiv, Ukraine
m.inomistova@gmail.com
Abstract:
72
Marija Mihailovic
European Institute of Oncology
Via Adamello 16
20139 Milan, Italy
marija.mihailovic@ieo.eu
Abstract:
77
16
Linda Minotti
University of Ferrara
Via Fossato di Mortara 70
c/o "CUBO"
44121 Ferrara, Italy
mntlnd@unife.it
Abstract:
103
Graziano Pesole
University of Bari and IBBE-CNR
via Orabona, 4
70125 Bari
graziano.pesole@uniba.it
Abstract:
83, 48
Irina Pinheiro
EMBL Monterotondo
Via Ramarini 32
00015 Monterotondo (Rome), Italy
irinasmp@gmail.com
Abstract:
84
Stefania Oliveto
INGM - Fondazione Istituto Nazionale
Genetica Molecolare
Via Francesco Sforza, 35,
20122 Milan, Italy
oliveto@ingm.org
Abstract:
78
Nicoletta Potenza
Second University of Naples
Department of Environmental,
Biological and Pharmaceutical Sciences
and Technologies
via Vivaldi 43
81100 Caserta, Italy
nicoletta.potenza@unina2.it
Abstract:
85
Massimiliano Pagani
Istituto Nazionale Genetica Molecolare
Via F. Sforza 35
20122 Milano, Italy
pagani@ingm.org
Abstract:
79
Valeria Ranzani
Istituto Nazionale Genetica Molecolare
(INGM)
Via Francesco Sforza 35
20122 Milano, Italy
ranzani@ingm.org
Abstract:
79
Sara Pagotto
Ageing Research Center (Ce.S.I.)
G. d'Annunzio University Foundation
Via Luigi Polacchi 11
66100 Chieti, Italy
sara.pag8@gmail.com
Abstract:
80, 82
Laura Ricci
Department of Clinical and Biological
Science
University of Turin, Italy
Regione Gonzole, 10
Orbassano, 10043 Turin, Italy
ricci.laura134@gmail.com
Abstract:
86
Olga Patutina
Institute of Chemical Biology and
Fundamental Medicine SB RAS
Lavrentiev Ave. 8
630090 Novosibirsk, Russia
olga.patutina@gmail.com
Abstract:
81
Giuseppina Roscigno
Department of Molecular Medicine and
Medical Biotechnology
University of Naples "Federico II"
Via Pansini, 5
80121 Naples, Italy
giusy_roscigno@yahoo.it
Abstract:
87, 64, 89
Felice Pepe
Ageing Research Center (Ce.S.I.)
G. d'Annunzio University Foundation
Via Luigi Polacchi 11
66100 Chieti, Italy
felice.pepe@unich.it
Abstract:
82
17
Arefeh Rouhi
University Hospital of Ulm
Department of Internal Medicine III
Albert-Einstein-Allee 23
89081 Ulm, Germany
arefeh.rouhi@uni-ulm.de
Abstract:
73
Ilaria Sciamanna
Istituto Superiore di Sanità
Viale Regina Elena 299
00161 Rome, Italy
ilaria.sciamanna@iss.it
Abstract:
91
Francesco Russo
Laboratory of Integrative Systems
Medicine (LISM)
IIT-IFC-CNR
Department of Computer Science
University of Pisa
Via Giuseppe Moruzzi 1
54124 Pisa, Italy
francesco.russo@iit.cnr.it
Abstract:
88
Rosa Maria Sepe
Stazione Zoologica Anton Dohrn
Villa Comunale
80121 Naples, Italy
ros.sepe@live.com
Abstract:
92
Sama Shamloo
Sapienza – Università di Roma
Department of Biology and Biotechnology
"Charles Darwin"
Piazzale Aldo Moro 5
00185 Rome, Italy
sama.shamloo@gmail.com
Abstract:
93
Valentina Russo
University of Naples "Federico II"
Via Pansini, 5
80100 Naples, Italy
valentina.russo86@alice.it
Abstract:
89, 64, 71, 87
Annamaria Salvati
University of Salerno
Laboratory of Molecular Medicine and
Genomics
Via S. Allende, 1
84081 Baronissi (SA), Italy
asalvati@unisa.it
Abstract:
90
Miroslawa Skupinska
Polish Academy of Sciences
Institute of Bioorganic Chemistry
ul. Noskowskiego 12/14
61-704 Poznań, Poland
mirkaj@ibch.poznan.pl
Abstract:
94
Edith Schneider
Ulm University
Department of Internal Medicine III
Albert-Einstein Allee 23
89081 Ulm, Germany
edith.schneider@uni-ulm.de
Abstract:
73
Mario Leonardo Squadrito
École Polytechnique Fédérale
de Lausanne (EPFL)
Station 19,
1015 Lausanne, Switzerland
mario.squadrito@epfl.ch
Abstract:
95
18
Antonietta Tarallo
TIGEM
Telethon Institute of Genetics and
Medicine
Via Campi Flegrei 34
80078 Pozzuoli (NA), Italy
tarallo@tigem.it
Abstract:
96
Ivan Zaporozhchenko
Institute of Chemical Biology and
Fundamental Medicine RAS
prosp. ac. Lavrentyev, 8
630090 Novosibirsk
Russia
ivanzap@niboch.nsc.ru
Abstract:
102
Valeria Tarallo
Institute of Genetics and Biophysics
“ABT”
National Research Council (CNR)
Via P. Castellino 111
80131 Naples, Italy
valeria.tarallo@igb.cnr.it
Abstract:
97
Carlotta Zerbinati
University of Ferrara
Via Fossato di Mortara 70 c/o CUBO
44121 Ferrara, Italy
zrbclt@unife.it
Abstract:
103
Consuelo Torrini
International Centre for Genetic
Engineering
and Biotechnology (ICGEB)
Padriciano, 99
34149 Trieste, Italy
torrini@icgeb.org
Abstract:
98
Vamshidhar Reddy Vangoor
University of Utrecht
Universiteitsweg 100
Utrecht, The Netherlands
v.vangoor@umcutrecht.nl
Abstract:
99, 67
Ivan Vannini
IRST S.r.l. IRCCS
via Piero Maroncelli 40
47014 Meldola, Italy
ivan.vannini@irst.emr.it
Abstract:
100, 62
Marianna Vitiello
Oncogenomics Unit
Core Research Laboratory
Istituto Toscano Tumori
(CRL-ITT)
Via Moruzzi 1
56124 Pisa, Italy
mariannavitiello@live.it
Abstract:
101
19
GRAZIELLA PERSICO
photo by Anna M. Aliperti
Plenary Lecture
The Non-Coding RNA Revolution in Cancer Research
Pier Paolo PANDOLFI
Cancer Center @ Beth Israel Deaconess Medical Center and
Harvard Medical School, Boston, MA, USA
We will discuss exciting new data regarding the role of pseudogenes,
lincRNAs and miRNAs in the pathogenesis of human cancer as also
studied in vivo in the mouse. We will also focus on competing endogenous
RNAs (ceRNAs), circular(circ)-ceRNAs and pseudo-ceRNAs and give
important attention to how understanding the ceRNA language will facilitate
efforts to deconvolute ceRNA networks and their role in tumorigenesis.
22
SPEAKERS
ABSTRACTS
Enhancer RNAs at the service of p53
Reuven AGAMI
Division of Biological Stress Response, The Netherlands Cancer Institute, The Netherlands
Enhancers are genomic domains that regulate transcription of distantly
located genes and that are characterized by specific chromatin signatures
of histone methylation and acetylation patterns. Interestingly, RNA
polymerase II binds to a subset of enhancers and produces transcripts,
called enhancer RNAs (eRNAs). It is unclear if eRNAs carry a
transcriptional function. p53 is a transcription factor and tumor suppressor
that is very frequently mutated in cancer. Chromatin-binding profiles reveal
specific interactions of p53 with promoter regions of nearby genes, within
genes, but also with remote regions located more than 50 kbps away from
any known gene, suggesting a role as enhancer factor. Indeed, many of
these remote regions possessed evolutionary highly conserved p53-binding
sites and all known hallmarks of enhancer regions, as well as binding of
RNAPII. While p53 binding to promoter regions locally activates expression
of one gene, its binding in the context of enhancers may affect several
distant genes in tissue-dependent manners. We found out that many
remote p53-bound domains are indeed p53-dependent eRNA-producing
enhancers, and, most importantly, eRNA production of at least some
regions seemed to be required for transcriptional induction of distal genes
and for p53-dependent cellular control 1.
Intriguingly, though many p53-induced enhancers contained p53-binding
site, many did not. As long-noncoding RNAs (lncRNAs) are prominent
regulators of chromatin function, we hypothesized that p53-induced
lncRNAs contribute to activation of enhancers by p53. We investigated the
link between p53, lncRNAs and enhancer activity using eRNA expression
analysis, and connect it to p53 function as a tumor suppressor.
Reference
1. Melo, C.A. et al. eRNAs are required for p53-dependent enhancer activity and gene
transcription. Molecular cell 49, 524-35 (2013).
25
Regulatory circuitries controlled by long non coding RNAs
Monica Ballarino, Mariangela Morlando, Ivano Legnini, Irene BOZZONI
Department of Biology and Biotechnology, University “La Sapienza”, Rome, Italy
In the RNA field we are witnessing a big effort in trying to integrate the
function of long non-coding RNAs (lncRNAs) in the molecular circuitries
controlling cell proliferation and differentiation. Along this direction, we
recently discovered linc-MD1, a cytoplasmic lncRNA, which regulates the
transition from early to late phases of differentiation by acting as a
competing endogenous RNA. By binding miR-133 and miR-135, linc-MD1
impacts on their activity on the corresponding mRNA targets. Notably, lincMD1 is also the host transcript of miR-133b and their biogenesis is mutually
exclusive.
Besides the already identified miRNA targets, Maml1 and Mef2C, we
described HuR as another component of the linc-MD1-regulated circuitry:
HuR is under the repressive control of miR-133 and, during early stage of
myogenesis, its expression is sustained by the ceRNA activity of linc-MD1.
Moreover, we have shown that, besides being a component of the linc-MD1
circuitry, HuR is also able to control linc-MD1 biogenesis and to favour its
accumulation at the expense of miR-133b biogenesis, thus establishing a
positive feed-forward control.
Notably, this positive loop between HuR and linc-MD1, operates in a
specific window of time; exit from this circuitry is insured by increased miR133 expression, at later stages of differentiation, due to the activation of
miR-133a-1 and miR-133a-2 transcribed from two unrelated genomic loci.
More recently, we have expanded the existing collection of murine myolncRNAs by using a strand-specific RNA sequencing approach on growing
C2C12
myoblasts
and
differentiated
myotubes.
A
preliminary
characterization of selected newly identify lncRNAs will be presented.
26
Understanding the relationship between microRNA
concentration and activity
Brian D. BROWN
Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine,
New York, USA
We are investigating the quantitative relationship between microRNA
concentration and target suppression, including questions related to how
changes in microRNA abundance affect the expression of a microRNA’s
network of targets. We are also examining how target affects microRNA
activity, and have uncovered evidence that target regulation can accelerate
the decay of a microRNA; providing a negative feedback mechanism for
control of microRNA activity.
27
Star roles for miRNAs in cancer drug resistance
David R. F. CARTER, Ryan R. Pink, Priya Samuel, Laura A. Jacobs
Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK.
Each year 125,000 women are killed by ovarian cancer, more than all other
gynaecological cancers combined. Ovarian cancer is associated with a
poor prognosis, with only around 40% of diagnosed women alive after 5
years. Treatment usually involves removal surgery and chemotherapy with
platinum-based compounds such as cisplatin. The majority of patients
respond well initially, but eventually, in most cases, a chemoresistant and
metastatic malignancy recurs, sometimes following multiple rounds of
treatment. Understanding the molecular basis of chemoresistance is
therefore imperative if we are to prolong the effective treatment of ovarian
cancer and to identify novel treatment strategies that may be effective in
resistant disease. Here we describe the characterisation of miRNA levels in
ovarian cancer cell lines that are either sensitive or resistant to cisplatin
treatment. Using loss or gain of function experiments we have identified two
miRNAs that directly contribute to the level of cisplatin resistance.
Characterisation of these miRNAs provides insight into the molecular
mechanisms that govern the acquisition of cisplatin resistance during
ovarian cancer progression.
28
Identification of temporally modulated lncRNAs
in cardiac hypertrophy and failure
1
Stirparo G, 1,2Greco C, 1,2Rossi P, 1,2Kunderfranco P, 1,2Carullo P, 1,2Serio S,
1,2
Papait R, 1,2,3CONDORELLI G
1
2
Humanitas Research Hospital, Rozzano (MI), Italy; Institute of Genetics and Biomedical
3
Research, National Research Council of Italy; University of Milan, Italy
Gene expression reprogramming in cardiac myocytes is a key feature of heart
hypertrophy and failure. Many mechanisms are involved in the control of gene
expression. Among these, non-coding RNAs (ncRNA), such as long ncRNA
(lncRNA), antisense RNA and pseudogenes, are gaining importance as
regulatory elements in several cellular process, such as cell growth, apoptosis
and development. Not surprisingly, the dysregulation of these RNAs has been
found to cause several human diseases, such as cancer (e.g., prostate and
breast cancer) and neurodevelopmental diseases (e.g., Alzheimer's disease and
spinocerebellar ataxia) and, more recently, to be implicated in cardiac
commitment. Despite this, little is known about their involvement in cardiac
hypertrophy and how they regulate gene expression in this pathology. The aim of
this study was to provide a map of ncRNAs modulated in cardiac hypertrophy. To
do this, we performed ribo-depleted RNA-sequencing on cardiomyocyte RNA
isolated from mice subjected to transverse aortic constriction (TAC) for 1, 2, 4
and 7 days. We identified ~130 lncRNA, which we divided into different classes
(antisense RNA, lincRNA, processed transcripts, etc.) according to the Ensembl
glossary. We found that the lncRNA signature was time-dependent modulated
and could play a central role in gene expression reprogramming during cardiac
hypertrophy. Furthermore, by clustering genes by their temporal expression
profiles (with Short Time-series Expression Miner), we are able to define how
biological process were modulated through time. For one cluster enriched for
processes involving methyltransferase, we found an over-representation of
lncRNA. This is in line with the idea that lncRNA could contribute to epigenetic
reprogramming by regulating or interacting with genes involved in this process.
Moreover, because identification of novel lncRNAs is critical for the
understanding of the intrinsic complexity of the transcriptome, we developed a
method to identify novel lincRNA, exploiting both epigenetic modification and
RNA-sequencing data, which allowed us to more accurately define transcribed
regions. These new lncRNAs are being tested and their role in cardiac
hypertrophy and failure defined.
29
Modified PNA as anti-miR
Roberto CORRADINI, Alex Manicardi
Department of Chemistry, University of Parma, Parma, Italy
Micro-RNA (miR) targeting is a process more and more important in the
development of new therapeutics.1 Peptide nucleic acids (PNAs) have been
extensively used for targeting mRNA in the antisense approach and DNA in
the anti-gene approach for the down-regulation of the expression of target
genes. More recently, PNAs have been used for the inhibition of miR
activity (anti-miR approach).
Modification of the PNA either by conjugation or by chemical modification at
the backbone or at the nucleobase have been extensively used in the last
years. The present lecture will deal with work carried out in our laboratory
for the synthesis of conjugated, backbone-modified, nucleobase-modified,
and polyfunctional PNA.
We have recently described the synthesis of anti-miR PNA either
conjugated with a carrier peptide or bearing modified residues along the
chain.2-5 PNA of high affinity and high specificity for miR210 and miR221,
involved in erythroid differentiation and tumor progression respectively,
were obtained. Modified PNA showed improved biovailability and effectively
entered into tumor cells and exerted anti-miR activity, leading to upregulation of genes.2-4 Backbone modified PNAs, bearing incorporated
arginine side chains showed improved cellular uptake, and higher
biostability than the peptide-conjugated, and effectiveness of these
compounds was shown to depend on the type of substitution and on the
distribution of charges within the PNA strand.5 More recently, several
strategies based on modification of the PNA structure during solid-phase
synthesis have been developed to allow precise positioning of multiple
functions (reporter groups, binding elements or catalytic moieties) within the
PNA chain, and either within the minor or the major groove of the
PNA:nucleic acid duplexes.6,7 The rationale of the design of these
multifunctional PNA and of multi-functional materials8 in the context of antimiR strategy will be discussed.
References
1. R. Gambari, et al Biochemical Pharm 2011 , 82, 1416-1429.
2. E. Fabbri et al ChemMedChem 2011, 6, 2192-2202.
3. E. Brognara et al. Int. J Oncol. 2012, 2119-2127.
4. E. Brognara et al. J Neurooncol. 2014, 118 19–28.
5. A. Manicardi et al ChemBiochem 2012, 13, 1327 – 1337.
6. A. Manicardi et al. Artificial DNA: PNA & XNA 2012, 3, 53-62.
7. A. Manicardi et al. Beilstein J. Org. Chem. 2014, 10, 1495-1503.
8. A. Bertucci et al. Adv. Healthcare Mater. 2014, doi: 10.1002/adhm.201
30
MicroRNA function in postnatal forebrain neurogenesis
Philipp Follert, Nathalie Coré, Antoine de Chevigny, Christophe Beclin and
Harold CREMER
Developmental Biology Institute of Marseille (IBDM), Campus de Luminy, Marseille, France
In the postnatal and adult brain neuronal stem cells along the walls of the
lateral ventricles generate permanently neuronal progenitors that migrate
into the olfactory bulb (OB) where they differentiate into interneurons.
These new neurons are highly heterogeneous with respect to
neurotransmitter phenotype (GABA, dopamine and glutamate), position in
their target layers and innervation pattern. Neuronal heterogeneity is based
on regionalized neural stem cells occupying defined positions along the
wall of the lateral ventricles, implying the existence of molecular gradients
that pattern the ventricular wall. Moreover, the progression of a neural stem
cell into a mature OB neuron is a highly controlled process with defined
differentiation intermediates. We studied the implication of microRNAs in
the control of both, stem cell regionalization and differentiation, in the
system.
We used brain electroporation and microarray approaches to generate
high-resolution microRNA and mRNA expression data in space and time.
Based thereon we investigated the interplay between microRNAs and
mRNAs at the level of stem cell determination and show that a gradient of a
defined microRNA, miR-7a, confines the expression of the transcription
factor Pax6 to dorsal neural stem cells. This interaction is essential for
controlled generation of dopaminergic neurons in the OB. At the level of
differentiation, we found that the miR-200 family of microRNAs is
specifically induced during late stages of the neurogenic process and
demonstrate its implication in the regulation of the terminal neuronal
differentiation.
31
Causes and consequences of microRNA
dysregulation in cancer
Carlo M. CROCE
Department of Molecular Virology, Immunology and Medical Genetics
The Ohio State University Medical Center
Since the discovery of miR-15a and miR-16-1 deletions in CLL, many
laboratories around the world have shown miRNA dysregulation in all
tumors studied, including the most common, such as lung, breast, prostate
and gastrointestinal cancers. Such dysregulation, like the dysregulation of
oncogenes and tumor suppressor genes, can be caused by multiple
mechanisms, such as deletion, amplification, mutation, transcriptional
dysregulation and epigenetic changes.
As miRNAs have multiple targets, their function in tumorigenesis could be
due to their regulation of a few specific targets, possibly even one, or many
targets. A future challenge will be to identify all of the targets of the miRNAs
involved in cancer and establish their contribution to malignant
transformation. An additional challenge will be the identification of all of the
miRNAs that are dysregulated by pathways that are consistently
dysregulated in various types of human cancers. This point is of particular
importance, as instead of focusing on specific alterations in protein-coding
oncogenes or tumour suppressor genes — which may be difficult to treat —
we could focus on their downstream miRNA targets. If these miRNA targets
are crucial for the expression of the malignant phenotype and the cancer
cells depend on their dysregulation for proliferation and survival, we can
expect that the use of miRNAs or anti-miRNAs will result in tumor
regression. Genomic analyses for alteration in miRNA genes or for copy
number alterations in various human tumors by deep sequencing is in
progress but has not been completed. These studies could provide
additional information concerning the involvements of miRNAs in cancer
and in many other diseases.
Over the past few years, we have observed a shift from conventional
chemotherapy to targeted therapies, and miRNAs and anti-miRNAs will
contribute extensively to the latter.
32
Long non-coding RNAs in cancer
Sven DIEDERICHS
“Molecular RNA Biology & Cancer”, German Cancer Research Center (DKFZ) &
Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany
The highly conserved long non-coding RNA MALAT1 was one of the first
lncRNAs associated with cancer (1) as a predictive marker for metastasis
development in lung cancer (2). However, its high abundance and nuclear
localization have hampered its functional analysis due to its inefficient knockdown by RNAi. To uncover its functional importance, we developed a MALAT1
knockout model in human lung tumor cells by genomically integrating RNA
destabilizing elements site-specifically into the MALAT1 locus using Zinc
Finger Nucleases (ZFN). This approach yielded a more than 1000-fold
silencing providing a unique loss-of-function model (3).
Proposed mechanisms of action of MALAT1 include regulation of splicing or
gene expression. In lung cancer, MALAT1 does not alter alternative splicing
but actively regulates gene expression inducing a signature of metastasisassociated genes. Consequently, MALAT1-deficient cells are impaired in
migration and form fewer tumor nodules in a mouse xenograft model.
Antisense Oligonucleotides (ASOs) blocked MALAT1 expression effectively in
the cell culture and in the animal. Notably, MALAT1-ASO treatment prevents
metastasis formation after tumor implantation. Thus, targeting MALAT1 with
antisense oligonucleotides provides a potential therapeutic approach to
prevent lung cancer metastasis with MALAT1 serving as both, predictive
marker and therapeutic target (4).
To discover novel cancer-associated lncRNAs, we defined their expression
landscape in lung, breast and liver cancer and normal tissue from these organs
(N=150). We provide a comprehensive map of 17000+ lncRNAs discovering
hundreds of new lncRNAs associated with three major tumor entities. We
exploited our expression map to generate an siRNA library specifically
targeting 638 tumor-induced lncRNAs. This comprehensive but focused library
elucidates the role of lncRNAs in tumorigenesis, cell viability, mitosis, migration
and the DNA damage response.
References:
1. T Gutschner et al. "The Hallmarks of Cancer: A long non-coding RNA point of view" RNA
Biology (2012) 9: 703-719
2. P Ji*, S Diederichs* et al. "MALAT-1, a novel non-coding RNA, and Thymosin b4 predict
Metastasis and Survival in early-stage Non-Small Cell Lung Cancer" Oncogene (2003)
22: 8031-8041
3. T Gutschner et al. "Non-coding RNA gene silencing through genomic integration of RNA
destabilizing elements using zinc finger nucleases" Genome Research (2011) 21: 19441954
4. T Gutschner et al. "The non-coding RNA MALAT1 is a critical regulator of the metastasis
phenotype of lung cancer cells" Cancer Research (2013) 73: 1180-1189
33
Non-coding RNAs in angiogenesis signaling
Stefanie DIMMELER
Institute of Cardiovascular Regeneration, Centre of Molecular Medicine,
Goethe-University Frankfurt, Germany
In the last years, it has become evident that the majority of the genome is
transcribed, while only about 2% codes for proteins. These so-called non-coding
RNAs gained increasing attention as multifactorial regulators of gene expression.
MicroRNAs (miRs) are small non-coding RNAs that bind to target mRNAs
thereby inducing degradation or translational repression, whereas long noncoding RNAs act as epigenetic regulators of gene expression or by modulating
splicing. Several miRs were shown to regulate vascular function, angiogenesis
and atherosclerosis. Particularly, we and others have shown that miR-92a
impairs endothelial cell functions (Bonauer et al, Science 2009) and its inhibition
improves the recovery after cardiac ischemia and prevents atherosclerotic lesion
formation. These findings were recently confirmed in large animal models and we
showed that LNA-based antimiRs improve heart function in pigs after
ischemia/reperfusion (Hinkel et al, Circulation 2013). The presentation will
summarize these findings and additionally will provide some recent insights into
the regulation and function of hypoxia regulated long non-coding RNAs in
endothelial cells.
34
miRceptors and exosomic microRNAs dictate
the biology of the tumor microenvironment
Muller FABBRI
Departments of Pediatrics and Molecular Microbiology & Immunology, University of
Southern California- Keck School of Medicine, Norris Comprehensive Cancer Center,
Children’s Center for Cancer and Blood Diseases, Children’s Hospital Los Angeles, Los
Angeles, CA, USA
MicroRNAs (miRNAs) regulate gene expression mainly (but not exclusively)
by binding to the 3’-untranslated region of target mRNAs. Recently, it has
been shown that miRNAs are also secreted by cells (including cancer cells)
within extracellular vesicles called exosomes. Through this mechanism
exosomic miRNAs participate to paracrine intercellular communication
between different cell populations of the tumor microenvironment. We have
recently discovered that in addition to their “traditional” mechanism of gene
expression regulators, exosomic miRNAs can also bind to and activate Tolllike receptors, triggering the NF-kB inflammatory pathway and the secretion
of pro-tumoral inflammatory cytokines. Intriguingly, the discovery of
receptors for miRNAs (which we called “miRceptors”) suggests that
miRNAs might have more profound implications in affecting the biology of
the tumor microenvironment than expected. This lecture will focus on these
aspects of miRNA biology and their implications in the identification of new
molecular targets for cancer patients.
35
Functional high throughput screenings to identify miRNAs inducing
cardiac regeneration and overcoming cellular senescence
Mauro GIACCA
International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
Lack of cellular replication is a major cause of most prevalent degenerative
conditions, including heart failure and neuronal degeneration, and a strong
correlate of aging. A major goal is thus to identify global regulators of cellular
replication that might revert cellular aging and promote tissue regeneration in vivo.
We first wanted to identify miRNA mimics able to the proliferation of cardiac
myocytes and thus stimulate cardiac repair. By high throughput screening using a
whole genome human miRNA mimics library, we found 40 miRNAs that stimulate
both neonatal and adult cardiomyocyte proliferation, as evaluated by EdU
incorporation (DNA synthesis), phospho-H3 positivity (G2/M progression) and
Aurora B staining in midbodies (karyokinesis). Deep sequencing of cardiac
miRNAs revealed that several of the identified miRNAs were expressed in
neonatal, replicating cardiomyocytes but not in adult cardiomyocytes. Two of the
identified miRNAs were tested in vivo and found to promote cardiac regeneration
after myocardial infarction in adult mice.
A second HTS was performed to search for miRNAs able to restore replication of
human diploid fibroblasts approaching senescence. Screening endpoints were the
simultaneous analysis of EdU incorporation and of the levels of the cell cycle
kinase inhibitor p21. We identified 20 miRNAs that exert a strong replicative effect
on senescent cells (up to 40% EdU-positive cells compared to a basal
incorporation of <10%) while markedly reducing p21 levels. Cells treated with
these miRNAs were less enlarged, had decreased expression of p16 and reverted
their senescence-associated secretory phenotype. Interestingly, all of the identified
miRNAs were capable of exerting their pro-proliferative action even in the absence
of serum.
There was only a partial overlap between the sets of miRNAs identified in the two
screens, underlying different mechanisms of action. In the case of miRNAs
inducing cardiomyocyte proliferation, fate mapping experiments in transgenic mice
indicated that most of the selected miRNAs directly act by promoting the
proliferation of differentiated cardiac cells and not that of undifferentiated stem
cells. Most of these miRNAs do so by targeting the mRNAs for several cytoskeletal
proteins, suggesting that assembly of the contractile apparatus, and not cellular
senescence, is a major inhibitor of proliferation for adult cardiac cells.
Remarkably, for both cardiomyocytes and senescent fibroblasts, stimulation of cell
proliferation by several of the identified miRNAs could also be achieved by using
the supernatants of the miRNA-treated cells, suggesting that the pro-proliferative
and anti-aging effects can be mimicked by secreted factors.
Both the identified miRNAs and the soluble factors mediating their effects might
represent leads for the development of novel biotherapeutics to combat tissue
degeneration in ageing.
36
The lncRNA components of the p53 network
Maite HUARTE
Oncology Department, CIMA, University of Navarra, Pamplona, Spain
How do cells coordinate and integrate information to produce the adequate
gene expression output? For decades genetics has focused in the study of
protein-coding genes that control this critical balance. However, cellular
networks are fine-tuned and maintained by the coordinated function of not
only proteins, but also non-coding RNAs (ncRNAs). In addition to the wellcharacterized protein-coding constituents, large non-coding RNAs are
emerging as important regulatory molecules in tumor-suppressor and
oncogenic pathways. Supporting this idea, we have found that the
transcription factor p53, which is crucial for the maintenance of cellular
homeostasis, specifically regulates the expression of dozens of large
intergenic non-coding RNA genes (lncRNAs). These lincRNAs are bonafide transcriptional targets of p53, and are induced by p53 to modulate
specific facets of the p53 cellular response, including the regulation of gene
expression through epigenetic mechanisms. Altogether, our work suggests
that large non-coding RNAs constitute an unknown layer of regulation of
the p53 cellular response that could represent future novel targets for
cancer treatments.
37
Non-coding RNAs in hematologic malignancies
Sakari KAUPPINEN1,2
1
Department of Clinical Medicine, Aalborg University, København SV, Denmark;
2
Department of Haematology, Aalborg University Hospital, Denmark
Non-coding RNAs play important regulatory roles in many biological
processes and are frequently dysregulated in human diseases, including
cancer. We are exploring the functions of dysregulated ncRNAs in a variety
of hematologic malignancies with the aim of translating our discoveries into
improved treatments of these diseases. We will provide an update on
genome-wide analysis of ncRNA expression in hematologic cancers and
describe recent progress in the discovery of ncRNA-based therapeutics for
the treatment of multiple myeloma.
38
Cancer crosstalk: miRNA transfer by exosomes between cancer cells
confers metastatic capability
Minh Le, Judy LIEBERMAN
Program in Cellular and Molecular Medicine, Boston Children’s Hospital and
Department of Pediatrics, Harvard Medical School
Not all cancer cells in a tumor are capable of metastasizing. The miR-200
microRNA family, which regulates the mesenchymal-to-epithelial transition,
is enriched in the serum of patients with metastatic cancers, and ectopic
expression of miR-200 can confer metastatic ability to poorly metastatic
tumor cells in some settings. We investigated whether metastatic capability
could be transferred between metastatic and non-metastatic triple negative
breast cancer cells via extracellular vesicles. Metastatic breast cancer cell
lines highly expressing miR-200 secrete miR-200 microRNAs in
extracellular vesicles and transfer them in vivo to otherwise weakly
metastatic cells either nearby or at distant sites and confer upon them the
ability to colonize distant tissues in a miR-200-dependent manner. Thus,
uptake of extracellular vesicles can transfer metastatic capability in vivo.
39
Targeting microRNAs for therapeutics
Susanna OBAD
Roche Innovation Center Copenhagen (RICC), Hørsholm, Denmark
MicroRNAs act as important post-transcriptional regulators of gene
expression by mediating mRNA degradation or translational repression.
There is now ample evidence that perturbations in the levels of individual or
entire families of miRNAs are associated with the development of a wide
variety of human diseases. Besides cancer, miRNAs have also been
implicated in viral infections, cardiovascular diseases and CNS disorders.
Thus, disease-associated miRNAs represent a potential new class of
targets for oligonucleotide-based therapeutics, which may yield patient
benefits unobtainable by other therapeutic approaches.
LNA is a bicyclic high-affinity RNA analogue, in which the ribose ring is
locked in a RNA-like, N-type (C3'-endo) conformation by the introduction of
a 2'-O,4'-C methylene bridge. Transfection of LNA-modified antimiR
oligonucleotides into cells results in potent and specific inhibition of miRNA
function with concomitant de-repression of direct target mRNAs. In addition,
systemically
delivered,
unconjugated
LNA-antimiRs
with
a
phosphorothioate backbone show high metabolic stability and uptake in
many tissues in mice, coinciding with long-term miRNA silencing in vivo.
These findings support the utility of LNA-antimiR oligonucleotides in the
development of therapeutic strategies aimed at pharmacological inhibition
of disease-associated miRNAs. We will describe recent progress in
targeting of miRNAs for therapeutics using LNA-antimiRs.
40
Regulatory RNAs
Nikolaus RAJEWSKY
Max Delbruck Center for Molecular Medicine, Division of Systems Biology and Berlin
Institute for Medical Systems Biology, Berlin, Germany
I will summarize our current efforts to understand more about biogenesis
and expression of circular RNAs. Biogenesis is analyzed using
computational and biochemical approaches. Expression is studied in cell
lines and human clinically relevant samples.
I will further discuss non-coding RNAs as possible competitors for binding
and present a quantitative model that makes testable predictions about
“sponge” or “cross-talk” effects.
41
Identification and delivery of anti-angiogenic miRNAs
N. Babae1, M. Bourajjaj2, Y. Liu3, J.R. van Beijnum4, F. Cerisoli2, P.V.
Scaria3, M. Verheul2, M.P. van Berkel4, E.H.E. Pieters1, R.J. van Haastert2,
A. Yousefi1, E. Mastrobattista1, G. Storm1, E. Berezikov5, E. Cuppen5, M.
Woodle3, R.Q.J. Schaapveld2, G.P. Prevost2, A.W. Griffioen4, P.I. van
Noort2, R.M. SCHIFFELERS
1
2
Utrecht Institute for Pharmaceutical Sciences, Utrecht, The Netherlands; InteRNA
3
Technologies B.V., Utrecht, The Netherlands; Aparna Biosciences Corporation, Rockville
4
5.
MD, USA ; VU University Medical Center, Amsterdam, The Netherlands; Hubrecht
6
Institute, Cancer Genomics Center, Utrecht, The Netherlands; Laboratory Clinical
Chemistry & Haematology, University Medical Center Utrecht, The Netherlands
Angiogenesis is one of the hallmarks of cancer. Therefore it constitutes an
attractive therapeutic target for cancer treatment. Several pharmaceuticals
have been successfully developed to inhibit tumor angiogenesis and are
currently approved for treatment of various types of cancer. But despite
their regulatory approval, the therapeutic benefit of these therapeutics
remains modest and resistance against these treatments emerges.
miRNA expression levels in endothelial cells (EC) change after angiogenic
stimulation shifting the expression profile of miRNAs towards proangiogenic miRNA species that inhibit anti-angiogenic genes. Our
approach is based on restoring the balance by delivery of anti-angiogenic
miRNAs
By screening a lentiviral miRNA library we identified anti-angiogenic
miRNAs, which were able to inhibit the proliferation of primary and
immortalized EC. These miRNAs were evaluated in in vitro and in vivo
studies. We confirmed the ability of several miRNAs to inhibit the
angiogenic properties of EC in various in vitro angiogenesis assays. Each
of these assays was focused on different features of the process, such as
migration, sprouting and two-dimensional tube formation.
The mechanism of action of the miRNAs was investigated by transcriptional
analysis using RNA-seq in combination with in silico target prediction
analysis. Depending on the mIRNA species we observed between 2002500 differentially expressed genes. Interestingly, many of these genes are
known to be involved in angiogenesis and against many of these proteins
drugs are in (advanced stages of) clinical trials. Finally, the applicability of
successful in vitro miRNAs was tested in three different in vivo models,
both after local and systemic administration. For local delivery, cell
membrane passage of the miRNAs was facilitated by electroporation,
whereas system delivery was achieved by an Arg-Gly-Asp targeted
sterically stabilized nanoparticle.
42
The Dlk1-Dio3 imprinted gene cluster noncoding RNAs are novel
candidate biomarkers for liver tumour promotion
John P THOMSON, Antonio Vitobello, Jonathan Moggs,
Remi Terranova, Richard R Meehan
MRC Human Genetics Unit, Western General Hospital, University of Edinburgh, Scotland
The molecular events which arise during toxicological insults - particularly in
cases resulting in the progression to cancerous states - are still poorly
understood and present a significant hurdle to the pharmaceutical and
research community. As part of a IMI funded European wide initiative (entitled
the “MARCAR project”) we are investigating molecular and pathological
perturbations which arise during the progression of at early stages of nongenotoxic carcinogenesis through phenobarbital (PB) mediated liver tumor
promotion in vivo. As non-genotoxic carcinogenesis arises without changes in
the DNA sequence this progression is largely driven through epigenetic
perturbations. We have previously shown that epigenetic DNA modification
(both 5-methly-cytosine and the recently characterized 5-hydroxymethylcytosine marks) and histone modification (H3K4me2, H3K27me3 and
H3K36me3) are reproducibly perturbed in a temporal manner following PB
exposure which allows us to define specific barcodes of drug exposure which
could ultimately result in the identification of novel biomarkers for nongenotoxic carcinogenesis. Interestingly molecular profiling (epigenetic as well
as mRNA and microRNA [miRNA]) of mouse liver samples during both short
(1-7 days) and long term (13 week) PB treatment revealed progressive
increases in hepatic expression of long noncoding RNAs (Meg3) and miRNAs
originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently
been associated with stem cell pluripotency in mice, targeting of repressive
chromatin complexes (PRC2/Jarid2) and various neoplasms in humans. PB
induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was
localized to glutamine synthetase-positive hepatocytes, suggesting a role for βcatenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic
relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo
genetic dependence on constitutive androstane receptor and β-catenin
pathways. We are currently investigating the potential consequences of Meg3
mis-expression in liver tumours with reference to H3K27me3 deposition.
References
1. Lempiainen et al (2012) Identification of Dlk1-Dio3 imprinted gene cluster noncoding
RNAs as novel candidate biomarkers for liver tumor promotion. Toxicol Sci. 2013
Feb;131(2):375-86
2. Thomson et al (2013) Dynamic changes in 5-hydroxymethylation signatures underpin
early and late events in drug exposed liver.Nucleic Acids Res. 2013 Jun;41(11):5639-54
3. Thomson et al (2012) Non-genotoxic carcinogen exposure induces defined changes in
the 5-hydroxymethylome. Genome Biol. 2012 Oct 3;13(10):R93
43
Do pluripotent stem cell miRNAs and
long non-coding RNAs have a role in cancer?
Stefano VOLINIA
Department of Morphology, Surgery and Experimental Medicine,
University of Ferrara, Ferrara, Italy
We studied microRNA and lncRNA profiles during differentiation of human
embryonic stem cells (n=26) and in breast cancer patients (n=33) and
human cell lines (n=35). Using in situ hybridization, we then investigated
MIR302 expression in 318 untreated breast cancer patients (test cohort,
n=22 and validation cohort, n=296). In parallel, using next-generation
sequencing data from breast cancer patients (n=684), we assessed
microRNA association with stem cell markers.
In healthy tissues, the MIR302 (high)/MIR203 (low) asymmetry was
exclusive for pluripotent stem cells. MIR302 was expressed in a small
population of cancer cells within invasive ductal carcinoma, but not in
normal breast (P<.001). Furthermore, MIR302 was expressed in the tumor
cells together with stem cell markers, such as CD44 and BMI1. Conversely,
MIR203 expression in 684 breast tumors negatively correlated with CD44
(Spearman correlation, Rho=-0.08, P=.04) and BMI1 (Rho=-0.11, P=.004),
but positively correlated with differentiation marker CD24 (Rho=0.15,
P<.001. Primary tumors with lymph node metastasis had cancer cells
showing scattered expression of MIR302 and widespread repression of
MIR203. Finally, overall survival was statistically significantly shorter in
patients with MIR302 positive cancer cells (P=.03).
We also studied long non-coding RNAs in embryonic and induced
pluripotent stem cells. Among normal tissues, the uc.283 lncRNA was
highly specific for pluripotent stem cells. Intriguingly, the uc.283-plus
lncRNA was highly expressed in some solid cancers, particularly in one of
the most untreatable types, glioma.
44
APPLICANTS
ABSTRACTS
Post transcriptional regulation of microRNAs in embryonic stem cells
Diana A. ALEXIEVA, Rupa Sarkar, Christos Karampelias, Nicholas Dibb
Department of Surgery and Cancer, Imperial College London, United Kingdom
MicroRNAs (miRNAs) are ~22 nucleotide-long RNAs that post
transcriptionally repress gene expression in metazoans and plants by
binding to mRNAs of protein coding genes. They are involved in all
physiological processes and are of a particular importance during the early
stages of development; disruption of their function has been associated
with various pathologies, especially cancer. Regulation of miRNA
biogenesis occurs at both transcriptional and post transcriptional level and
this ensures their tissue and developmental specific expression. Previous
results in our group have shown prevalence of post transcriptional
regulation of miRNAs in embryonic stem cells (ESCs) compared to
differentiated cell types. Lin28 was not found to be associated with miRNAs
outside the let-7 family. However, a recent publication showed that Lin28 is
involved in the post transcriptional regulation of mir-9 (1). We are currently
working on determining what cell types of mouse and human origin express
and do not express the mature form of the miRNAs found to be post
transcriptionally regulated in ESCs. miRNAs not detected in these cell
types are of particular interest and we are aiming to assess whether the
primary and precursor transcripts are present. This will classify them as
transcriptionally (if no primary transcript is evident) or post transcriptionally
(if either transcript is detected) regulated. Our aim is to estimate the
proportion of miRNAs which are transcribed, but not fully processed in
other cell types compared to ESCs. We will determine which miRNAs are
associated with Lin28 and focusing on the ones that are not, we will work
on identifying other potential miRNA regulator proteins.
Reference:
1. Nowak JS, Choudhury NR, de Lima Alves F, Rappsilber J, Michlewski G. Lin28a
regulates neuronal differentiation and controls miR-9 production. Nature
communications 2014;5.
46
miR-194:a key player in differentiation commitment and
cell cycle in acute myeloid leukaemias
Carmela DELL’AVERSANA1,2, Ilaria Lepore2, Loredana D’Amato2, Roberta
Sarno2, Cristina Giorgio1,2, Annamaria Carissimo2, Mariarosaria Conte2,
Matthias Nees3, Francesco Paolo Tambaro2, Valeria Belsito Petrizzi4,
Alfonso Maria D'Arco4, Lucia ALTUCCI1,2,*
1
2
Institute of Genetics and Biophysics “ABT”, CNR, Naples, Italy; Department of General
3
Pathology, Second University of Naples, Italy; Medical Biotechnology Knowledge Centre,
4
VTT Technical Research Centre of Finland, Turku, Finland; Umberto I Hospital, San
Francesco 1, Nocera Inferiore, Salerno, Italy
*
To whom correspondence should be addressed
MicroRNAs are endogenous, single-stranded, non-coding RNA molecules
approximately 18 to 22 nucleotides long. miRNAs are frequently expressed as
polycistronic transcripts and made functional by the miRNA processing machinery
(Bartel, 2004; Carthew and Sontheimer, 2009; Krol et al, 2010). They modulate
post-transcriptional expression of direct and secondary target genes, not
completely shut down their gene expression rather fine-tuning their expression
(Bartel, 2009). Deregulation of miRNAs expression and/or function may contribute
to initiation and maintenance of cancer, thus suggesting tumor-suppressorgene
(TSG) or oncogene(OG) functions, respectively (Volinia et al, 2006; Lu et al, 2005).
Epigenetic modulators (the so-called epidrugs), such as histone deacetylase
inhibitors (HDACis), are currently used in several anticancer therapies for their
antiproliferative, proapoptotic and differentiative action (Humeniuk et al, 2009;
Fandy et al, 2005). By comparative analysis of gene and miRNA expression
profiles in different acute myeloid leukaemia (AML) cell lines treated with the wellknown HDACi, Vorinostat, miR-194 has emerged to be up-regulated and to
negatively modulate its newly identified target gene, BCL2-associated transcription
factor 1gene (bclaf1). Our data identify and characterize that miR-194 is essential
in modulating myeloid lineage differentiation, cell cycle progression and apoptosis
in AML cells.
In particular, we demonstrate the power of miR-194 to reorganize the chromatin
arrangement addressing to a better and specific apoptotic and differentiation
response to epi-drugs. Moreover, miR-194 would be able to regulate the
expression of its new target, bclaf1, and its “locus dependent” function.
Furthermore, our findings ex-vivo suggest that the expression levels of miR-194,
and its target gene bclaf1, and their opposite modulation in response to Vorinostat
might be exploited as readout of epi-based anti-leukaemia therapy.
Keywords: bclaf1/differentiation/HDACi/leukaemia/miR-194/epigenetics
References
1. Bartel DP. (2009). MicroRNAs: target recognition and regulatory functions. Cell136: 215–
233
2. Bartel DP. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function.
Cell.116: 281–972.
3. Carthew RW, Sontheimer EJ. (2009) Origins and Mechanisms of miRNAs and siRNAs.
Cell 136: 642-55. Review
47
Detection of miRNAs associated with Alzheimer's disease in brain
tissues using next-generation sequencing technology
Anita ANNESE1, Caterina Manzari2, Matteo Chiara3, Gaia Zaffaroni3,
Ernesto Picardi1, Italia Aiello1, Alessio Valletti2, David S. Horner3,
Anna Maria D’Erchia1, Graziano PESOLE1,2
1
Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Italy;
2
Institute of Biomembranes and Bioenergetics, National Research Council, Bari, Italy;
3
Department of Biomolecular Science and Biotechnology, University of Milan, Milan, Italy
Neurodegenerative diseases are an heterogeneous group of hereditary and
sporadic conditions characterized by a progressive dysfunction of specific
subsets of neurons in specific functional anatomic systems including
Alzheimer's disease (AD), that affects the daily life of about 40 million of people
in the world. Current drugs help mask the symptoms of Alzheimer's, but do not
stop or delay the cell damage causing the disease. Changes in gene
expression and splicing patterns have been proposed as mechanisms involved
in the molecular pathogenesis of AD investigated using traditional profiling
methods (i.e. qRT-PCR and Microarray) and transgenic animals models of AD
or patient-derived cell lines. In this work, we have used post-mortem brain
tissue and miRNA-Seq technology to investigate the pathological mechanisms
of AD and to uncover new diagnostic and prognostic markers for the disease
and new potential drug targets.
Frozen post-mortem brain tissues from the middle temporal gyrus (10 AD
patients and 6 sex and age-matched controls) and the hippocampus (6 AD
patients and 6 sex and age-matched controls), were provided by the
Netherland Brain Bank and the NICHD Brain and Tissue Bank for
Developmental Disorders. miRNA libraries were constructed following
Illumina's TruSeq Small RNA Sample Preparation Protocol and subjected to a
single-end 1x50 nt sequencing on the Illumina MiSeq platform. Following a
suitable bioinformatics protocol we detected several differentially expressed
miRNAs, their potential target genes and assessed their impact on the
biological pathways. In total, we have detected 1567 and 1460 different
miRNAs in the middle temporal gyrus and in the hippocampus, respectively.
miRNA-Seq data analysis highlighted 30 differently expressed miRNAs (23
down-regulated and 7 up-regulated) in the middle temporal gyrus and 5
differently expressed miRNAs, all down-regulated, in the hippocampus of AD
patients. In these two brain areas of AD patients, our analysis highlighted
previously uncharacterized differently expressed miRNAs potentially involved,
according to DIANA-mirPath, in the regulation of brain functions, including
neurotrophin signaling pathway, axon guidance, long-term potentiation and
homeostasis. In conclusion, changes in miRNAs expression may contribute to
AD progression through aberrant regulation of mRNA targets, whose
identification will be fundamental to understand the molecular mechanisms
leading to the disease.
48
Identification and characterization of miRNAs involved in midbrain
dopaminergic neurons differentiation and function
Roberto DE GREGORIO1, Salvatore Pulcrano1, Claudia De Sanctis1,
Floriana Volpicelli1,2, Roberta Esposito1, Carla Perrone Capano1,2,
Umberto di Porzio1, Meng Li3, Gian Carlo BELLENCHI1
1
2
Institute of Genetic and Biophysics “ABT”, CNR, Naples, Italy; Dept. of Pharmacy,
3
University of Naples “Federico II”, Naples, Italy; MRC-CSC, London, UK
Midbrain dopamineregic neurons (mDA) development is a complex and still
not fully understood phenomenon. Many studies till now concentrated their
attention on the role played by several specific and well known transcription
factors. Our aim is to focus the attention on a relatively new class of posttrascriptional regulators, microRNAs (miRNAs), a class of small non-coding
RNA (~21 nucleotides) able to regulate gene expression by targeting
partially complementary sequences in the 3' untranslated regions (UTRs) of
the target mRNAs.
To investigate the role played by miRNAs during mDA differentiation we
choose to analyse the miRNA expression profile by using miRNA Array
platforms. To this purpose we used an optimised protocol from mouse
epiblast stem cells (epiSC) in order to differentiate DA neurons in vitro (1;
Jeager, I., et al 2011). By bioinformatics analysis of the array data we were
able to select few candidates most likely implicated in the DA neurons
differentiation and function.
Four miRNAs, mir-218, miR-34c and miR-204 and miR-210 were confirmed
being enriched in E14 midbrain embryos by qPCR. By luciferase assay
Nurr1, a well described transcription factor involved in mDA differentiation,
results as a potential target of miR-204 and miR-34c, while miR-210 when
over-expressed in A1 cells, upregulates the dopaminergic transporter DAT.
Taken together our results identify a group of miRNAs involved in
mDA neurons development and function.
Reference
1. Jaeger, I., C. Arber, J. R. Risner-Janiczek, J. Kuechler, D. Pritzsche, I. C. Chen, T.
Naveenan, M. A. Ungless and M. Li (2011). "Temporally controlled modulation of
FGF/ERK signaling directs midbrain dopaminergic neural progenitor fate in mouse and
human pluripotent stem cells." Development138(20): 4363-4374.
49
The study of subtle structural changes of miR21 RNA and pre-miR21
RNA - the basic markers of the brain tumors in human
Agnieszka BELTER, Dorota Gudanis, Katarzyna Rolle, Monika Piwecka
Mirosława Nskręt-Barciszewska, Jan Barciszewski
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
Glioblastoma multiforme is the most frequent and malignant brain tumors
with extremely poor prognosis. Despite the recent advances in therapeutic
strategies, such as surgical resection and adjuvant radiotherapy and
chemotherapy, the prognosis for patients with brain tumors remains poor.
New therapies that provide highly specific tumor cell killing and complete
eradication of cancer cells are urgently needed.
MicroRNAs (miRNAs), a class of non-protein-coding RNAs, are singlestranded, ~22 nucleotides molecules. They regulate gene expression by
affecting stability and/or translation of mRNAs. miRNAs display precise
tissue-specific expression patterns, which change during cell development
and differentiation. They mediate gene regulation in many key biological
processes, such as cell proliferation, embryonic development, apoptosis
and gene expression. In pathologic conditions, in case of miRNA
biogenesis dysfunction, miRNA dysregulation and their mutations, miRNA
expression can be significantly altered, typical for particular disease.
miRNA were identified as risk, diagnosis, and prognosis predictors and as
being potentially useful in monitoring high-risk individuals.
Our expression profiling studies revealed that the level of several miRNAs
are significantly altered in glioblastoma cells. miR21 was showed the
highest over-expression and is most prominent in high grade tumors. It has
been shown that the sequence-specific functional inhibition of miR-21 in
glioma cells in vitro leads to reduction in cell viability and increase cell
death. It implies attractive opportunities for miR-21 targeting in brain tumor
treatment.
We are focused on determining the structure of miR21 and pre-miR21
RNAs. Determining the structures of these RNAs and their subtle changes
indicated by RNA interactions with proteins and other RNAs will help to
understand molecular background of miR21-associeted diseases and will
be useful while designing new therapeutics. Our preliminary studies on
potential anti-pre-miRNA therapeutics for brain tumors therapies indicate
that the cleavage efficiency of RNA with anti-mRNA ribozymes highly
depends on the structure of pre-miRNA molecules.
50
miRNA and gene regulatory pathway of stage I epithelial ovarian
cancer: reconstructing cancer circuits
Enrica CALURA1, Gabriele Sales1, Paolo Martini1, Robert Fruscio2 Eliana
Bignotti4, Antonella Ravaggi4, Lara Paracchini3, Mariacristina Di Marino3,
Laura Zanotti4, Dionyssios Katsaros5, Germana Tognon6, Enrico Sartori6,
Sergio Pecorelli4,6, Maurizio D’Incalci3, Sergio Marchini3, Chiara Romualdi1
1
2
Department of Biology, University of Padova; Clinic of Obstetrics and Gynecology,
3
University of Milano-Bicocca, San Gerardo Hospital, Milano, Italy; Division of Gynecologic
Oncology, “Angelo Nocivelli” Institute of Molecular Medicine, University of Brescia, Brescia,
3
5
Italy; Department of Oncology, IRCCS - “Mario Negri” Institute, Milano, Italy; Department
6
of Gynecology/Oncology, S. Anna Hospital, University of Torino, Torino, Italy; Department
of Obstetrics and Gynecology, University of Brescia, Brescia, Italy
It is becoming increasingly evident that the current difficulties in improving the
clinical management of epithelial ovarian cancer (EOC) reside within its complex
nature that hampers the possibility to identify the critical circuits driving tumour
growth and therapy response. Stage I EOC is an infrequent disease characterized
by a good prognosis since more than 80% patients survive five years the end of
chemotherapy. However, despite the therapy results, we are currently unable to
predict, at diagnosis, who will relapse or not, and neither evaluate with efficacy the
severity of the disease. Studies performed so far by our laboratory demonstrated
that altered expression of miR-200c is an independent prognostic factor (1),
suggesting that defects in the mechanisms of transcription regulation are
associated with patient outcome. Following this idea, we decide to analyse miRNA
expression integrated in gene circuit developing a dedicated new system able to
integrate miRNAs in gene pathways and identify circuits of functionally related
genes and miRNAs showing coordinated expression changes (2). We used this
new approach to study EOC progression within the stage I EOC. 257 snap-frozen
stage I EOC biopsies have been collected from three independent tumour tissue
collections. A subset of samples have been profiled for gene and miRNA
expression and used for the integrated analyses of circuits identifying a pathway
composed of 44 functionally related elements. The expression values of all the
circuit elements have been assessed in all the 257 collected biopsies and
expression levels have been used to perform univariate and multivariate survival
analyses. We identified 15 miRNAs and 9 genes with prognostic value that
compose a barcode specific for each patient, useful to evaluate the patient-specific
level of risk. Specifically, we elaborated an index representing the activation state
of the studied circuit in each patient. The index has been used to efficiently stratify
patients in high, medium or low risk classes resulting in a prediction of patients
outcome with a sensitivity=88% and specificity=91%. Finally, the same criteria
have been applied to evaluate the risk in an external and independent validation
set of 50 patients confirming the powerful predictive value of risk assessment of the
identified circuit. Our results present the first pathway, entirely composed of
elements with prognostic value, described in stage I EOC.
References
1. Marchini et al, Lancet Oncology, 2011;
2. Calura et al, Nucleic Acid Research, 2014
51
Chemical modifications in the seed region of miRNA 221/222 increase
silencing performances in GIST cancer
Montano Durso1-2, Maria Gaglione3, Linda Piras4, M. Emilia Mercurio3
Sara Terreri1-4, Michele Olivieri1, Paolo Grieco4, Anna Messere3,
Giancarlo Tonon2, Amelia CIMMINO1
1
Institute of Genetics and Biophysics “A. Buzzati Traverso”, National Research Council
2
3
(CNR) Naples, Italy; Bioker srl multimedica spa, Naples, Italy; Dipartimento Scienze e
Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli, Caserta,
4
Italy; Dipartimento di Chimica Farmaceutica e Tossicologica, Università
“Federico II”, Naples, Italy
GIST is an uncommon tumor in the digestive system, most often in the wall
of the stomach1. These tumors start in very early forms of special cells
found in the wall of the GI tract, called the interstitial cells of Cajal (ICCs).
GIST are most common in people between 50 and 70 years old.
Approximately 85% GISTs are associated with an abnormal c-kit pathway.
C-KIT is a gene that encodes for a transmembrane receptor termed stem
cell factor (scf). The c-kit receptor comprises a long extracellular domain, a
transmembrane segment, and an intracellular part. The c-kit
product/CD117 is expressed on ICCs and a large number of other cells, in
particular bone marrow cells, mast cells, melanocytes and several others.
Preliminary studies have shown that the in vitro transfection of primary cells
GIST with several commercially available miRNA 221/222 strongly inhibited
the level of expression of Kit and the tumor proliferation.
In order to improve the silencing performances of miRNAs 221/222, new
miRNA mimics were designed. Synthetic miRNAs containing modified
guide strands with 3’-terminal phosphorothioates (PS) and/or 2’-O-methyl
RNA (2’-OMe) in the seed region and outside, were prepared following
standard protocols2. Natural and modified guide strands were annealed
with the natural passenger strand to prepare the miRNA duplexes. Then,
we evaluated the positional effect of the chemical modifications on miRNAs
activities. Our results show that miRNA mimics with alternate 2’-OMe-PS
nucleotides in the seed region are the most effective inhibitors of the
expression of c-Kit gene, of the tumor proliferation and cell migration even if
compared to several commercially available mimics.
References
1. Ashwin Rammohan, Jeswanth Sathyanesan, Kamalakannan Rajendran, Anbalagan
Pitchaimuthu, Senthil-Kumar Perumal, UP Srinivasan, Ravi Ramasamy, Ravichandran
Palaniappan, and Manoharan Govindan. A gist of gastrointestinal stromal tumors: A
review. World J Gastrointest Oncol. Jun 15, 2013; 5(6): 102–112.
2. Serge L. Beaucage, Colin B. Reese. Current Protocols in Nucleic Acid Chemistry: Recent
Advances in the Chemical Synthesis of RNA. Unit Number: Unit 2.16. DOI:
10.1002/0471142700.nc0216s38. Online Posting Date: September, 2009
52
Fingerprinting of ultra conserved long noncoding RNAs in bladder
cancer analysis reveals a network between
non-coding RNA and microRNA
Michele Olivieri1, Matteo Ferro2, Montano Durso1,3, Sara Terreri1,
Alessandra Romanelli4, Anna Messere5, Muller FABBRI6, Daniela
Terracciano7, Ferdinando Febbraio8, George A. Calin9, Amelia CIMMINO1
1
Institute of Genetics and Biophysics “A. Buzzati Traverso”, National Research Council
2
(CNR), Naples, Italy; Division of Urology, European Institute of Oncology, Milan, Italy;
3
4
Bioker Srl Multimedica Spa, Naples, Italy; Università di Napoli “Federico II” Dipartimento di
5
Farmacia, Naples, Italy; Dipartimento Scienze e Tecnologie Ambientali, Biologiche e
6
Farmaceutiche Seconda Università degli Studi di Napoli, Naples, Italy; Dept. of Pediatrics
and Molecular Microbiology & Immunology, Norris Comprehensive Cancer Center, Keck
7
School of Medicine, University of Southern California, USA; Dept. of Translational Medical
8
Sciences, University of Naples “Federico II”, Naples, Italy; Institute of Protein Biochemistry,
9
National Research Council (CNR), Naples, Italy; Dept. of Experimental Therapeutics and
Leukemia, and the Center for small interfering RNA and non-codingRNAs, University of
Texas MD Anderson Cancer Center, Houston, TX, USA
Transcribed ultraconserved regions (T-UCRs) are pieces of human genome
located both intra- and inter-genic that are conserved between orthologous
regions of the human, rat and mouse genomes. They are frequently located
at fragile sites and genomic regions involved in cancers [1]. By using
genome-wide profiling, we identified 289 ucRNAs de-regulated in patients
with bladder cancer (BC) compared to normal control (24 tumors, 4
control). The greatest change was noted for ultraconserved element 8
(uc.8+), which was increased in expression by 5.45 ± 0.9-fold (P = 0.001),
and for ultraconserved element 388 (uc.388+), which was decreased in
expression by 0.23-fold. Expression level of the most deregulated T-UCRs
was validated in 60 patients and 16 normal donors. We showed that uc.8+
functions as a natural decoy RNA for miR-596 in BC. As result, matrix
metallopeptidase 9 (MMP9), a direct target of these microRNAs is upregulated, promoting cancer growth and migration. We also observed that
either the up regulated, as well as the most down-regulated T-UCRs in BC,
showed putative binding sites for the mir-596 with a mfe < -30 kcal/mol for
each duplex formed. Our studies have found and experimentally validated
an extensive and dynamic regulatory network based on the RNA signaling
that explains how the perturbation of a single T-UCR can affect on whole TUCRs network of interactions.
Reference
1. Calin GA, Liu CG, Ferracin M, Hyslop T, Spizzo R, Sevignani C, Fabbri M, Cimmino A,
Lee EJ, Wojcik SE, Shimizu M, Tili E, Rossi S, Taccioli C, Pichiorri F, Liu X, Zupo S,
Herlea V, Gramantieri L, Lanza G, Alder H, Rassenti L, Volinia S, Schmittgen TD, Kipps
TJ, Negrini M, Croce CM. 2007. Ultraconserved regions encoding ncRNAs are altered in
human leukemias and carcinomas. Cancer Cell 12:215-229.
53
Extra-vesicular miRNA profiling of in vitro & in vivo models
of therapy resistant neuroblastoma
CONLON R A1, Harvey H1, Piskareva O1, Miller-Delaney S1,
Bray I1, Stallings R L1
1
Cancer Genetics Group, Department of Molecular and Cellular Therapeutics,
RCSI, Dublin 2, Ireland
The acquisition of drug resistance is the principal obstacle to the successful
treatment of neuroblastoma. Thus, the elucidation of mechanisms involved
in multidrug resistance is vital for the discovery of novel biomarkers and
therapeutics. The aim of our work is to ascertain the contribution of miRNAs
in the development of multi-drug resistance in neuroblastoma. To this end we
have developed three cell lines, KellyCis83, CHP212Cis100 and SK-NASCis24 that are significantly resistant to cisplatin and other agents.
MicroRNA expression profiling of the lines identified a panel of miRNAs
predicted to target genes involved with drug resistance, providing a basis for
testing our hypothesis that these miRNAs modulate drug resistance. It has
previously been demonstrated that extra-vesicular miRNAs are present in
the bloodstream and these circulating miRNAs are remarkably stable, here
we demonstrate here that both drug resistant and parental neuroblastoma
cell lines export a significant amount of these miRNAs into their culture
medium. MiRNAs were profiled with Taqman® low density arrays and we
identified a panel of miRNAs differentially expressed in the 3 cell lines,
when compared to their parental counterparts. Using the KellyCis83 cell
line in an orthotopic xenograft murine model of neuroblastoma, we
established tumours in Athymic Nude-Foxn1nu mice and profiled plasma
to validate the panel of extra-vesicular miRNAs. The list includes miRNAs
which have previously been implicated in neuroblastoma pathogenesis and
also those with prior associations with an acquired drug resistance
phenotype in cancer. Our findings also lend further support to the idea of a
novel miRNA trafficking system concurrent with the cell–cell communication
hypothesis.
54
Statistical validation of a comprehensive gene/miRNA expression
profile dataset for miRNA:mRNA interactome analysis
Claudia CORONNELLO1,2, Giovanni Perconti1, Patrizia Rubino1,
Flavia Contino3, Serena Bivona3, Salvatore Feo1,3, Agata Giallongo1
1
2
Istituto di Biomedicina ed Immunologia Molecolare (IBIM) CNR, Palermo, Italy;
3
Fondazione Ri.MED, Palermo Italy; Dipartimento di Scienze e Tecnologie Biologiche
Chimiche e Farmaceutiche, Universitá degli Studi di Palermo, Italy
MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the
translational repression and degradation of target mRNAs in the cell.
Mature miRNAs are used as a template by the RNA-induced silencing
complex (RISC) to recognize the complementary mRNAs to be regulated.
Up to 60% of human genes are putative targets of one or more miRNAs.
Several prediction tools are available to suggest putative miRNA targets,
however, only a small part of them has been validated by experimental
approaches. In addition, none of these tools does take into account the
network structure of miRNA-mRNA interactions, which involve competition
effects [1] crucial to efficiently predict the miRNA regulation effects in a
specific cellular context. We undertook this issue and started to increase
the complexity of the miRNA target prediction approach by developing a
tool designed to predict the targets of co-expressed miRNA, ComiR [2].
We aim to model the miRNA-mRNA interaction network (interactome), by
considering all the miRNAs and mRNAs endogenously expressed in any
cellular condition. Out test bed has been breast cancer MCF-7 cells. We
collected several miRNA and mRNA expression profiles, by using the
Agilent microarray platforms. We analyzed samples derived from the
immunoprecipitation (IP) of two RISC proteins, AGO2 and GW182, and
correspondent input and flow-through as well. The expression level of the
top expressed miRNAs has been validated by real time PCR.
Due to the singularity of our dataset, we used non-standard bioinformatics
techniques to preprocess and analyze the obtained expression profiles. As
result, we validated the sample extraction technique, by obtaining
expression profile clustering and regression results consistent with the
experimental design. Our dataset can then be useful to further investigate
on miRNA-mRNA interactions.
References
1. Y Tay, J Rinn and PP Pandolfi, The multilayered complexity of ceRNA crosstalk and
competition (2014) NATURE 505, 344-352
2. C Coronnello, PV Benos, ComiR: Combinatorial microRNA target prediction tool (2013),
Nucleic Acid Research, 41(Web Server issue):W159-64
55
Differential miRNA-mRNA co-expression networks
in colorectal cancer
Teresa M. CREANZA1,2, A. Piepoli3, N. Ancona1
1
Institute of Intelligent Systems for Automation, National Research Council, CNR-ISSIA,
2
Bari, Italy; Center for Complex Systems in Molecular Biology and Medicine, University of
3
Torino, Torino, Italy; Department of Medical Sciences, Division and Laboratory of
Gastroenterology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy
Colorectal cancer (CRC) is one of the most common neoplasms in the world
and its molecular biology is one of the most intensively and successfully
studied. Altered expression of miRNAs is associated with the development and
progression of CRC by regulating the translation of oncogenes and tumor
suppressor genes (1). Moreover, miRNA-expression profiling has been
exploited to predict the functions of the deregulated miRNAs by detecting the
enriched pathways of their target genes. In cancer, genetic variants in miRNA
genes and mRNA targets can alter miRNA-mediated repression (2). We
studied changes in miRNA-mRNA CRC interactions in terms of differential coexpressions relative to normal condition. The aim was to capture alterations
resulting from the aforementioned modifications that influence miRNA activity
on gene transcription. By using paired miRNA-mRNA expression profiles,
correlations between miRNA and gene expressions were estimated for both
tumor and normal tissues. Correlation changes between the two conditions
were incorporated into scores of predefined gene sets in order to identify
signaling pathways and biological processes with an altered miRNA-mediated
control. Compared with other types of miRNA-mRNA interaction scores, using
co-expression coefficients with any a priori information has several
advantages: a complete coverage of the human genes on the chip, little bias
due to the knowledge obtained from the published literature, and the ability to
infer condition specific relationships. Notably, our integrative analysis
suggested an alteration in CRC tissues in the interplay between miRNAs and
the eukaryotic translation initiation factor 3 (eIF3) which has a central role in
recruiting both mRNAs and the cellular translation machinery to form
translation initiation complexes (3). Unveiling differential miRNA-mRNA coexpression properties allows to gain insights into miRNA-mediated molecular
mechanisms underlying the pathogenesis of the disease and may suggest
novel drug targets to be validated.
References
1. Calin GA, Croce CM., MicroRNA signatures in human cancers. Nat Rev Cancer
(2006);6:857–66.
2. Ryan BM, Robles AI, Harris CC., Genetic variation in microRNA networks: the
implications for cancer research. Nature Reviews Cancer (2010); 389-402.
3. Jackson RJ, Hellen CU, Pestova TV, The mechanism of eukaryotic translation initiation
and principles of its regulation. Nat Rev Mol Cell Biol (2010) 11:113–127.
56
Micro-RNA in hematopoietic stem cell self-renewal
Laura CRISAFULLI, Paolo Uva, Michael Cleary,
Anna Villa, Francesca Ficara
UOS Milan Unit, Istituto di Ricerca Genetica e Biomedica (IRGB), CNR, Milan, and
Humanitas Clinical and Research Center, Rozzano, Milan, Italy
The ability of balancing self-renewal and multi-potent differentiation is a key
hallmark of somatic stem cells; however, the molecular pathways underlying
this regulation are not completely understood, including any role for microRNAs (miRNAs).
Pbx1 is a homeodomain transcription factor that positively regulates HSC
quiescence. Its absence in post-natal HSCs causes an excessive proliferation
that ultimately leads to their exhaustion, indicating a profound self-renewal
defect, and a premature myeloid differentiation at the expenses of the lymphoid
one. Cell cycle regulation and differentiation, both abnormal in Pbx1-null HSCs,
are two major roles of miRNAs. Therefore the study of Pbx1-null HSCs
provides the unique opportunity to identify miRNAs involved in the
maintenance of HSC identity.
We employed Pbx1-conditional knockout mice (and controls) to perform
miRNA profiling of highly purified HSCs and of their immediate downstream
progeny named multi-potent progenitors (MPPs). The profiles were then
compared to our previously described microarray mRNA data, relative to the
same populations, to search for miRNA predicted targets (PT) whose change
in expression inversely correlates with those of miRNAs. Unsupervised
hierarchical clustering indicates a clear distinction between HSCs and MPPs at
the level of miRNA expression, suggesting that miRNAs might regulate the first
transition step in the adult hematopoietic development. Within each group,
mutant and control cells cluster separately, linking miRNAs to self-renewal
impairment. More in detail, SAM analysis showed 48 miRNAs differentially
expressed (DE) between Pbx1-null and wt HSCs, among which 84% have anticorrelated PT within the list of DE mRNAs. A similar analysis on wt cells
revealed 71 DE miRNAs during the physiological HSCs-to-MPP transition, of
which 31 are concordantly DE in the Pbx1-null HSCs, in accordance with the
hypothesis that miRNAs are involved in HSC self-renewal. Among those 31, by
applying very stringent selection criteria we have selected few miRNA
candidates. Within this short list the miRNA that is most DE both in Pbx1-null
HSCs and in the normal HSC-to-MPP transition is particularly interesting since
it is strongly HSC-specific, being quickly down regulated in Flk2-MPPs, and not
re-expressed further down in the hematopoietic hierarchy. We are currently
dissecting its role through gain and loss of function experiments, with the future
aim of studying its mechanism(s) of function and its role in regulating HSC selfrenewal.
57
Long non-coding RNAs explosion: novel implications in
Neurotrophins during the development of the nervous system
Ylenia D’AGOSTINO, Elena De Felice, Margherita Branno, Annamaria
Locascio, Antonietta Spagnuolo, Filomena Ristoratore, Laura Zanetti,
Paolo Sordino, Salvatore D’Aniello
Cellular and Developmental Biology, Stazione Zoologica "Anton Dohrn", Naples, Italy
Neurotrophins (NT) (BDNF, NGF, NT3, NT4/5 and NT6/7) are growth
factors that control development, differentiation, synaptic plasticity and
survival of several types of neuronal and glial cells in the embryonic and
adult central nervous system. Recently, the possible role of a new class of
molecules, such as long non-coding RNAs (lncRNAs), in the regulation of
these processes is emerging.
LncRNAs are a new group of regulatory molecules that have been shown
to be involved in almost all biological phenomena, especially in the
development and physiology of the nervous system.
To this respect, using Zebrafish (Danio rerio) as model system, we aim to
extend our knowledge on the genetic factors and the functional processes
of the NT molecular pathways, with emphasis on the characterization of
long non-coding RNAs that are involved in the regulation of NTs and their
receptors (NTRs).
The project consists of a multidisciplinary study based on bioinformatic,
molecular, genetic and behavioural approaches, with the goal to acquire
new insights on the NT regulatory networks and on the function of lncRNAs
during the correct CNS development, in conditions of thermal, social and
nutritional stress.
The main objectives of the project are:
1) The bioinformatic identification and genetic characterization of lncRNAs
linked to the NT and NTR genes, thanks to the availability of the zebrafish
genome and transcriptome, and study of the function of a short list of
candidates during embryonic development;
2) The generation of NTs zebrafish knockout, through the new
CRISPR/CAS9 technology.
In conclusion, we expect that the spectrum of approaches and objectives
planned in this study will be instrumental to the development of new model
systems for the study of lncRNAs and to improve our understanding on the
role played by NTs during embryonic development and adult brain
physiology, with particular attention to the biomedical impact in terms of
diagnosis and treatment of neurological diseases with neurodegenerative
and/or psychological traits.
58
A long non-coding RNA regulated in development and cancer
Barbara D’Andrea1,3, Angela Pascarella1, Miriane De Oliveira1, Sara
Credendino1, Elena Amendola1, Pina Marotta2, Marzia Scarfò2,
Roberto Di Lauro1, Gabriella DE VITA1
1
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita’ Federico II,
2
Naples, Italy; Istituto di Ricerche Genetiche G.Salvatore, Biogem s.c.ar.l,
Ariano Irpino, Avellino, Italy
To search for genes involved in thyroid morphogenesis we performed an
unbiased screening for transcripts enriched in the early mouse thyroid bud
(E10.5). At the top of the list we found a gene for a long non-coding RNA
(lncRNA) whose genomic locus shows a partially overlapping head-to-head
antisense arrangement with that of another gene present in our list, the
protein encoding gene KLHL14. The lncRNA (herein named KLHL14-AS)
shows strong and highly specific expression in mouse thyroid both during
development and in the adult. It is worth noting that KLHL14-AS
encompasses one of the Human Accelerated Regions (HAR), genomic
regions conserved across vertebrates that have acquired many sequence
changes in humans. KLHL14 is a poorly characterized gene, whose
expression in thyroid has not been previously described. To shed light on
the function of this overlapping gene pair, we analyzed their expression in a
panel of adult mouse tissues. Interestingly, these genes resulted invariably
co-expressed in several tissues, thus suggesting either a common
regulation or a reciprocal regulation. To check if there is a cross-regulation
between the two genes, we performed KLHL14-AS and KLHL14 lossoffunction experiments in thyroid cells in vitro, which did not reveal
reciprocal effects on expression levels of each gene. Nevertheless, both
genes resulted strongly downregulated during oncogene-induced dedifferentiation of thyroid cells. To investigate the function of KLHL14-AS in
vivo, we generated a floxed allele in order to obtain both constitutive and
conditional KO mouse lines. Phenotype of KLHL14-AS KO mouse lines is
currently under investigation and will provide definitive informations on the
role of such gene in development. These mice will be also useful to study
the possible involvement of KLHL14-AS in cancer.
59
Identification, characterization, and determination of molecular
function of long non coding RNAs in breast cancer, by
transcriptomics and epigenomics analysis
Valentina DEL MONACO1, Valeria D’Argenio1,2, Massimiliano D’Aiuto3,
Donatella Montanaro1, Fatima De Palma1,2, Giuseppina Liguori3, Giuseppe
D’Aiuto3, Gerardo Botti3, Alfonso Baldi1, Raffaele Calogero4,
Francesco Salvatore1,2,5
1
2
CEINGE-Biotecnologie Avanzate, Naples, Italy; Department of Molecular Medicine and
3
Medical Biotechnologies, University of Naples, Federico II, Italy; Istituto Nazionale Tumori –
4
IRCCS Fondazione Pascale, Naples, Italy; University of Torino, Bioinformatics & Genomics
5
unit, Molecular Biotechnology Center, Turin, Italy; IRCCS-Fondazione SDN, Naples, Italy
A large portion of the human genome is transcribed into RNAs without known
protein-coding functions, far outnumbering coding transcription units [1; 2].
Transcriptome sequencing studies have contributed to the discovery of an intricate
interplay among diverse RNA species, which are also implicated in numerous
diseases such as cancer [3]. Cancer is thought to arise and progress due to
genetic alterations that disrupt processes required for maintaining normal cellular
homeostasis, such as cell cycle, DNA damage response, survival, and migration.
Misregulated long non-coding (lncRNAs) that affect each of these processes have
been identified in cancer [4]. Recent analyses have also revealed the aberrant
expression of lncRNAs in breast cancer (BC) [5]. BC is a major cause of cancerrelated mortality, despite advances in early detection and treatment. To date, the
major challenge is to define the type of BC and its biology.
By massively RNA-sequencing, we obtained 9 billion reads from 66 healthy and
tumour breast tissues belonging to Basal, HER2-positive and Luminal BCs, with or
without hereditary predisposition. In order to analyse the transcriptomes in specific
tumour cells, we used Laser Capture Microdissection. Furthermore, we performed
a RiboZero-based rRNA depletion. Stranded RNA sample prep kit (Illumina) and
paired-end sequencing allow us to maintain strandness and directionality of each
transcript. Comparative transcriptomic analyses highlighted differentially expressed
transcripts, among the different BC groups, identifying also transcripts which may
be putative modulators. We found 956 transcripts differentially expressed between
Basal-like and Luminal A BCs. Furthermore, we found 177 long non-coding RNAs
differentially expressed between these two groups.
Identification of these lncRNAs should reveal additional subtype- and stagespecific diagnostic and therapeutic targets in BC. With this study will help to
determine the role of specific lncRNAs in BC, as well as the molecular mechanisms
by which they act.
References
1. Trends Biochem Sci. 2014 Apr;39(4):170-82
2. Nature. 2014 Jan 16;505(7483):344-52
3. Annu Rev Biochem. 2012;81:145-66
4. Oncogene. 2012 Oct 25;31(43):4577-87
5. Int J Biochem Cell Biol. 2014 Mar 26
60
Relationships between MeCP2 and pericentric heterochromatin
factors during neural differentiation
Floriana DELLA RAGIONE1,2, Carmela Zarrillo1, Maria R. MATARAZZO1,
Maurizio D'Esposito1,2
1
2
Institute of Genetics and Biophysics ‘‘A Buzzati Traverso’’, Naples, Italy, Istituto di
Ricovero e Cura a Carattere Scientifico Neuromed, Pozzilli, Italy
The methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcription factor
predominantly expressed in the brain and mutated in Rett syndrome (OMIM
#321750), a progressive neurodevelopmental disorder. Neuronal MeCP2 genomewide binding tracks methyl-CpG density and its absence results in large-scale
changes in chromatin structures, suggesting a global regulatory role. In mouse
cells MeCP2 accumulates at pericentric heterochromatin (PCH), composed by
major satellite DNA of different chromosomes that aggregate during development
to form chromocenters, structures possibly critical for the establishment of silent
1
compartments . Several proteins and ncRNAs [e.g. HP1a and maj sat forward
transcript (MSFT)] seem to be relevant for establishment and maintenance of PCH.
Recently, we established a versatile model of murine embryonic stem cells lacking
MeCP2 (MeCP2-/y), capable to differentiate to neurons and astroglia. There, we
highlighted a crucial role of MeCP2 in the PCH re-organization during neural
differentiation, supporting the view of MeCP2 as a multifunctional chromatin
2
organizing factor .
To unravel the molecular mechanism by which MeCP2 regulates the PCH reorganization we investigated the expression and the spatial distribution of MSFT
and HP1a during neural differentiation of wt and MeCP2-/y cells. MSFT expression
increases during differentiation of both wt and MeCP2-/y cells, without significant
changes among the two cell lines. On the other hand, RNA-FISH analyses
revealed the presence of very strong MSFT signals at chromocenters of wt cells,
whereas the fluorescence intensity appeared weaker in MeCP2-/y nuclei.
Noteworthy, the foci number in wt nuclei is significantly greater compared to
MeCP2-/y nuclei at each time point of differentiation. Furthermore, MeCP2 colocalizes with MSFT and physically associates with it in differentiated wt cells.
These data suggest an involvement of MeCP2 in the localization of MSFT at
chromocenters, while in mutated nuclei this phenomenon is partially impaired.
Moreover, MeCP2 co-localizes with HP1a at chromocenters, suggesting possible
functional interactions in PCH organization. A deeper analysis of HP1a sub-nuclear
distribution in MeCP2-/y cells is currently under investigation. Our preliminary
results allow to hypothesise that MeCP2 may cooperate with MSFT and, possibly,
HP1a, at chromocenters for the pericentric heterochromatin structural organization.
References
1. Della Ragione, F., et al.. (2012). Front Genet Sep 11;3:181;
2. Bertulat, B., De Bonis, M.L., Della Ragione, F. et al., (2012). PLoS One 7(10).
61
A signature of ultraconserved regions (UCRs) harbors prognostic
implications in clear cell renal cell carcinoma (ccRCC)
Francesca FANINI1, Ivan VANNINI1, Cecilia Fernandez-Cymering2,
Dino Amadori1, Carlo Maria CROCE2, Muller FABBRI1,3
1
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRCCS, Meldola (FC),
2
Italy; Department of Molecular Virology, Immunology and Medical Genetics,
Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA;
3
Departments of Pediatrics and Molecular Microbiology & Immunology, Keck School of
Medicine, Norris Comprehensive Cancer Center, University of Southern California, Saban
Research Institute, Children's Center for Cancer and Blood Diseases, Children's Hospital
Los Angeles, CA, USA
Renal cell carcinoma (RCC) accounts for approximately 2% of all adult cancer
cases in Europe, by representing the ninth most common malignancy and the most
common cancer in the adult kidney (85% of the kidney cancer cases). The clearcell variant (ccRCC), one of the three major subtypes, occurs in 75% of all RCC
cases. Although the advent of new target-oriented therapies, ccRCC responds
poorly to chemotherapy and radiotherapy, then radical surgery still represents the
best choice for patients diagnosed with ccRCC, but the prognosis remains severe
in case of disseminated disease. One of the main challenges in the management
of ccRCC is related to stage I patients. At present there is no valid biomarker able
to differentiate those patients who will benefit from surgical tumor dissection from
those who will recur and eventually develop a disseminated disease. Such a
biomarker would be of great value, since it will select a population to be tested for
adjuvant treatments and/or for a more aggressive “follow-up” plan. The transcribed
ultraconserved regions (UCRs) are a family of ncRNAs highly conserved (100%)
between orthologous regions of the human, rat, and mouse genomes. UCR genes
are frequently located at genomic regions involved with cancers, and their
expression is dys-regulated in several types of solid and hematological
malignancies. The role of UCRs as prognostic biomarkers is still unexplored. In this
study we collected 136 frozen paired normal and adjacent non-tumoral tissues
from stage I ccRCC patients. We assessed the expression of all 482 identified
UCRs in primary tumors and in the adjacent non-tumoral tissues from the same
patient, by high-throughput microarray profiling. We found a differential expression
of 30 UCRs between the two groups, and we validated the most dys-regulated
UCRs by qRT-PCR. We then correlated the expression of these UCRs, with the
overall survival (OS) of our patients by Kaplan-Meier analysis. We observed that
the expression of 8 UCRs (namely, uc.115A, uc.466, uc.282, uc.395A, uc.203A,
uc.1, uc.332, and uc.243A) significantly correlated with prognosis in stage I ccRCC
patients with shorter OS, with respect to patients with better OS. In particular, the
expression in "good" prognosis stage I ccRCC was high for uc.115A and uc.466,
and low for uc.282, uc.395A, uc.203A, uc.1, uc.332, and uc.243A. Overall, this
study identifies a prognostic signature of UCRs, correlating with survival in stage I
ccRCC patients, and could lead to the identification of a new biomarker for a better
stratification of ccRCC patients eligible for adjuvant treatments and/or a closer
post-surgery clinical follow-up.
62
Control of pluripotency and cell lineage determination mediated by
ultra-conserved non-coding RNA in Embryonic Stem Cells
Alessandro FIORENZANO, Emilia Pascale, Gennaro Andolfi,
Marco Galasso, Cristian Taccioli, Amelia CIMMINO,
Gabriella Minchiotti, Annalisa FICO
Institute of Genetics and Biophysics “A. Buzzati-Traverso”, Stem Cell Fate Laboratory,
CNR, Naples, Italy
Despite extensive studies, how the cellular, molecular and epigenetic
mechanisms integrate to control cell lineage specification in mammals is
still unclear. Cell identity is ensured by distinct gene expression programs,
which allow highly specialized functions. The cell lineage commitment is the
result of complex regulatory interactions between transcription factors,
chromatin remodelling proteins and cell signalling. More recently,
modulation of long noncoding RNAs (lncRNAs) has emerged as an
additional level of regulation. Interestingly, emerging evidence points to an
extensive expression of lncRNAs in mouse embryonic stem cells (mESC),
and their involvement in controlling pluripotency and differentiation
programs. Within the long noncoding RNA class, the function of the subset
including the transcribed ultraconserved elements (T-UCE) is largely
unknown.
Being a highly conserved class of non-coding RNA, the T-UCEs might be
key regulators of pluripotency and cell lineage determination, by triggering
transcription and chromatin factors to both nearby and distant target genes
and/or by recognizing specific chromatin signatures.
We identified the T-UCEs expression profile during differentiation of ESCs
into neurons, by genome-wide transcriptional profiling, and we found 10 upregulated and 33 down-regulated T-UCEs in undifferentiated cells (day 0)
as compared to neurons (day 12). Among them, uc.170 has been validated
by quantitative PCR and molecularly characterized. The transcript is
approximately 4,5 Kb in length, it presents a polyA and is located in the
intron 6 of the host gene FAM172. Moreover, in the ultraconserved region
of such transcript there is the seed sequence for the binding to miR-9 that
has been already linked to neurogenesis, although in another biological
system. By performing in vitro experiments we also showed the direct
binding between the uc.170 and miR-9-3p/5p, raising the possibility of a
regulatory mechanism of uc.170 on miR-9. All together, our preliminary
results suggest the involvement of the UCEs in the balance selfrenewal/differentiation of ESCs and pave the way for a better
understanding of the complex molecular machinary controlling the stem cell
fate determination.
63
Survival in glioblastoma cancer patients is predicted by miR-340, that
regulates key cancer hallmarks by inhibiting NRAS
Danilo FIORE1, Cristina Quintavalle1, Elvira Donnarumma2, Giuseppina
ROSCIGNO1,3, Margherita IABONI1, Valentina RUSSO1, Assunta Adamo1,
Fabio De Martino1, Giulia Romano5, Soini Ylermi4, Carlo Maria CROCE5,
Gerolama CONDORELLI1,2
1
2
Department of Molecular Medicine, University of Naples, Italy; IRCCS-SDN Foundation,
3
4
Naples, Italy; IEOS, CNR, Naples, Italy; Department of Pathology and Forensic Medicine,
Institute of Clinical Medicine, Pathology and Forensic Medicine, School of Medicine, Cancer
5
Center of Eastern Finland, University of Eastern Finland, Kuopio, Finland; Department of
Molecular Virology, Immunology and Medical Genetics, Human Cancer Genetics Program,
Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA
Glioblastoma is the most frequent brain tumor in adults and is the most
lethal form of human cancer. Despite the improvements in treatments,
survival of patients affected by this disease remains poor. Nevertheless, a
subset of patients survives longer than 3 years and they are classified as
long-term survivors (LTS). The molecular and cellular mechanisms
underlying the rare phenomenon of long-term survivors in glioblastoma
patients are not well known. MicroRNAs (miRNAs or miRs) are a class of
endogenous non-coding RNA of 19–24 nucleotides in length that has an
important role in the negative regulation of gene expression. Deregulation
of miRNAs is fundamental to the pathogenesis of many types of cancer.
Nowdays, there is a great interest in discovery new prognostic and
therapeutic miRNAs involved in LTS phenotype in glioblastoma.
In this study, we reported the identification of miR-340 as a novel
prognostic tumor-suppressor-miR in glioblastoma. miR-340 expression was
increased in LTS compared to short term survivors in glioblastoma.
Moreover, low levels of miR-340 were a risk indicator for glioblastoma
patients survival. Functionally, miR-340 overexpression decreased cell
proliferation, anchorage independent cell growth, cell cycle, motility and
response to temozolomide in glioblastoma cells. We identified NRAS as a
direct target of miR-340. Rescue experiments showed its essential role in
mediating the onco-suppressive activity of miR-340. The overexpression of
miR-340 decreased the activation of ERKs and AKT, the main pathways
downstream NRAS. Furthermore, glioblastoma cells stably infected with a
lentivirus encoding miR-340 exhibited a drastic reduction of tumor growth in
nude mice.
In conclusion, our findings reveal miR-340 as a new tumor-suppressor
miRNA up-regulated in LTS in glioblastoma. Its expression correlates with
survival of glioblastoma patients. miR-340 is able to regulate multiple
tumorigenic features of glioblastoma cells, offering a novel potential
prognostic and therapeutical target for glioblastoma.
64
Cripto is required for Embryonic Stem Cell (ESC) to Epiblast Stem Cell
(EpiSC) transition and regulates DLK1-Dio3 cluster expression
Alessandro FIORENZANO, Emilia Pascale, Cristina D’Aniello, Gilda
Cobellis, Mauro Biffoni, Annalisa FICO, Gabriella Minchiotti
Institute of Genetics and Biophysics “A. Buzzati-Traverso”,
Stem Cell Fate Laboratory, CNR, Naples, Italy
Increasing evidence indicate that mouse embryonic stem cells (mESCs)
fluctuate between a naive inner cell mass-like state and a primed epiblastlike state when cultured in serum. This metastable condition ensures both
self-renewal and predisposes ESCs for differentiation to Epiblast stem cells
(EpiSCs). The transition from naive to primed cells represents a pivotal
event in cellular differentiation; the extracellular signals that control this
fundamental process remain still unclear. Our findings indicate that the
extracellular protein Cripto plays a crucial role in this complex scenario. We
demonstrate that Cripto is metastable in mESCs and that separation of
ESCs on the basis of Cripto expression levels yields to cell populations
characterized by different stemness-associated features. Unexpectedly,
genome-wide transcriptional profiling shows a significant downregulation of
the DLK1-Dio3 cluster in Cripto KO ESCs. Interestingly, recent evidences
indicate that expression of DLK1-Dio3, which is the largest microRNA
cluster in the mammalian genome, correlates with pluripotency in ESCs
and induced Pluripotent Stem Cells (iPS). According to the trancriptome
analysis, microRNAome profiling shows that >50% of the microRNAs
deregulated in Cripto KO ESCs belong to the DLK1-Dio3 cluster. In line
with the idea that high level of Cripto correlates with “full pluripotency”, a
combination of loss-and-gain of function approaches show that Cripto
regulates ESC-to-EpiSC transition, and is required both to induce and to
maintain the EpiSC-like state. Together our preliminary data suggest a
novel role for Cripto in EpiSC self-renewal, likely through modulation of the
Dlk1-Dio3 cluster.
65
Regulation of microRNA processing by mutp53 in colon cancer
Francesca GARIBALDI, Emmanuela Falcone, Daniela Trisciuoglio,
Gianluca Bossi, Giulia Piaggio, Aymone GURTNER
Experimental Oncology Department, Regina Elena National Cancer Institute, Rome, Italy
Human cancers are characterized by deregulated miRNA expression and
defects in miRNA biogenesis promoting cellular transformation and
tumorigenesis; still, the mechanisms through which miRNAs are regulated
in cancer remain unclear.
Tumor suppressor p53, mutated in approximately 50% of human cancers,
can acquire GOF activities favouring tumor induction, maintenance,
spreading; miRNAs can be regulated by wtp53 at transcriptional and posttranscriptional level but few data about mutp53-dependent miRNA
expression are available yet.
Our results, coming from a genome wide analysis, reveal 34 miRNAs upregulated after interference of mutp53 in human colon adenocarcinoma
cells.
Validation of microarray’s analysis for mature miRNAs, pri-miRNAs and
pre-miRNAs shows that mutp53 regulates the majority of miRNAs at posttranscriptional level, thus suggesting a mut-p53-mediated miRNA
processing inhibition.
The in vivo association between the target pri-miRNAs and Drosha is
significantly attenuated in the presence of mut-p53. We found that the
endogenous mut-p53 inhibits the interaction between Drosha and DEADbox RNA helicase (DDX17 alias p72), a regulatory factor of the
microprocessor complex, by binding and sequestering it. Indeed, DEADbox RNA helicases overexpression increases mutp53–dependent miRNA
levels.
Moreover, we found that 5 of the post-transcriptional regulated miRNAs
show tumor suppressive properties, playing a role in apoptosis, cell cycle
arrest, EMT repression and migration inhibition.
These data support the idea that mutp53 is one of the key factors leading to
the decreased expression of miRNAs in human cancers by interfering with
Drosha-mediated miRNA processing.
66
Unravelling the nuclear activity of microRNAs in mTLE
Giuliano GIULIANI, Vamshidhar R. VANGOOR, Rosanne C. Ausems,
Pierre N. de Graan, R. Jeroen Pasterkamp
Department of Translational Neuroscience, Brain Center Rudolf Magnus,
University Medical Center Utrecht, The Netherlands
Mesial temporal lobe epilepsy (mTLE) is a chronic neurological condition
characterized by recurrent seizures (epilepsy) which originate in the
temporal lobe of the human brain. Current therapy relies upon classical
anti-epileptic drugs; however, ~40% of mTLE patients do not respond well
to available treatment. The only alternative being surgical intervention with
removal of the seizure focus at the hippocampus. Recently, in a genomewide microRNA screening of hippocampi from patients with mTLE, Kan et
al., (2012) identified a number of microRNAs associated with different
clinical stages of the disease, and among these a subset were found to
modulate posttranscriptional regulation of genes involved in the
inflammatory pathway. In addition, a number of microRNAs showed
subcellular mislocalization, with aberrant enrichment in the nucleus (Kan et
al.,2012). In light of this, the aim of our study is to investigate the
mechanism-of-action of these microRNAs in the nucleus and their
contribution towards aspects of the disease. As a starting point, we
performed RNA-seq on brain tissue to compare the nuclear and
cytoplasmic distribution of microRNAs and other RNAs both in controls and
different classes of mTLE patients (mTLE patients with hippocampal
sclerosis (W4) and without hippocampal sclerosis (W0)). In further
experiments, we will use a combination of mTLE animal models, cellular
systems and computational approaches to further characterize the role of
the nuclear mislocalization of microRNAs in the pathogenesis of mTLE.
Reference
1. Kan AA et al., (2012) Genome-wide microRNA profiling of human temporal lobe epilepsy
identifies modulators of the immune response. Cell Mol Life Sci. 69 (18): 3127-45.
67
Genomic association of the imprinting maintenance factor ZFP57 and
associated chromatin regulators in murine embryonal stem cells at
imprinted and non imprinted loci and its expression in the developing
mouse embryo
S. LAD1, I. Defeis2, M. Cammisa1,3, G. Verde1,3, V. Riso1,3,
G. Manganelli1,4, S. Filosa1,4, G. BELLENCHI1, A. SIMEONE1,4, A. Riccio1,3,
C. Angelini2, G. GRIMALDI1
1
;2
Istituto di Genetica e Biofisica "Adriano Buzzati Traverso", CNR, Naples, Italy Istituto per
3
le Applicazioni del Calcolo "Mauro Picone", CNR, Naples, Italy; Dip.to Scienze e
Tecnologie Ambientali Biologiche e Farmaceutiche, Seconda Università degli Studi di
4
Napoli, Italy; IRCCS, Neuromed, Pozzilli (IS), Italy
Several lines of evidence have provided clues for the pivotal role played by
the KRAB-containing zinc finger protein ZFP57 in the maintenance of the
parent of origin-dependent epigenetic state, DNA methylation and histone
tails specific modifications, of the hundreds imprinted loci in mouse which
include several non coding RNAs. ZFP57 associates with KAP1, a general
co-repressor of the abundant KRAB-ZFPs family, which acts as a scaffold
for the recruitment of chromatin modifiers that include the histone H3
methyltransferase SETDB1 and heterochromatin protein-1 (HP1) isoforms,
thus promoting H3 trimethylation at lysine9 and chromoproteins recruitment
at targeted sites. DNA methyltransferases are also present in KAP1
complexes. This finding correlates with the requirement for ZFP57 to
maintain DNA methylation at imprinting control regions (ICRs).
In this work we established the genome wide occupancy of ZFP57, along
with associated chromatin modifiers KAP1, Hp1 and tri-methyl H3K9 and
correlated it with the DNA methylation profile in murine embryonal stem
cells (ESC). The data show that the majority of ZFP57 bound sites reside at
DNA methylated non-imprinted loci, of which several encompass noncoding
RNAs, implying a wider role for this factor in the shaping of the epigenome,
notably at germ-line DNA methylated CpG islands and repeated
retrotrasposons. Further, we show that ZFP57, while strongly expressed in
ES cells and down regulated during differentiation, is also expressed at
several sites during embryo development. The strong and sustained
expression in the nervous system of the developing murine embryo is
paralleled by association of the factor to ICRs extending its potential role
beyond that of imprints maintenance at pre-implantation. We will report also
on data suggesting a potential role for ZFP57 in the in vitro differentiation of
ES cells toward the neural fate.
68
NF-Y regulates E-cadherin expression by
miRNA-200 family in colon cancer
Emmanuela Falcone, Francesca GARIBALDI, Giulia Piaggio,
Aymone GURTNER
Experimental Oncology Department, Regina Elena National Cancer Institute, Rome, Italy
miRNAs, small noncoding RNAs, have emerged as key post-transcriptional
regulators of gene expression by causing transcript degradation or
translational repression and a number of tools have been developed for
predicting miRNA targets. Their activities are implicated in cancer
progression or suppression by affecting growth, transformation, invasion,
metastasis and response to treatment. A widespread deregulation of
miRNAs is commonly observed in human cancers and promotes
tumorigenesis. Thus, miRNAs may be potential targets for cancer therapy;
still, the mechanisms through which miRNAs are regulated in cancer
remain unclear.
Recently, we catalogued, from literature, a set of common miRNAs
deregulated in colon cancer. This search retrieved 118 deregulated
miRNAs. Through a computational analysis on 5,5 kb around the TSS, we
identified conserved NF-Y consensus motif in 39 (corresponding to 55
miRNAs) of the 118 miRNA’s promoters, among them the two promoters of
miR-200 family. Being potent inhibitors of epithelial-mesenchymal transition
(EMT), the members of miR-200 family are down-regulated in aggressive
human cancers. By ChIP experiments performed in human colon cancer
cells we observed that NF-Y directly binds the CCAAT-box of miR-200
family promoters and this binding correlates with the appearance of open
chromatin marks. Consistent with this, NF-Y silencing and overexpression
experiments indicate that NF-Y sustains E-cadherin protein expression
regulating miRNA-200 family expression at transcriptional level. Moreover,
we find that the over-expression of the regulatory subunit of the trimer, NFYA, inhibits cell migration. Finally, through a computational analysis we
identified NF-Y as putative target of miR-200 family members.
These data strongly suggest a main role for NF-Y in the regulation of miR200 family members.
69
Identification of long non-coding RNAs expression profile
in human breast cancer cells
Adnan HASHIM, Giorgio Giurato, Antonio Rinaldi,
Gianluca Santamaria, Alessandro Weisz
Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery,
University of Salerno, Baronissi (SA), Italy
With the advent and advancement of high-throughput transcriptomics, the
evidence that at least 90% of human genome, referred to as “junk DNA”, is
actively transcribed is revealing the involvement of non-coding RNAs
(ncRNAs), including in particular long ncRNAs (lncRNAs), in multiple
genetic and epigenetic pathways. Initially thought to be transcriptional
noise, lncRNAs are now known to mediate critical cellular functions, such
as transcriptional and post-transcriptional gene regulation, epigenome
remodeling and cellular stemness and differentiation, but the molecular
function of most lncRNAs is still to be elucidated. Recently, roles for
lncRNAs as drivers of tumor suppression and oncogenicity have been
discovered in prevalent cancer types, such as breast cancer, suggesting
that a better understanding of the mechanisms that control synthesis and
activity of these RNAs opens new venues in molecular oncology. To
investigate the implications of lncRNAs in hormone responsive breast
cancer, we performed an extensive transcriptome analysis in MCF7 cells,
that express the oncogenic Estrogen Receptor α (ERα), and in a clone of
these cells expressing also the oncosuppressor ERβ, under different in
vitro growth conditions. Bioinformatics analysis identified a pattern of
lncRNA expression that is significantly affected by ERα and, notably, also
by ERβ. When combined with the known functions of responsive RNAs,
these results indicate that lncRNAs expression is significantly influenced by
either one or both ERs, suggesting that control of hormonal breast
carcinogenesis and breast cancer phenotype by these transcription factors
is mediated also by the activity of a subset of lncRNAs.
RESEARCH SUPPORTED BY: AIRC (Grant IG-13176), MIUR (PRIN 2010LC747T_002),
University of Salerno (FARB 2011-2012), CNR (EPIGEN Flagship Project). A.H. is a PhD
student of the Research Doctorate ‘Experimental Physiopathology and Neurosciences’ of
the Second University of Napoli.
70
Aptamer-miRNA-212 conjugate sensitizes NSCLC cells to TRAIL
IABONI M.1, RUSSO V.1, Fontanella R.2, FIORE D.1, ROSCIGNO G.2,
Donnarumma E.3, Giangrande P.H.4, de Franciscis V.2,
CONDORELLI G.1,2
1
Department of Molecular Medicine and Medical Biotechnology, ‘Federico II’ University of
2
3
Naples, Naples, Italy; IEOS, CNR, Naples, Italy; IRCCS-SDN Foundation, Naples, Italy
4
Department of Internal Medicine, University of Iowa, Iowa City, IA, USA
Aptamers are single-stranded RNAs able to form different threedimensional structures, which allows them to specifically recognize their
molecular targets. Aptamers were first utilized for their ability to bind and
inhibit the activity of their target protein, including extracellular ligands and
cell surface proteins. More recently, aptamers were also used as delivery
agents. Thanks to their ability to be endocytosed, aptamers have been
incorporated into novel constructs involving siRNAs, chemotherapeutic
agents, cell toxins and nanoparticles, in which they function as delivery
agents for therapeutic cargoes.
In order to modulate TRAIL therapeutic response in non-small cell lung
cancer (NSCLC) cells, we designed an aptamer-microRNA chimera
molecule in which the aptamer is the delivery vehicle for a specific
microRNA. The use of microRNAs represents a challenging approach in
cancer therapy since they are able to regulate the expression of cellular
proteins modulating different pathways. However, to date the absence of
reliable means that permit the specific delivery of microRNAs to the
appropriate tissue represents an obstacle to the success of this approach.
We selected an internalizing aptamer, GL21.T, that specifically binds to the
Axl receptor, overexpressed in many types of cancer cells. We linked this
aptamer to miR-212. This miR is considered a tumour-suppressor miR
because negatively regulates the anti-apoptotic protein PED found
overexpressed in many tumors and involved in resistance to therapeutics,
included TRAIL. In A549 cells, TRAIL-resistant NSCLC cells
overexpressing Axl, the chimera was able to enter within the cells and carry
the miR to the processing machinery. We then demonstrated that miR-212,
subsequently targets PED, since PED protein levels were decreased upon
chimera treatment. Moreover, chimera incubation into TRAIL resistant cells
resulted in increasing TRAIL sensitization.
In conclusion, we demonstrate that the specific delivery of miR-212 into
NSCLC cells increases TRAIL-induced apoptosis, and, therefore, aptamermiR-212 conjugate may represent a promising therapeutic molecule.
71
miR-885-5p expression level in childhood neuroblastoma
Mariia V. INOMISTOVA, Nataliia M. Svergun, Natalia M. Khranovska,
Grygorii I. Klymniuk, Oksana V. Skachkova, Nataliia V. Kliukovska,
Oleksandr I. Gorbach
Laboratory of Experimental Oncology, National Cancer Institute, Kyiv, Ukraine
Neuroblastoma (NB) is the most frequent childhood malignant solid tumor
characterized by high clinical heterogeneity indicating the diversity of tumor
biology. p53/MDM2 pathway is often inactivated in NB by different
mechanisms and is also involved in MYCN oncogene overexpression which
has a critical role in determining the clinical behavior of NB [1]. MicroRNA
885-5p has a tumor suppressive role interfering with cell cycle progression
and cell survival. miR-885-5p leads to the accumulation of p53 protein and
activates the p53 pathway, resulting in upregulation of p53 targets [2]. The
aim of our study was to determine the miR-885-5p expression level (EL) in
NB tumors with different clinical and biological characteristics.
We analyzed miR-885-5p and MDM2 EL, and MYCN amplification in tumor
tissue obtained from 64 patients with NB: 51 biopsies – from primary NB
tumors, 6 – recurrent tumors, 7 – metastatic lymph nodes. 25% of tumor
samples were MYCN amplified.
Detection of miR-885-5p and MDM2 expression was conducted with realtime RT-PCR and their EL were evaluated by the ΔΔCt method. MYCN
amplification was detected with FISH method.
We established that the value of miR-885-5p expression in tumor samples
from NB patients varied in wide limits particularly depending on MYCN
status, MDM2 EL and disease stage. The lowest miR-885-5p EL was
noticed in recurrent tumors and metastatic foci of NB in comparison to
primary tumors (p<0.05). miR-885-5p EL was significantly lower in primary
tumors with high MDM2 EL than with low MDM2 EL (p<0.01). Low level of
miR-885-5p expression was observed more often in MYCN amplified
tumors versus non-amplified (p<0.05). Also, in advanced NB stages (III-IV)
significantly lower miR-885-5p EL was detected comparing to early disease
stages (p<0.02).
Our results suggest the possibility that miR-885-5p is involved in
MDM2/P53 pathway regulation in NB cells and might influence the clinical
behavior of NB.
References
1. Barone G, Tweddle DA, Shohet JM, et al. MDM2-p53 interaction in paediatric solid
tumours: preclinical rationale, biomarkers and resistance. // Curr Drug Targets. 2014;
15(1): 114-23.
2. Afanasyeva EA, Mestdagh P, Kumps C, et al. MicroRNA miR-885-5p targets CDK2 and
MCM5, activates p53 and inhibits proliferation and survival. // Cell Death Differ. 2011;
18(6): 974-84.
72
Transcriptional regulation of microRNAs through Meis1 in the
pathogenesis of acute myeloid leukemia
Edith SCHNEIDER1, Anna Staffas2, Arefeh ROUHI1, Lars Palmqvist2,
Konstanze Döhner1, Hartmut Döhner1, Florian KUCHENBAUER1
1
2
Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany; Department
of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital,
Gothenburg, Sweden
Deregulation of the HOX-gene cofactor MEIS1 is a commonly observed
phenomenon in acute leukemias. The oncogenic potential of aberrant
Meis1 expression has been previously shown in a murine Hox-gene based
acute myeloid leukemia model (AML). However, the relevant downstream
factors of MEIS1-induced leukemogensis are still not completely
understood. In order to dissect functionally relevant Meis1 induced
miRNAs, we used a Hoxa9-Meis1 AML progression model, allowing us to
quantify miRNAs at a pre-leukemic and leukemic stage by miRNA
microarrays. Of the differentially expressed miRNA species, we could
independently validate the overexpression of miR-155 by qRT-PCR.
To further determine the occurence of miR-155 we performed expression
profiling studies of all murine hematopoietic subpopulations. In accordance
with the known elevated levels of Hoxa9 and Meis1 in primitive bone
marrow cells, we confirmed an enrichment of miR-155 in primitive myeloide
cells.
In vitro functional analysis studies coupled with in vivo tranplantation
assays of mouse bone marrow cells co-overexpressing Hoxa9 and miR155 were used to elucidate the function of miR-155. Our results revealed
that overexpression of miR-155, in combination with Hoxa9, led to
enhanced proliferation/self-renewal, with a slight block of differentiation in
vitro, compared to cells only overexpressing Hoxa9. Mouse bone marrow
transduction experiments showed that co-overexpression of Hoxa9 and
miR-155 results in 70% engraftment four weeks post transplantation,
compared to 90% engraftment levels with the Hoxa9+Meis1 phenotype and
0-5% with Hoxa9 alone. In contrast to the agressive AML induced with
Hoxa9 and Meis1 co-overexpression, Hoxa9+miR-155 induces a
myeloproliferative disease-like leukemia 10-15 weeks post transplantation,
partially recapitulating the Hoxa9+Meis1 phneotype.
Our results highlight the role of miR-155 as relevant downstream target in
Meis1-induced leukemic transformations and make it a potential
therapeutical target.
73
The role of tumor-derived miRNAs in exosomes-mediated
suppression of NK cell cytotoxicity
Alireza LABANI MOTLAGH, Eva Dehlin, Olga Nagaeva, Ivan Nagaev,
Lucia Mincheva-Nilsson
Department of Clinical Microbiology, University of Umea, Umea, Sweden
Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer
and the one with the worst 5-year survival amongst all gynaecological tumors.
Its etiology and pathogenesis are to a great extent still unknown. The
establishment and metastatic spreading of EOC is associated with strong and
progressive derangement of the patients’ immune system. Although tumorinfiltrating lymphocytes are correlated with improved patient survival, the
immunosuppressive signals are often dominant in the tumor environment and
prevent effective elimination of cancer cells. Some of those signals are spread
by secretion of tumor exosomes, nanometer-sized extracellular vesicles with
endosomal origin that are constitutively produced and secreted by a huge
variety of cancers. Tumor exosomes, thanks to their cargo of bioactive
molecules and their ability to circulate in body fluids, have emerged as an
efficient tool used by cancer cells to influence the host immune responses
without the need for a cell-to-cell contact. Exosomes carry on their surface and
inside a variety of proteins, lipids and genetic materials (e.g. miRNAs).
Although NK cell cytotoxicity has a role in the immune defence against tumors,
malignant cells escape immune responses by releasing exosomes with
immunosuppressive ability. We have shown that NKG2D ligand-bearing
exosomes are able to impair NK cell function and cytotoxicity in vitro(1). At
present we study expression of the NKG2D ligands MICA, MICB, ULBP1-3 and
the DNAM-1 ligands nectin-4 and PVR in EOC tumors and the EOC cell lines,
OVCAR3 and SKOV3, at protein- and gene- expression levels. Furthermore,
we study the expression of these molecules on exosomes isolated from EOC
ascitic fluid and secreted by EOC cell lines and tumor explant cultures. We
plan to characterize the miRNAs carried by EOC exosomes and compare to
the miRNA profiles of the EOC cells that secrete them. We hypothesize that
EOC-derived exosomes carry functional miRNAs which are posttranscriptionally able to down-regulate the expression of various proteins
involved in NK cell-cytotoxicity. It is of great interest for future therapeutic
interventions to study the interactions between exosomal tumor-derived
miRNAs and the 3’-untranslated regions of genes involved in regulation of NK
cell cytotoxicity, e.g. receptors and proteins regulating cell proliferation.
Reference
1. Hedlund M., Nagaeva O., Kargl D., Baranov V., and Mincheva-Nilsson L. 2011. Thermaland oxidative stress causes enhanced release of NKG2D ligand-bearing
immunosuppressive exosomes in leukemia/lymphoma T and B cells. PLoS ONE. 6:
e16899.
74
miRNA-mediated co-regulation of urokinase receptor
and CXCR4 expression
1
Daniela Alfano, 2Anna Gorrasi, 2Anna LI SANTI, 3Patrizia Ricci,
4
Nunzia Montuori, 5Carmine Selleri, 2Pia Ragno
1
Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", CNR, Naples, Italy;
3
Department of Chemistry and Biology, University of Salerno, Italy; Department of Clinical
4
Medicine and Surgery and Department of Translational Medical Sciences, “Federico II”
5
University, Naples, Italy; Department of Medicine and Surgery, University of Salerno, Italy
2
MicroRNAs (miRs) play key roles in many biological processes. MiRs are
frequently deregulated in acute myeloid leukemia (AML) and could
represent useful biomarkers and targets in clinics.
CXCR4, the receptor for the SDF1 chemokine, is crucial in the retention of
hematopoietic stem cells (HSCs) in the bone marrow. CXCR4 activity can
be regulated by the urokinase receptor (uPAR), which is also involved in
HSC mobilization. Both uPAR and CXCR4 are expressed in AML, with a
lower expression in undifferentiated and myeloid subsets, and higher
expression in myelomonocytic and promyelocytic subsets. We
hypothesized a miR-mediated co-regulation of uPAR and CXCR4
expression in AML, which could allow their cross-talk.
We identified three miRs regulating uPAR and CXCR4 expression in AML
cell lines. Indeed, these miRs directly target the 3’untranslated region
(3’UTR) of both uPAR- and CXCR4-mRNAs; accordingly, uPAR/CXCR4
expression was reduced by their over-expression in AML cells and
increased by their specific inhibitors. Over-expression of all three miRs
inhibited migration and proliferation of myelomonocytic cells. Interestingly,
we observed an inverse relationship between uPAR/CXCR4 expression
and selected miRs in AML blasts, suggesting their possible role in the
regulation of uPAR/CXCR4 expression also in vivo.
75
Oleate regulates miRNA expression in
androgen-indipendent prostate cancer cells
Giorgio E.1, Olivieri M.2, Cosimato V.1, MARINO A.1, Perruolo G.3,
Ferro M.4, Perdonà S.5, Beguinot F.1,3, Formisano P.1,3,
CIMMINO A.2, Terracciano D.1
1
2
DiSMeT, University of Study of Naples, Italy; IGB, Institute of Genetics and Biophysics
3
4
“Adriano Buzzati-Traverso”(CNR) Naples, Italy; IEOS/CNR, Naples, Italy; IEO, European
5
Oncologic Institute, Milan, Italy; INT, IRCCS “Fondazione G. Pascale”, Naples, Italy
Prostate cancer (PCa) is the most frequently diagnosed malignancy in men
and the second leading cause of cancer deaths in western countries1.
Epidemiological studies suggest an association between dietary fat intake,
prostate cancer risk and bad prognosis2. We have showed that oleate is
able to promote proliferation and motility and to impair drug response in
PCa cells. Little is known about the mechanisms regulating the effects of
fatty acids on prostate cancer cells. We used a relatively new approach, the
micro-RNA (miRNAs), small non-coding RNA sequences able to regulate
the expression of genes linked to cancer3. We evaluated whether oleate,
the main circulating free fatty acid may affect miRNA expression in
androgen-indipendent (AI) prostate cancer cells. In particular, we
investigated a series of miRNA related to prostate cancer aggressiveness:
miRNA-32 (resistance of PCa cells to apoptotic stimuli by downregulation of
gene BIM)4; miRNA-34a (PCa cells chemo-resistance by inhibition target
gene SIRT-1)5; miRNA-200b (epithelial-mesenchymal transition of PCa
cells by inhibition of ZEB1 and ZEB2 genes)6; miR-221 and miR-222
(proliferation potential of human PCa cell lines by targeting p27Kip1)7;
miRNA-370 (proliferation of human PCa cells by downregulating the
transcription factor FOXO)8.
We incubated AI PCa cell lines DU145 with oleate 200µM for 48h and we
observed a significant downregulation of miRNA-32, miRNA-34a, miRNA200b, miRNA-221, miRNA-222 and miRNA-370 expressions by real-time
PCR compared to control cells.
In conclusion, oleate seems to regulate miRNA expression in AI PCa cells,
suggesting a potential mechanism linking dietary factors and PCa
malignant phenotype.
References
1. Nelson WG et al (2003). N Engl J Med; 349:366-381.
2. Crowe FL et al (2008). Am J Clin Nutr.; 87(5):1405-13.
3. Bagga S et al (2005). Cell.;122(4):553-63.
76
miR-17-92 counterbalances MYC and fine-tunes MYC-centred
regulatory networks in lymphoma maintenance
Marija MIHAILOVICH, Michael Bremang, Gabriele Varano, Federico
Zambelli, Giulio Pavesi, Stefano Casola, Tiziana Bonaldi
Dept. of Experimental Oncology, European Institute of Oncology, Milan, Italy
The functional effect of miRNA depends on their molecular environment,
having the potential to play different roles in cancer development and
maintenance. Although the synergism between the transcription factor cMYC and miR-17-19b, a truncated version of the miR-17-92 cluster, has
been demonstrated in tumor initiation, little is known about the role of these
miRNAs in full-blown lymphoma.
Here we investigate the role of miR-17-19b in an aggressive model of cMYC driven B cell lymphoma. Our data demonstrate that the functional
interplay between c-MYC and miR-17-19b is highly plastic and changes
dynamically during tumor development as a consequence of the plasticity
of the tumor’s transcriptome. We observed an extensive 3’ UTR shortening
in full-blown lymphoma, which correlates with a distinct outcome of miR-1719b activity relative to tumorigenesis. Hence, in an established lymphoma,
a mild increase in miR-17-19b levels reduces MYC oncogenic potential,
interfering with tumor aggressiveness, both in vitro and in vivo. Proteomicsbased approach revealed more than a hundred novel miR-17-19b targets,
out of which 40% are shared with c-MYC, indicating a role of these miRNAs
in fine-tuning c-MYC transcriptional activity. We also show that miR-17-19b
negatively regulates translation of c-MYC through the novel target Chek2.
Modulation of Chek2-dependent phosphorylation of the RNA-binding
protein HuR results in increased binding of HuR to c-MYC mRNA. HuR, in
turn, recruits the miRNA-loaded RISC complex to c-MYC mRNA, thus
providing a tight regulation of its translation.
Taken together, our data suggest that while miR-17-92 potentiates c-MYC
pro-tumorigenic function during lymphomagenesis, it dampens its
oncogenic activity in established tumors by exerting a fine control over cMYC expression and functions. Thus, the cluster preserves cancer cell
hemostasis, which guarantees the right balance between proliferation and
apoptosis and, thus sustain tumor maintenance and progression.
77
High-throughput analysis of microRNAs in
Malignant Pleural Mesothelioma
Stefania OLIVETO,2, Pierluigi Gasparini3, Luciano Mutti4,
Carlo M. CROCE3, Stefano Biffo1,2
1
2
3
INGM, “Romeo ed Enrica Invernizzi”, Milano, Italy, DISIT, Alessandria, Italy; Human
Cancer Genetics Program, Ohio State University Comprehensive Cancer Center, Columbus,
4
OH, USA; Laboratory of Clinical Oncology, Vercelli National Health Trust, Vercelli, Italy
MicroRNAs (miRNAs) are 19-25 nucleotide-long non coding RNA
molecules. miRNAs negatively regulate the expression of several genes
both at messenger RNA (mRNA) level, degrading mRNA target, and at
protein level, blocking translation. miRNAs are involved in different cellular
processes, including proliferation, development, differentiation, apoptosis
and stress response. One of the most recent advances is the
understanding of their role in oncogenesis. Alterations in miRNAs
expression are involved in the initiation, progression and metastasis of
different tumors, playing a relevant role as oncogene or tumor suppressor
gene during tumorigenesis. MiRNAs are aberrantly expressed in several
neoplasm, including breast, colon, prostate and lung cancers. Although
abnormal expression of miRNAs has been linked to cancer progression, the
mechanism of this dysregulation is yet unknown. Malignant pleural
mesothelioma (MPM) is an aggressive and lethal asbestos-related cancer
resistant to conventional therapies and with a dramatic low survival. We
used a microarray approach to analize miRNA profile in different
mesothelioma cell lines, representative of the three histopatologic subtypes
(epithelioid, biphasic and sarcomatoid type). We found an high variability in
the expression levels of miRNAs, reflecting the heterogeneity of MPM, in
particular we observed a substantial difference between REN and MM98
representing respectively the less and the most aggressive form of MPM.
miRNAs may inhibit the translation of target mRNAs at the initiation stage
of protein synthesis or at the postinitiation phase. In order to differentiate,
we performed a sucrose density gradient analysis of REN cells allowing
mRNAs to be separated, based on the number of polysomes associated.
Then, to identify the localization of miRNAs in RNA subpopulations, we
analyzed miRNAs distribution both in monosomes and active polysomes.
We found that, after dissociation of polyribosomes into single subunits,
some miRNAs showed a significant change of localization, whereas others
were stable, suggesting that some miRNAs could be associated to binding
partners ,such as, for istance, ribonucleoprotein complexes. Future studies
will clarify what structures are involved in polysomal miRNAs distribution.
78
LincRNAs landscape in human lymphocytes highlights regulation
of T cell differentiation by linc-MAF-4
Valeria RANZANI, Grazisa Rossetti, Ilaria Panzeri, Alberto Arrigoni, Raoul
JP Bonnal, Serena Curti, Paola Gruarin, Elena Provasi, Elisa Sugliano,
Raffaele De Francesco, Jens Geginat, Beatrice Bodega,
Sergio Abrignani, Massimiliano PAGANI
Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi”, Milano, Italy
Long intergenic non-coding RNAs are a novel class of regulatory RNAs
with a high cellular and temporal specificity of expression in defined
developmental stages. Recent studies demonstrated the involvement of
lincRNAs in cell fate determination and maintenance in diverse biological
contexts. Nevertheless only few studies have been published regarding
their function in the human immune system. In our study we investigated
long intergenic non-coding-RNAs (lincRNAs) in thirteen T and B
lymphocyte subsets by RNA-seq analysis and de-novo transcriptome
reconstruction and we defined a set of lincRNAs that are specifically
expressed in these subsets. In particular, we focused on the CD4+ Th1
lincRNAs signature and investigated the functional role of linc-MAF-4,
located next to MAF gene (150 Kb): a Th2-associated transcription factor.
The expression of linc-MAF-4 is anti-correlated with the expression of MAF
and its down-regulation skews T cell differentiation toward a Th2-like fate.
We identified a long-distance intra-chromosome interaction between lincMAF-4 and MAF genomic regions and demonstrated the enrichment of lincMAF-4 transcript in the chromatin fraction. Furthermore we showed that
linc-MAF-4 associates with LSD1 and EZH2, thus likely acting as a recruiter
of chromatin modifiers for MAF promoter resetting in Th1 cells. Our results
highlight the functional importance of non-coding RNAs as drivers of human
T lymphocytes differentiation processes.
79
miR-155 regulates BUB1, CENP-F and ZW10 affecting
cellular chromosomal stability
Sara PAGOTTO1,2, Serena Veschi, Alessandra Soranno, Paola Lanuti,
Mirco Di Marco, Marco Vincenzo Russo, Simone Guarnieri, Marilena Iorio,
Nicola Valeri, Marco Marchisio, Sebastiano Miscia, Paolo Emilio Guanciali
Franchi, Giandomenico Palka, Veronica Balatti, Luca Laurenti, Renato
Mariani Costantini, Carlo M. CROCE, Angelo Veronese, Rosa Visone1,2
1
Department of Medical, Oral and Biotechnological Sciences, G. d'Annunzio University,
2
Chieti, Italy; Unit of General Pathology, Aging Research Center (Ce.S.I.), Chieti, Italy
Defects in spindle checkpoint contribute to chromosome instability (CIN)
and subsequent production of aneuploid daughter cells. These defects
represent an hallmark of many tumors including chronic lymphocytic
leukemia. In the attempt to better clarify this process in CLL, we investigate
the putative role of the miR-155 in CIN. MiR-155 has been selected since it
is up-regulated in CLL and many solid tumors and its overexpression has
been linked to other genetic mechanisms, such as the genomic instability
and DNA repair. Here we describe a new role of miR-155 in mitotic
chromosome congression that is dependent of its newly identified spindle
checkpoint regulatory targets such as BUB1, CENP-F and ZW10.
Moreover, the mutational status of BUB1, an activator of the spindle
checkpoint found mutated and down-regulated in several tumors, has been
assessed in patients with CLL as well. We observed two missense
mutations into the BUB1 coding sequence in two CLL samples out of 30
aggressive CLL tumor samples (6,7%). To provide evidence of miR-155
capacity to induce misaligned chromosomes, we studied by
immunofluorescence analysis, the morphology of cellular metaphases and
errors in chromosome congression were counted. We found in two
independent experiments a decreased number of misaligned chromosomes
in immortalized HDF infected with an LV anti-miR-155 (Exp1 p<0,001; Exp2
p:0,028) and conversely an increase of errors in U2OS cells infected with
LV miR-155 (p:0,0038). Since BUB1, CENP-F and ZW10 aberrations are
linked to chromosome miss-segregation and tumorigenesis via CIN
generation, we performed a karyotype characterization in immortalized LV
miR-155AS and control HDF at different passage numbers in two
independent experiments. The analysis shows as LV miR-155AS HDF
maintained near-diploid karyotype compared with the control cultures which
resulted in a marked early aneuploidy and chromosomal instability (fisher's
exact test Exp1 p:0,04; Exp2 p:0,002). These findings provide a direct
mechanism linking miR-155 and CIN.
80
RNase A inhibits metastasis spreading upon tumor progression due
to reorganization of regulatory networks of miRNAs
Olga A. PATUTINA, Nadezhda L. Mironova, Evgeniy V. Brenner, Alexander
M. Kurilshikov, Valentin V. Vlassov, Marina A. Zenkova
Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia
MicroRNAs have been recently defined as key players in tumor progression
and invasion and thus represent attractive targets for the use of nucleasebased therapy. Antitumor and antimetastatic properties of pancreatic
RNase A have been studied and proved [1]. It was found that observed
antimetastatic effect of RNase A was accompanied by a reduction in
pathologically elevated levels of extracellular RNAs and an increase in
nuclease activity of blood plasma of tumor-bearing animals. By highthroughput SOLiD sequencing technology we performed an analysis of
genome-wide profiles of miRNAs in the tumor and serum of mice with
Lewis lung carcinoma (LLC) after treatment with RNase A. Sequencing
data revealed that RNase A therapy resulted in the boost of 116
microRNAs in the tumor tissue and a significant drop of 137 microRNAs in
the bloodstream, that was validated by qPCR [2]. Analysis of miRNA profile
showed that the pool of miRNA with the most significantly changed level
contains a considerable number of ascertained tumor-associated miRNAs,
such as miRNAs from let-7 family, miR-21, miR-10b, miR-145, miR-451a,
miR-29b1, miR-17a, miR-18a and others. The presumable mechanism of
antimetastatic effect of RNase A is a generation of short RNA fragments
from long circulating noncoding RNAs such as tRNA, rRNA and snoRNA,
that competitively displace oncogenic miRNAs from circulating Ago2
nucleoprotein complexes, ensuring accessibility of miRNAs for RNase
cleavage.
Owing to multiplicity and complexity of cancer-associated disturbances in
miRNA production, the impact of nuclease therapy on the entire regulatory
networks of miRNAs represents a promising therapeutic approach.
This work was supported by RAS programs “Molecular and cellular biology” and “Sciences
to medicine 2012-24”, RFBR 14-04-0107a, SS № 1350.2014.4 and Fellowship 2307.2012.4.
References
1. Patutina O, Mironova N, Ryabchikova E, Popova N, Nikolin V, Kaledin V, Vlassov V,
Zenkova M. Inhibition of metastasis development by daily administration of ultralow
doses of RNase A and DNase I. Biochimie. 2011,93(4):689-96.
2. Mironova N, Patutina O, Brenner E, Kurilshikov A, Vlassov V, Zenkova M. MicroRNA
drop in the bloodstream and microRNA boost in the tumour caused by treatment with
ribonuclease A leads to an attenuation of tumour malignancy. PLoS One. 2013,
8(12):e83482.
81
Regulation of miR 483-3p by cellular glucose uptake and drug
resistance in hepatoma cancer cells
Felice PEPE1, Sara PAGOTTO, Chiara Braconi, Laura Lupini, Massimo
Negrini, Renato Mariani-Costantini, Rosa Visone, Veronese Angel
1
Unit of General Pathology, Aging Research Center (Ce.S.I.), G. d'Annunzio University
Foundation, Chieti, Italy
The hepatocellular carcinoma (HCC) is one of the most common cause of
tumor-related death in the world, and it is characterized by significant
chemoresistance. We have previously shown that the miR-483-3p is overexpressed in about 30% of HCC and could have a role in the tumor
chemoresistance by targeting the p53 upregulated modulator of apoptosis
protein (BBC3/PUMA). This miR is transcriptionally regulated by the factors
CTNNB1 and USF1 at the E-Box DNA sequence located upstream the
miR. Since it is reported that CTNNB1 and USF1 respond to the cellular
glucose concentration, we hypothesized that either glucose depletion, or
drugs that inhibits glucose metabolism (2-deoxy-D-glucose, 2-DG), could
reduce the expression of the miR-483-3p. Therefore we cultured two HCC
cell lines, HepG2 and Hep3B, p53 wild type and null respectively, in either
no-glucose conditions or 2-DG, and we saw a reduction of the miR in both
cases. Then, by luciferase assay, we identify the CTNNB1/USF1 E-box as
the responsive element to glucose media concentration. Since it is reported
that CTNNB1 is O-GlcNAcylatated by the O-linked N-acetylglucosamine
transferase (OGT), that uses derivative of glucose, we studied the
involvement of this pathway on the miR-483-3p expression. By silencing
OGT or using Azaserine, a specific OGT-pathway inhibitor drug, we saw
the reduction of the miR expression, suggesting that this pathway could be
the link between glucose concentration and miR-483-3p. Finally, to study if
the 2-DG could be useful to improve the drug therapy by the reduction of
the miR, we treated the HepG2 cells with either 2-DG, or the drug 5fluorouracil (5-FU) or both the drugs. Surprisingly, we saw that the drug
combination had less apoptotic response than 5-FU treatment alone. We
repeated the treatment after transfection of the anti-miR 483-3p, and we
saw that the reduction of the miR-483-3p improved significantly the drugsinduced apoptosis, reverting the protective effect of the 2-DG in
combination with the 5-FU. The sensitivity to 5-FU, linked to the miR-4833p expression, was less evident in Hep3B cells, and was not observed for
other drugs (doxorubicin and sorafenib). These data show that the 2-DG
and the miR 483-3p reduction could be useful in a certain tumor genetic
context (p53 wild type) to improve the efficacy of 5-fluorouracil and hinting
the re-enter of this drug in the management of HCC patients.
82
Exploring RNA editing alterations in ncRNAs
of tumor samples through REDItools
Ernesto Picardi1, Claudia Lionetti1, Caterina Manzari2, Italia Aiello1, Anita
ANNESE1, Bruno Fosso2, Bachir Balech2, Anna Maria D’Erchia1,
Graziano PESOLE1,2
1
Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Italy
2
Institute of Biomembranes and Bioenergetics (IBBE), CNR, Bari, Italy
RNA editing is a post-transcriptional molecular phenomenon whereby a
genetic message is modified from the corresponding DNA template by
means of substitutions, insertions and/or deletions (PMID: 11092837). In
eukaryotes, it mainly involves the deamination of adenosines to inosines by
the family of ADAR enzymes acting on double RNA strands (PMID:
20192758). A-to-I RNA editing has a plethora of biological effects
depending on the RNA region involved in the modification. Changes in
UTRs can lead to altered expression, whereas modifications in coding
protein regions can induce amino acid replacements with more or less
severe functional consequences (PMID: 22988838). In addition, RNA
editing deregulation has been linked to a variety of human diseases
including neurodegenerative disorders and cancer (PMID: 24289319).
Recent genomic-scale investigations has revealed that A-to-I changes
occur mainly in non-coding protein regions (PMID: 22484847).
Consequently, diverse ncRNAs may be subjected to RNA editing with
potential effects on the modulation of gene expression.
Nowadays RNA editing events can be investigated by NGS technology
through REDItools (http://code.google.com/p/reditools/), a suite of python
scripts aimed to the genome-wide detection of RNA editing changes by
using RNA-Seq and DNA-Seq data or RNA-Seq data alone (PMID:
23742983).
Here we show the results of REDItools application to explore RNA editing
alterations in ncRNAs of tumor samples from The Cancer Genome Atlas
(TCGA) database (http://cancergenome.nih.gov/).
83
The role of microRNAs in Endothelial to Hematopoietic Transition
Irina S. M. PINHEIRO1, Kerstin Ganter1, Harpreet K.Saini2,
Anton J. Enright2, Christophe Lancrin1
1
European Molecular Biology Laboratory (EMBL) Mouse Biology Unit, Monterotondo, Italy;
2
European Bioinformatics Institute, Hinxton, Cambridgeshire, UK
The Endothelial to Hematopoietic Transition (EHT) constitutes a key step in
the formation of hematopoietic stem cells. Even though recent discoveries
have identified several transcription factors involved in this process, many
things remain to be discovered about EHT.
MicroRNAs differential expression observed in specific cell types suggests
that they could regulate lineage commitment [1,2]. Nevertheless, their role
in early hematopoietic mouse development remains unclear. We have
recently identified miR-142-3p as a potential regulator of EHT. miR142-3p
has also been reported to be an important regulator of the formation and
differentiation of hematopoietic stem cells [3]. Validation of miR-142-3p
function and expression as well as identification of its potential targets, both
in vitro and in vivo, is therefore necessary.
In addition, abrogating microRNA pathways in vivo will give us insight into
the general importance of microRNAs during blood stem cell and
progenitors generation.
References
1. Petriv, O. I., Kuchenbauer, F., Delaney, a D., Lecault, V., White, a, Kent, D., Marmolejo,
L., et al. (2010). Comprehensive microRNA expression profiling of the hematopoietic
hierarchy. Proceedings of the National Academy of Sciences of the United States of
America, 107(35), 15443–8.
2. Mildner, A., Chapnik, E., Manor, O., Yona, S., Kim, K.-W., Aychek, T., Varol, D., et al.
(2013). Mononuclear phagocyte miRNome analysis identifies miR-142 as critical
regulator of murine dendritic cell homeostasis. Blood, 121(6), 1016–27.
3. Lu, X., Li, X., He, Q., Gao, J., Gao, Y., Liu, B., & Liu, F. (2013). miR-142-3p regulates the
formation and differentiation of hematopoietic stem cells in vertebrates. Cell Research,
23(12), 1356–68.
84
Role of human miR-125a-5p in hepatitis B virus infection
progressing to severe livere disease and hepatocarcinoma
Nicoletta POTENZA, Nicola Mosca, Filomena Castiello,
Nicola Coppola, Aniello Russo
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies,
Second University of Naples, Caserta, Italy
The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV
infection is a major risk factor for hepatocellular carcinoma (HCC). Some
cellular microRNAs are emerging as important regulators of virus–host
interaction, indirectly or directly modulating HBV replication and pathogenesis.
miR-125a binds the viral transcript encoding the surface antigen and interferes
with its expression, thus inhibiting viral replication (1, 2). Intriguingly, liver miR125a expression has been found increased in patients with high levels of
hepatic HBV-DNA (3), suggesting that liver exposure to HBV induces the
expression of miR-125a. This hypothesis was explored by focusing the
attention on the viral X protein (HBx), a transcriptional transactivtor of viral and
cellular promoters and enhancers. The analyses were first performed on liver
biopsies from HBV patients, showing that the expression of HBx paralleled the
increase of miR-125a expression. Moreover, a clear correlation between high
HBx levels and the severity of the liver disease was observed, supporting the
role of HBx in the hepatocarcinogenesis. Then, transfection of HCC cell lines
with an HBx-expressing vector showed a substantial increase of miR-125a
expression (4). Overall, the available data depict a self-inhibitory feedback loop
in which HBV, through HBx, increases the expression of miR-125a, that in turn
interferes with expression of HBV surface antigen, thus repressing viral
replication; fine-tuning of viral replication would be beneficial for both the host
and the virus, because it possibly leads to a persistent infection with a long
host survival and a high spread of the virus in the human population.
Taking into account that miR-125a has also a number of oncogenic targets
relevent for HCC, the regulatory circuit highlited in this study has potential
investigational and clinical implications both for HBV infection and for
hepatocarcinogenesis, since an outcome re-direction from a persistent
infection, possibly progressing to HCC, to a complete recovery may be
envisaged by unbalancing the molecules involved in the virus-host
coexistence.
References
1. Potenza, N.; Papa, U.; Mosca, N.; Zerbini, F.; Nobile, V.; Russo, A. Nucleic Acids Res.
2011, 39, 5157-5163
2. Park, S.O.; Kumar, M.; Gupta, S. PLoS One 2012, 7, e39276.
3. Coppola, N.; Potenza, N.; Pisaturo, M.; Mosca, N.; Tonziello, G.; Signoriello, G.;
Messina, V.; Sagnelli, C.; Russo, A.; Sagnelli, E. PLoS One 2013, 8(7), e65336
4. Mosca, N.; Castiello, F.; Coppola, N.; Trotta, M.C.; Sagnelli, C.; Pisaturo, M.; Sagnelli,
E.; Russo, A.; Potenza, N. Biochem Biophys Res Commun. 2014, 449, 141-145
85
Detection and role of circular RNAs in breast cancer
L. RICCI, 2, G. Ferrero1, 2, F. Cordero1,3, L. Annaratone4, I. Gregnanin5,
G. Chiorino5, A. Sapino4, Michele De Bortoli1, 2
1
2
Center for Molecular Systems Biology, University of Turin, Italy, Department of Clinical and
3
Biological Science, University of Turin, Italy, Department of Computer Science, University
4
of Turin, Italy, Department of Medical Science University of Turin, Italy,
5
Fondo Edo Tempia, Biella, Italy
Circular RNAs (circRNAs) are a class of endogenous, abundant and stable
noncoding RNAs recently revealed in mammalian cells, but their function has
remained elusive. Recent studies have suggested that some circRNAs may
regulate microRNAs function and control transcription (Memczak et al. 2013;
Jeck et al. 2013). To delineate their potential role as regulators of cellular
behaviour in disease, we identified and characterized circRNAs in breast
cancer. Using datasets from ENCODE RNA-Seq on MCF-7, we predicted a
total of 433 circRNAs: of these, 83 were specific to the MCF-7 cell line,
whereas 350 overlapped with genomic coordinates of predicted circRNAs
published in different cell lines (Salzman et al. 2013). The majority of predicted
MCF-7 specific (MCF7s) circRNAs overlapped with exons of protein coding
genes with an ontological enrichment relative to “membrane organization” and
“formation of plasma membrane projection” terms, suggesting that MCF7s
circRNAs could be involved in specific processes. We experimentally validated
our predicted circRNAs through quantitative PCR, using divergent primers that
amplify the backsplice junction sequence of the RNA molecule that circularize,
as well as RNase R, an exonuclease that leaves intact circular molecules. In a
panel of 24 predicted circRNAs, 19 showed a fair level of expression in MCF-7
and were resistant to RNase R treatment. Several circRNAs showed variable
levels of expression across different breast cancer cell lines, suggesting that
circRNAs have a cell-type specific abundance. Finally, we investigated the
expression of circRNAs in breast cancer tissues and plasma collected from
breast cancer patients with different clinical phenotypes. We highlighted
differences in expression between various subtypes of samples, underlining
their potential role as molecular markers of breast cancer phenotypes. Ongoing
work consists in further discovering and characterizing circRNAs by poly(A)RNA-Seq on MCF-7 cells after estrogen receptor-α depletion and estradiol
treatment, in order to determine circRNAs that could define a Luminal A breast
cancer phenotype.
References
1. Memczak S et al. Circular RNAs are a large class of animal RNAs with regulatory
potency. Nature. 2013 Mar 21;495(7441):333-8.
2. Jeck WR et al. Circular RNAs are abundant, conserved, and associated with ALU
Repeats. RNA. 2013 Feb;19(2):141-57
3. Salzman J et al. Cell-type specific features of circular RNA expression. PLoS Genet.
2013;9(9):e1003777.
86
MiR-221 acts as stemness promoter in breast cancer cells
by targeting DNMT3b
Giuseppina ROSCIGNO1,2, C. Quintavalle1,2, M. IABONI1, I. Puoti1,
E. Donnarumma3, D. FIORE1, V. RUSSO1, M. Todaro4, G. Stassi4,
G. Romano5, C. CROCE5, R. Thomas6, G. Cortino6, S. Piscuoglio7,
L. Terracciano7, G. CONDORELLI1,2,3
1
Department of Molecular Medicine and Medical Biotechnology, “Federico II” University of
2
3
Naples, Italy; IEOS, CNR, Naples, Italy; IRCCS-SDN Foundation, Naples, Italy;
4
Department of Surgical and Oncological Sciences, Cellular and Molecular Pathophysiology
5
Laboratory, University of Palermo, Italy; Department of Molecular Virology, Immunology
and Medical Genetics, Human Cancer Genetics Program, Comprehensive Cancer Center,
6
The Ohio State University, Columbus, OH, USA; Department of Surgical and Oncology,
7
Clinica Mediterranea, Naples, Italy; Molecular Pathology Division, Institute of Pathology,
University Hospital, Basel, Switzerland
Multiples studies report that within the cancer there is a subpopulation of stem-celllike cells with the ability to self-renew and to generate the bulk of differentiated
cells that form the tumor. This population of cancer cells, called cancer stem cells
(CSC), is responsible for sustaining the tumor growth and is able to disseminate
and migrate giving metastases to distant organs. Furthermore, CSCs have shown
to be more resistant to anti-tumor treatments than the differentiated cells,
suggesting that surviving CSCs could be responsible for tumor relapse after
therapy. Nevertheless, the molecular mechanisms underlying the cancer stem-like
properties are not well characterized yet. MicroRNAs (miRs) are small, noncoding
RNAs (20-25 nucleotidies) that play a crucial role in biological processes including
development, proliferation, and apoptosis. Previous investigations have linked
miRs to the control of self-renewal and differentiation of normal stem cells. The aim
of this study was to test the functional role of miRs in human Breast Cancer Stem
Cells (BCSCs) also named mammospheres. We analyzed, by miR-Array, the miRs
differentially expressed in BCSCs and their differentiated counterpart. Among
several miRs, we focused our attention on miR-221 that was found increased in
mammospheres. In order to validate data achieved in primary cultures, we
obtained mammospheres from T47D, an immortalized breast cancer cell line.
Interestingly, like primary cultures mammospheres, also T47D mammospheres
exhibited increased levels of miR-221 compared to T47D differentiated cells.
Moreover, the overexpression of miR-221 by a miR mimic in T47D differentiated
cells was able to increase the number of mammospheres and the expression of
stem cell protein markers. Among miR-221 targets, we demonstrated, by
luciferase-assay, that miR-221 targets the 3' untranslated region of DNMT3b, a
DNA Methyl Transferase. Furthermore, our data showed that DNMT3b was able to
repress the expression of some stemness genes, such as Nanog and Oct3/4, and
mammospheres formation partially reverting miR-221 mediated effects on
stemness properties. In conclusion, we provided evidence that miR-221 may
contribute to breast cancer tumorigenicity regulating the stemness properties
through DNMT3b expression.
87
miRandola: towards a comprehensive resource
for non-invasive biomarkers
Francesco RUSSO1,2, Sebastiano Di Bella3, Giovanni Nigita4,
Alessandro Laganà4, Alfredo Pulvirenti5, Rosalba Giugno5,
Marco Pellegrini1, Alfredo Ferro5
1
Laboratory of Integrative Systems Medicine (LISM), IIT-IFC-CNR, Pisa, Italy;
2
3
Department of Computer Science, University of Pisa, Pisa, Italy; Nerviano
4
Medical Sciences; Department of Molecular Virology, Immunology and
Medical Genetics, Comprehensive Cancer Center, The Ohio State University,
5
Columbus, Ohio, USA; Department of Clinical and Molecular Biomedicine,
University of Catania, Catania, Italy
Non-coding RNAs (ncRNAs) such as for example microRNAs (miRNAs)
are frequently dysregulated in cancer and have shown great potential as
tissue-based markers for cancer classification and prognostication. ncRNAs
are present in membrane-bound vesicles, such as exosomes, in
extracellular human body fluids. Circulating miRNAs are also present in
human plasma and serum cofractionate with the Argonaute2 (Ago2) protein
and the High-density lipoprotein (HDL). Since miRNAs and the other
ncRNAs circulate in the bloodstream in a highly stable, extracellular forms,
they may be used as blood-based biomarkers for cancer and other
diseases. A knowledge base of non-invasive biomarkers is a fundamental
tool for biomedical research. Here, we present an updated version of the
miRandola database, a comprehensive manually curated collection and
classification of extracellular circulating miRNAs. miRandola contains 2366
entries, with 599 unique mature miRNAs and 23 types of samples,
extracted from 139 papers. miRNAs are classified into four categories,
based on their extracellular form: miRNA-Ago2 (173 entries), miRNAexosome (862 entries), miRNA-HDL (20 entries) and miRNA-circulating
(1311 entries). Moreover, the database contains several tools, allowing
users to infer the potential biological functions of circulating miRNAs, their
connections with phenotypes and the drug effects on cellular and
extracellular miRNAs. The future direction of the database is to be a
resource for all the potential non-invasive biomarkers such as lncRNAs,
cell-free DNA and circulating tumor cells (CTCs). miRandola is available
online at: http://atlas.dmi.unict.it/mirandola/
References
1. Russo et al. (2012) miRandola: Extracellular Circulating MicroRNAs Database. PLoS
ONE 7(10): e47786
2. Russo et al. (2014) A knowledge base for the discovery of function, diagnostic
potential and drug effects on cellular and extracellular miRNAs. BMC Genomics
15(Suppl 3):S4
88
Innovative therapies based on the use of non-coding RNA for nonsmall cell lung cancer (NSCLC)
RUSSO V.1, IABONI M.1, ROSCIGNO G. 1,2, Donnarumma E.3,
FIORE D.1, Esposito C. L.2, de Franciscis V.2, CONDORELLI G.1,2
1
Department of Molecular Medicine and Medical Biotechnology, "Federico II” University of
2
3
Naples, Italy; IEOS, CNR, Naples, Italy; Fondazione IRCCS SDN, Naples, Italy
Recent studies have shown the great potential of microRNA (miRNA)
mimics or miRNA inhibitors (antimiRs) as cancer therapeutics. However, a
major obstacle to their translation to clinic is actually represented by the
lack of a robust and reliable way to selectively deliver them to the target
malignant tumor cells.
An expanding new class of biomolecules that is revealing as highly
promising for the specific delivery of RNA-based therapeutics are nucleicacid aptamers.
We intend to validate these aptamers as cell-specific delivery molecules for
“therapeutic” miRNAs. We identified two tumor-suppressive miRs in nonsmall cell lung cancer (NSCLC), miR-34c and miR-126. We validated that
the expression of both miRs is low in NCSCL and when transfected into cell
lines are able to impact on cell survival. By applying methods successfully
used in our laboratory to conjugate aptamer to miRNAs, we have generated
two different “chimeras”. Chimeras have been generated via stick-end
annealing. With this procedure, the aptamer and a single chain anti-miRNA
(or the passenger strand of the miRNA) are annealed by the mean of
complementary sticky ends elongated at the 3’ end of the aptamer and at
the 5’ end of the single chain of the miRNA, respectively. We conjugated
either miRNA-34c or miR-126 to a nucleic acid aptamer, that selectively
recognize the Axl receptor and is rapidly internalized (GL21.T).
We demonstrated that the chimera is able to bind NSCLC cells and to carry
the miR within the cells. Interestingly, we demonstrated that one of the miR34c target is the Axl receptor. Thus, the GL21.T- miR-34c chimera is able
to exert a dual inhibition of Axl, either at functional or at transcriptional level.
We are thus evaluating the functional effect of the chimeras in the
regulation of proliferation and tumor regression in NSCLC that selectively
express Axl receptor.
89
In vitro platelet activation affects microRNA expression profile
inducing proteome reorganization
Annamaria SALVATI1, Giovanni Nassa1, Giovanni Cimmino2,
Giorgio Giurato1, Maria R. De Filippo1,3, Maria Ravo1, Francesca Rizzo1,
Giuseppina Bruno1, Stefano Conte2, Grazia Pellegrino4, Plinio Cirillo4,
Paolo Calabrò2, Tiina Öhman5, Nyman A. Tuula5, Alessandro Weisz1,6,
Paolo Golino2, Roberta Tarallo1
1
Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery,
2
University of Salerno, Baronissi (SA), Italy; Department of Cardiothoracic and Respiratory
3
Science, Second University of Naples, Italy; Department of Pathology, Memorial Sloan
4
Kettering Cancer Center, New York, USA; Department of Advanced Biomedical Sciences,
5
University of Naples Federico II, Italy; Institute of Biotechnology, University of Helsinki,
6
Finland; Division of Molecular Pathology and Medical Genomics, “SS. Giovanni di Dio e
Ruggi d’Aragona – Scuola Medica Salernitana” University of Salerno Hospital, Salerno, Italy;
Platelets are anucleate cytoplasmic fragments, derived from megakaryocytes, containing specific megakaryocytes-derived mRNAs and
microRNAs (miRNAs) and the machinery for de novo proteins synthesis1-2.
Given their role in severe pathologies, including the acute coronary
syndrome, understanding the functional changes that characterize platelet
responses to activating stimuli can provide new ways to control these
diseases. To this aim, purified platelet preparations from healthy volunteers
were exposed in vitro to different activating stimuli (ADP, collagen and
TRAP). Global analysis of mRNA expression patterns by RNA-Seq before
and after activation showed only few changes in relative mRNA abundance,
most of which consisting in RNA down-regulation. On the other hand,
quantitative proteomics (iTRAQ) revealed that almost half of >700
quantified proteins were modulated under the same conditions. To clarify
the reasons for the observed discrepancy between platelet transcriptome
and proteome responses to activation, we investigated by smallRNA-Seq
the possibility that the changes in proteome reflect changes of platelet
miRNA expression. This analysis revealed that activating stimuli indeed
induce changes in mature miRNAs expression that, in turn, promote the
proteome response observed and thereby can affect multiple pathways
control key normal and pathological platelet functions.
Work supported by MIUR (FIRB RBFR12W5V5_003), Univ. Salerno (FARB 2013) and CNR
(InterOmics Flagship Project)
References
1. Kieffer, N. et al. Biosynthesis of major platelet proteins in human blood platelets. Eur J
Biochem.1987;164:189-195
2. Landry, P. et al. Existence of a microrna pathway in anucleate platelets. Nat Struct Mol
Biol. 2009;16:961-966
90
The LINE-1 retrotransposon-encoded reverse transcriptase regulates
the biogenesis of small regulatory RNAs in human transformed cells
Ilaria SCIAMANNA , Alberto Gualtieri , Cristina Cossetti ,
Patrizia Vitullo , Corrado Spadafora1
1
1,2
1,2
1,2
1
2
Istituto Superiore di Sanità S.B.G.S.A., Rome, Italy; Università “Tor Vergata”,
Department of Experimental Medicine and Surgery, Rome, Italy
Full-length LINE-1 elements encode the reverse transcriptase (RT) enzyme
required for their own RNA-dependent retrotransposition as well as that of
non-autonomous retrotrasposons (e.g., Alu elements). RT is abundantly
expressed in transformed cells and in progressive stages during
tumorigenesis, but not in normal cells. We previously showed that
decreasing RT activity in cancer cells, by either RNA interference to active
LINE-1-elements, or by RT inhibitory drugs, reduces cell proliferation,
promotes differentiation, and also inhibits tumor growth in animal models.
We have now investigated how RT exerts these global regulatory functions.
Here we show that treatment of human melanoma cells with the RT
inhibitor efavirenz (EFV) causes a global reprogramming of the expression
profile of protein-coding genes, microRNAs (miRNAs) and ultra conserved
regions (UCRs). Subpopulations of RT-sensitive miRNAs and UCRs are
significantly enriched in Alus. Furthermore, we have identified Alu- and
LINE-1-containing RNA:DNA hybrid molecules as constitutive components
of tumor cells, which are absent in normal cells and are down-regulated
upon EFV-mediated RT inhibition. We propose a mechanism whereby the
LINE-1-encoded RT governs the balance between single-stranded and
double-stranded RNA production. In cancer cells, the abundant RT reversetranscribes retroelement-derived mRNAs, resulting in RNA:DNA hybrid
formation. We propose that this impairs the formation of double-stranded
RNAs and the ensuing production of small regulatory RNAs, with a direct
impact on global gene expression. RT inhibition restores the ‘normal’ small
RNA profile and the regulatory networks that depend on them. Thus, the
retrotransposon-encoded RT drives a previously unrecognized mechanism
crucial to the transformed state of tumor cells.
91
A functional study of long non-coding RNA (lncRNA) antisense to
KLHL14 protein-encoding gene during
zebrafish embryonic development
Rosa Maria SEPE1, Swaraj Basu1, Remo Sanges1, Elena Amendola2,
Elena De Felice1, Roberto Di Lauro2, Paolo Sordino1,3, Gabriella DE VITA2
1
Laboratory of Cellular and Developmental Biology, Stazione Zoologica Anton Dohrn,
2
Naples, Italy; Department of Molecular Medicine and Medical Biotechnology University of
3
Naples "Federico II"; Institute for Mediterranean and Forestal System, Catania, Italy
KLHL14 belongs to the Kelch-like (KLHL) gene family, encoding a group of
proteins that generally possess:
• a BTB/POZ domain, that facilitate protein binding and dimerization;
• a BACK domain, that is of functional importance since mutations in this
domain are associated with disease;
• five to six Kelch motifs, that form a tertiary structure of β-propellers with a
role in extracellular functions, morphology, and binding to other proteins1.
The KLHL family is conserved throughout evolution and KLHL genes are
responsible for several Mendelian diseases and have been associated with
cancer. In particular, the Comparative Toxicogenomics Database highlights
that KLHL14 is associated with nervous system, skin, cardiovascular and
endocrine system diseases.
In our bioinformatics analysis of the KLHL14 gene in zebrafish, mouse and
human, we have identified a syntenic conservation that includes a long noncoding antisense gene (KLHL14-AS) that contains a sequence highly
conserved throughout vertebrates. Interestingly, the conserved region of the
antisense gene in all the analyzed species contains binding sites for the
transcription factor Pax2, and for two microRNAs, miR-182 and miR-20a. In
mammals, Pax2 and both miR-182 and miR-20a are expressed in the thyroid
gland. Moreover, both miRs are able to target the thyroid-enriched gene Dirc2
in rat2.
Using zebrafish as model system, we aim to study the role of KLHL14-AS
during vertebrate development, with a focus on the formation of the thyroid
gland. In particular, we will first describe the KLHL14-AS spatial and temporal
pattern of expression, and then we will analyze its functions by studying the
effects of transcriptional alteration achieved through morpholino-mediated
knock-down technology. In summary, our study aims to provide new insights
on the role of the long non coding antisense KLHL14-AS, and will likely provide
valuable informations into basic developmental biology and human diseases.
References
1. Dhanoa et al., “Update on the Kelch-like (KLHL) gene family”, Human Genomics, 2013
2. Vejnar et al., “miRmap web: Comprehensive microRNA target prediction online”, Nucleic
Acids Research, 2013
92
Analysis of RNP structure of long non-coding RNAs
with a relevant function in muscle differentiation
Sama SHAMLOO, Julie Martone, Monica Ballarino, Irene BOZZONI
Department of Biology and Biotechnology “Charles Darwin”,
Sapienza Università di Roma, Rome, Italy
In vitro muscle differentiation is a powerful system for studying complex
regulatory circuitries involved in the control of cell differentiation.
Moreover,the network of transcription factors involved in different stages of
muscle differentiation is well known and evolutionary conserved. Recent
studies have shown that ncRNAs are also part of these regulatory
networks. miRNAs are the most extensively studied and characterized,
however, in the last years lncRNAs were also shown to be critical
regulators of muscle differentiation1.
In our group systematic identification of lncRNAs during differentiation of
murine myoblasts has been performed by using RNA-seq and
computational analysis; a group of not annotated lncRNAs differentially
expressed during muscle differentiation were identified.
For understating the role of a lncRNA, it is critical to investigate its
associated partners; with this aim, we started CLIP analysis for specific
RNA-binding proteins that have been well-defined as ncRNA partners
and/or have important role in myogenesis. Ago2, HuR, TNRC6, MSI2 and
Mll12 have been initially tested Several cytoplasmic lncRNA species were
identified that were able to specifically bind Ago2 and TNRC6. Since
miRNA sponging activity has been described as one possible function for
cytoplasmic lncRNAs, we are now testing whether these species indeed act
as sponges for specific miRNAs whose MRE have been detected on their
sequence. Specific experiments have been designed in order to verify this
hypothesis.
Further characterization of the RNP nature of the identified lncRNAs will
require RNA pull-down followed by Mass spectrometry.
References
1. Cesana M, et al. A long noncoding RNA controls muscle differentiation by functioning as
a competing endogenous RNA. Cell. 2011 Oct 14;147(2):358-69.
2. Legnini I, et al. A feedforward regulatory loop between HuR and the long noncoding RNA
linc-MD1 controls early phases of myogenesis. Mol Cell. 2014 Feb 6;53(3):506-14.
93
Natural inhibitors of aminoacyl-tRNA synthetases
as antimicrobial agents
Mirosława SKUPIŃSKA 1,2, Małgorzata Giel-Pietraszuk1, Piotr Stępniak2,
Leszek Rychlewski2, Mirosława Barciszewska1, Jan Barciszewski1
1
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego Poznań,
2
Poland; BioInfoBank Institute, Poznań, Poland
The aminoacyl-tRNA synthetases (aaRSs) are essential proteins found
in all living organisms. They catalyze the attachment of the correct amino
acid to its cognate transfer RNA (tRNA), and thus play a fundamental role
in translating the genetic code thus linking the RNA world with protein
world. The first stage of acylation reaction involves the formation of an
enzyme bound aminoacyl-adenylate, which is then resolved into the 2’ or
3’-hydoxyl of the terminal ribose of the tRNA. Inhibition of one of these two
enzymatic steps disrupts tRNA charging, which, in turn, stalls elongation of
growing polypeptide chains. aaRSs as the essential enzymes for protein
synthesis represent the promising targets for development of cures against
pathogenic species.
Several additional features highlight the suitability of aaRSs for drug
discovery: a) the divergence between prokaryotic and eukaryotic aaRSs, b)
phylogenetical conservation, and potential of the antimicrobials that target a
particular bacterial aaRS to inhibit homologous enzymes from a range of
other bacteria and c) existence of twenty distinct aaRSs in most bacterial
species representing an independent target for drug discovery.
Flavonoids are a group of natural products with many biological and
pharmacological activities; antibacterial, antiviral, antioxidant, and
antimutagenic effects and inhibition of several enzymes have been
demonstrated.
We show the results of analysis of the effects of different flavonoids on
activity of TyrRS from E.coli and S.aureus. The choice of bacteria against
which the inhibitors were selected was dictated by their role in the
epidemiology of infections in humans. Methicillin resistant S.aureus is the
reason for severe infections resistant to previously used antibiotics.
Inhibitory activity of compounds was analyzed in in vitro aminoacylation
reaction. The most potent inhibitors are acacetin, kaempferide, biochanin A
(IC50 - 5.12, 25,14 and 25,8 µM, respectively). Structure – function
relationship analysis shows that for Tyr-RS inhibition hydroxyl group at
position 5 and 7 as well as methoxy group 4’ are necessary. Flavones
showed slightly higher inhibitory activity than flavonols. Glycosylation of
aglycone weakens the interaction with the enzyme.
94
Endogenous RNAs modulate microRNA sorting to exosomes
and transfer to acceptor cells
Mario Leonardo SQUADRITO1, Caroline Baer1, Frédéric Burdet2,
Claudio Maderna1, Gregor D. Gilfillan3, Robert Lyle3, Mark Ibberson2,
Michele De Palma1
1
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École
2
Polytechnique Fédérale de Lausanne (EPFL), Switzerland; Vital-IT, Swiss Institute of
3
Bioinformatics (SIB), Lausanne, Switzerland; Department of Medical Genetics and
Norwegian High-Throughput Sequencing Centre (NSC), Oslo University Hospital, Norway
Macrophages modulate endothelial cell (EC) behavior by secreting a
variety of soluble mediators. Recent studies suggest that exosomes
mediate cell-to-cell communication via microRNA (miR) transfer. However,
both the mechanisms whereby miRs are loaded into exosomes and the
functional consequences of miR transfer to ECs are little understood. Here,
we investigated the significance of macrophage-derived exosomes (“exomacs”) and their miR cargo (“exo-miRs”) for macrophage-EC
communication. We performed TaqMan-based miR arrays of macrophages
polarized with distinct cytokines and their secreted exo-macs, as well as
deep-sequencing of the cells’ transcriptomes. We also employed
lentivectors to either overexpress or squelch individual miRs, or distinct
types of miR target sequences, in the macrophages. By these strategies,
we discovered that miR sorting to exo-macs depends on the nature and
abundance of miR targets, which are modulated by cell activation. By
biologically or artificially increasing target gene-transcripts, selected miRs
are enriched in the cell cytoplasm/P-bodies (sites of miR activity) and are
decreased in multivesicular-bodies (sites of exosome biogenesis), thus
limiting their sorting to exo-macs. These findings suggest a model for exomiR biogenesis whereby cell-activation dependent changes in target
transcript abundance finely regulate miR output to exosomes. We also
asked whether functional exo-miRs are transferred from macrophages to
ECs. To this aim, we generated exo-macs from either Dicer-deficient or
proficient macrophages, and a cell line of Dicer-knockout ECs. We then
employed a lentivector-based reporter system to measured the miR activity
of ~30 distinct miRs in Dicer-knockout ECs after exo-mac treatment or
coculture with macrophages. We found that several exo-miRs were
functionally transferred from macrophages to ECs in-vitro. Among these,
miR-142-3p and miR-188-5p were also transferred from macrophages to
Dicer-knockout ECs in vivo to modulate the expression of target
sequences. These results suggest that macrophages may modulate tumor
angiogensis by transferring functional miRs to ECs.
95
Analysis of circulating and tissue specific
microRNAs in Pompe Disease
A. TARALLO1,2, M. Karali1, A. Carissimo1, M. Mutarelli1, F. Gatto1,
O. Musumeci3, A. Toscano3, S. Fecarotta2, E. Acampora2,
S. Banfi1, G. Parenti1,2
1
2
Telethon Institute of Genetics and Medicine, Naples, Italy; Department of Translational
3
Medical Sciences, Federico II University, Naples, Italy; Department of Neurosciences,
University of Messina
Pompe Disease (PD) is a metabolic myopathy caused by the deficiency of acidalpha-glucosidase (GAA) that results in generalized tissue glycogen accumulation
and secondary cardiac and skeletal muscle destruction.
At present enzyme replacement therapy (ERT) with recombinant human GAA
(rhGAA), is the only available therapeutic approach to PD.
Despite progress in treatment, PD remains associated with unmet medical needs:
variability in clinical outcome in response to ERT; lack of markers of disease
progression and markers of ERT efficacy; need for new therapeutic targets.
Aim: Identification of differentially expressed miRNAs (DE-miRNAs) as new
biomarkers for PD and as tool to follow disease progression and therapeutic
efficacy.
Results: We have performed pilot experiments of miRNA profiling in plasma and
tissues from PD mice with the aim to validate our procedure for the identification of
“disease-specific” miRNAs. Next-generation sequencing screening in the PD
mouse model was performed at 3 and 9 months (two stages of disease
progression). Preliminary results showed one DE-miRNA in plasma at 9 months
with statistical significance (FDR< 0.05). We also found 219 DE-miRNAs in muscle
(gastrocnemius), and 35 DE-miRNAs in heart. In total, 104 miRNAs were
differentially expressed at 3 months, 109 at 9 months, 42 were differentially
expressed at both ages.
Some DE-miRNAs were selected for further evaluation; some of them are already
known to modulate the expression of genes involved in pathways such as
autophagy, muscle regeneration, inflammation that may be relevant for PD
pathophysiology.
Conclusions: Our results indicate that circulating miRNA and specific tissues DEmiRNAs can provide new/valuable biomarkers to monitor disease progression and
the effects of therapeutic intervention in PD. Additional studies in patients will be
suggested an important role of these DE-miRNAs for PD pathophysiology.
References
1. van der PLoeg AT, Reuser AJ. Pompe’s disease. Lancet. 2008, Oct11;372(9646):134253.
2. Ling H, Fabbri M, Calin GA: MicroRNAs and other non-coding RNAs as targets for
anticancer drug development. Nature reviews Drug discovery 2013, 12(11):847-865
3. Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, Qin YW, Jing Q: Circulating microRNA: a
novel potential biomarker for early diagnosis of acute myocardial infarction in humans.
European heart journal 2010, 31(6):659-666.
96
Unraveling the role of Alu RNA in cancer
Valeria TARALLO
Institute of Genetics and Biophysics “Adriano Buzzati Traverso”, CNR, Naples, Italy
The Alu repeat elements are the most abundant short interspersed
repeated elements (SINEs) in human genome1. Recently, it has been
showed that the accumulation of 300nt long Alu RNA transcripts is
responsible of a human disease, the dry form of age-related macular
degeneration (AMD), the Geographic disease (GA). Alu RNA is
enzymatically degraded by DICER1, and their accumulation in the Retina
Pigmented Epithelium (RPE) cells of human eyes, followed by DICER1
deficit, is responsible for GA2,3. To date, this is the first example of a role of
Alu RNAs in a human disease via direct RNA cytotoxicity. Interestingly,
increased levels of Alu RNAs and reduced DICER1 expression have been
observed in several cancers4-8, although no correlation has yet been
established. Overall, these data suggest that Alu RNA could have a
potential role in cancer disease.
In support of this hypothesis, I observed that Alu RNA is accumulated while
DICER1 transcript is reduced in two different human cancer cell lines,
HCT116 and H460, derived from colon and lung carcinoma, respectively. In
this frame, the main objective of the present proposal is to determine the
potential role of Alu RNAs accumulation in tumor formation and
progression.
Overall, these findings will provide new insights into the importance of Alu
RNA sequences and the non-canonical DICER1 signaling in cancer
development and progression, and potentially yield additional, new
therapeutic targets.
References
1. Batzer, MA & Deininger, PL. Nature reviews. Genetics 3, 370-379, (2002).
2. Kaneko, H et al. Nature 471, 325-330, (2011).
3. Tarallo, V et al. Cell 149, 847-859, (2012).
4. Merritt, WM et al. The New England journal of medicine 359, 2641-2650, (2008).
5. Karube, Y et al. Cancer science 96, 111-115, (2005).
6. Grelier, G et al. British journal of cancer 101, 673-683, (2009).
7. Tang, RB et al. Molecular carcinogenesis 42, 93-96, (2005).
8. Lin, RJ et al. Cancer research 70, 7841-7850, (2010).
97
Functional screening identified microRNAs
inducing cardiac regeneration through the recapitulation
of cardiac developmental programs
Consuelo TORRINI, Lorena Zentilin, Miguel Mano, Maria Ines Gutierrez,
Serena Zacchigna, Mauro GIACCA
Molecular Medicine Laboratory - International Centre for Genetic Engineering and
Biotechnology (ICGEB), Trieste, Italy
In mammals, cardiomyocytes rapidly divide during embryonic heart
development. Proliferation, however, rapidly stops after birth. As a
consequence, the adult myocardium is substantially unable to regenerate
itself following injury and cardiac repair occurs through a scarring
mechanism.
To identify the miRNAs that regulate cardiomyocyte proliferation, we
performed a high-content, fluorescence microscopy-based high-throughput
screening in rat neonatal cardiomyocytes using a library of microRNA
mimics corresponding to all the annotated human microRNAs. We
identified 40 miRNAs able to increase cardiomyocyte proliferation, as
evaluated by analyzing EdU incorporation (DNA synthesis), G2/M phase of
the cell cycle (phospho-H3 positivity) and karyokinesis (Aurora B staining in
midbodies).
Two of the most effective microRNAs, hsa-miR-590-3p and hsa-miR-199a3p, were tested in vivo by both injecting the synthetic microRNAs
intracardiacally in newborn rats and delivering their genes using AAV9
vectors into the infarct border zone in adult mice. In the former setting,
these microRNAs induced marked proliferation of cardiomyocytes while, in
the latter, also reduced the infarct area and improved cardiac function.
Genetic fate mapping experiments now indicate that miR-590-3p and miR199a-3p directly act by promoting the proliferation of differentiated, adult
cardiomyocytes. Analysis of the transcriptome after miR-590-3p or miR199a-3p treatment reveals the specific downregulation of genes involved in
the assembly of the cytoskeleton. In particular, the specific siRNA
knockdown of over 600 genes downregulated by the two microRNAs shows
that 45 siRNAs are capable to induce in vitro cardiomyocyte proliferation at
least twofold.
The actual targets of both miR-199a-3p and miR-590-3p include known
regulators of Hippo pathway; consistently, cardiomyocyte treatment with
pro-proliferative miRNAs determines the activation of the YAP1/TAZ
transcriptional coactivators, the final effectors of the Hippo pathway.
This information can pave the way to the possible clinical translation of
these miRNAs as deliverable drugs for the treatment of myocardial
infarction and heart failure.
98
Deregulation of microRNAs in human temporal lobe epilepsy:
molecular mechanisms
Vamshidhar R. VANGOOR, Ketharini S. Kumar, Pierre N. E. de Graan, R.
Jeroen Pasterkamp
Department of Translational Neuroscience, Rudolf Magnus Institute of Neuroscience, UMC
Utrecht, The Netherlands
Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disorder
characterized by recurrent seizures. The pathogenic mechanisms
underlying mTLE may involve defects in the post-transcriptional regulation
of gene expression. MicroRNAs (miRNAs) are small (18-25nt long) noncoding RNAs that control the expression of genes at the post-transcriptional
level. In a recent study (Kan et al., 2012), we have shown that a significant
number of miRNAs are up- or down-regulated in hippocampal tissue of
human mTLE patients. Several of these miRNAs (miR-92b, -20a, -374a, 135a) target a key transcription factor known as Myocyte enhancer factor
(MEF)2. By using bioinformatics tools we have predicted the presence of
highly conserved sites complementary to the seed regions in the 3′
untranslated region (UTR) of the MEF2 mRNA. MEF2 proteins are a family
of transcription factors which mediate activity-dependent synaptic
development, and are activated by neurotrophin stimulation and calcium
influx resulting from increased neurotransmitter release at synapses
(Flavell et al., 2008). Using luciferase reporter assays, we have verified
binding of these miRNAs to MEF2. Furthermore, the expression of MEF2
was downregulated in an animal model of mTLE and also in human mTLE
patients with hippocampal sclerosis where the expression of miRNAs is
upregulated. MEF2 negatively regulates synaptic density (Flavell et al.,
2006), and loss of MEF2 in mTLE could lead to abnormal spine formation
and contribute to aberrant firing pattern and cell death observed in epilepsy.
By using miRNA mimics and expression vectors encoding miRNAs we are
determining if these miRNAs (via MEF2) have an impact on neuronal
morphology and the pathogenesis of mTLE.
References
1. Flavell, S.W. et al., 2006. Activity-dependent regulation of MEF2 transcription factors
suppresses excitatory synapse number. Science 311, 1008–1012.
2. Flavell, S.W. et al., 2008. Genome-wide analysis of MEF2 transcriptional program reveals
synaptic target genes and neuronal activity-dependent polyadenylation site selection.
Neuron 60, 1022–1038.
3. Kan, A.A., et al., 2012. Genome-wide microRNA profiling of human temporal lobe
epilepsy identifies modulators of the immune response. Cell. Mol. Life Sci. 69, 3127–
3145.
99
Transcribed ultraconserved region 339 stimulates carcinogenesis by
trapping tumor suppressor microRNAs
Ivan VANNINI1, Kishore B. Challagundla2, Giorgia Paliaga1, Meropi
Plousiou1, Francesca FANINI1, Melissa Crawford3, Manuela Ferracin,4
Ramana V. Davuluri,5 Zhihi Guo,6 Maria Angelica Cortez,6 Cristina Ivan,6
Leng Han,7 Petra M. Wise,2 Silvia Carloni,1 Hui Ling,6 Mariam Murtadha,2
Barbara J. Gitlitz,8 Ite A. Laird-Offringa,8 Patrick Nana-Sinkam,3 Massimo
Negrini,4 Han Liang,7 Dino Amadori,1 Amelia CIMMINO,9
George A. Calin,6, 10, Muller FABBRI2
1
Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRCCS, Meldola (FC),
2
Italy; Depts. of Pediatrics and Molecular Microbiology & Immunology, Norris
Comprehensive Cancer Center, Keck School of Medicine, Univ. of Southern California,
Children’s Center for Cancer and Blood Diseases and The Saban Research Institute,
3
Children’s Hospital, Los Angeles, USA; Dept. of Internal Medicine, Division of Pulmonary,
Allergy, Critical Care and Sleep Medicine, The Ohio State University, Columbus, OH, USA;
4
Dept. of Morphology, Surgery and Experimental Medicine and Laboratory for Technologies
5
of Advanced Therapies (LTTA), Univ. of Ferrara, Italy; Depts. of Preventive Medicine and
Neurological Surgery, Northwestern University-Feinberg School of Medicine, Chicago, IL,
6
USA; Dept. of Experimental Therapeutics and The Center for RNA Interference and NonCoding RNAs, The University of Texas MD Anderson Cancer Center, Houston, TX, USA;
7
Dept. of Bioinformatics and Computational Biology, The University of Texas MD Anderson
8
Cancer Center, Houston, TX, USA; Division of Medical Oncology, Norris Comprehensive
Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles,
9
CA, USA; Institute of Genetics and Biophysics, CNR and Fondazione IRCCS SDN, Naples,
10
Italy; The Center for RNA Interference and Non-coding RNAs, The University of Texas MD
Anderson Cancer Center, Houston, TX, USA
The discovery of new human carcinogenic mechanisms allows to improve the
personalized medicine identifying cancer molecular targets that can reduce side
effects and drug resistance. The transcribed ultraconserved regions (T-UCRs) are
a class of long non-coding RNAs that are completely conserved (100%) between
orthologous regions of the human, rat, and mouse genomes. T-UCRs are dysregulated in human cancers and the mechanism of action of T-UCRs and the
molecules regulating their expression in human tumors are unknown. In this project
we observed that high expression of uc.339 is associated with lower survival in 204
non-small cell lung cancer (NSCLC) patients. Furthermore we discovered that
uc.339 up-regulated in archival NSCLC samples, sequestered miR-339-3p, -6633p and -95-5p acting as a decoy RNA for these miRNAs. miR-339-3p, -663-3p and
-95-5p have a common target CCNE2, that is up-regulated after uc.339 overexpression, stimulating cancer growth and migration. Moreover, we observed that
uc.339 is directly down-regulated by the tumor suppressor TP53, which is able to
contrast a uc.339-mediated oncogenic effect.
In summary, this study shows that uc.339 has an oncogenic function in NSCLC, by
sequestering miR-339-3p, -663-3p and -95-5p and blocking their targeting of
CCNE2, inducing a increased tumor growth and migration. Our study shows that
uc.339 is a key TP53-modulated regulator involved in lung cancer and a promising
anti-cancer target.
100
BRAFV600E-regulated microRNAs in melanoma
Marianna VITIELLO1,3, Andrea Tuccoli1,2, Romina D’Aurizio4,
Andrea Marranci3, Elisa Mercoledì2, Laura Poliseno1,2,4
1
2
Oncogenomics Unit of Istituto Toscano Tumori (ITT), Pisa, Italy; Istituto di Fisiologia
3
Clinica, Consiglio Nazionale delle Ricerche (CNR), Pisa, Italy; Doctorate School in
4
Biochemistry and Molecular Biology, University of Siena, Italy; Laboratory for Integrative
System Medicine, IIT-IFC, CNR, Pisa, Italy
Malignant melanoma is the most aggressive form of skin cancer and its
incidence increases every year. In more than 90% of melanoma cases, the
mutations driving the genesis and progression of the tumor are in the MAPK
pathway, which becomes constitutively active and leads to uncontrolled cell
proliferation/survival. The most common mutation is a nucleotide substitution at
the V600 residue of BRAF kinase (V600E). This event renders BRAF able to
act as a monomer and independently of RAS activation. Few years ago, a new
class of drugs that selectively bind and inhibit BRAF in its mutant forms have
been developed (BRAF inhibitors, BRAFi) and among them there is
vemurafenib (vem). BRAFi outperform conventional chemotherapeutic agents
in terms of both response rate and progression free survival/overall survival.
However, patients treated with BRAFi develop acquired resistance within 6
months of treatment on average and in about 75% of the cases this is due to
the reactivation of the MAPK pathway by various mechanisms.
The ultimate aim of this project is to increase the basic knowledge on BRAF
functioning and hence to identify new molecular entities that might improve the
outcome of melanoma patients when targeted in combination with
BRAFV600E. In particular, we have focused our efforts on the identification of
BRAFV600E-regulated microRNAs. We have performed deep sequencing
analysis upon vem treatment of one parental sensitive line and one vemresistant clone. In this way, we have identified 7 microRNAs up-regulated and
7 down-regulated upon BRAFV600E inhibition in the sensitive line, but not in
the resistant clone. The dependency of such microRNA family upon the MAPK
pathway has been confirmed by using additional chemical and genetic
inhibitors of the pathway itself. Furthermore, we have found that the overexpression of all the family members is able to cooperate with vem in
increasing melanin production. These results confirm the potentially
oncosuppressive role exerted by these microRNAs. However, we also
observed that the inhibition of 1 member of the family results in a dramatic
increase in apoptotic cell death, an effect that might be related with its marked
lineage specificity.
Our current research activities are mainly taking 3 directions: 1. The
identification of the targets that the family members share, as well as those that
are specific for each of them. 2. The study of their cooperation with vem. 3.
The assessment of their clinical relevance.
101
Plasma microRNA as a noninvasive biomarker for diagnostics and
post-treatment follow-up of lung cancer patients
Ivan A. ZAPOROZHCHENKO1, Evgeniy S. Morozkin1, Anastasiya A.
Ponomaryova2, Tatyana E. Skvortsova1, Nadezhda V. Cherdyntseva2,
Pavel P. Laktionov1
1
Laboratory of Molecular Medicine, SB RAS Institute of Chemical Biology and Fundamental
2
Medicine, Novosibirsk, Russia; Immunology Laboratory, RAMS Tomsk Cancer Research
Institute, Tomsk, Russia
Early tumour detection and prediction of response to therapy literally means
life or death in case of lung cancer. Circulating miRNAs have been shown
to reflect the progression of disease and thus offer a potential solution to
the cancer diagnostics and theranostics problem. To date a handful of
potential candidate miRNAs for testing has been identified.
The intent of this study was to investigate changes in expression of five
miRNA signature in plasma of lung cancer patients in response to
chemotherapy and surgical treatment. The miRNAs comprising the panel
were drawn from previously published data as potential indicators of
treatment efficiency and disease progression.
Blood plasma samples were obtained from Tomsk Cancer Research
Center (Tomsk, Russia) with informed consent provided by all participants.
In total 39 blood samples taken at the tumour detection, after receiving
nonadjuvant chemotherapy and after undergoing surgery were included in
the study.
MiRNA was isolated from blood plasma using a newly developed
phenolfree protocol. Expression of five miRNAs (miR-19b, miR-126, miR25, miR- 205, miR-125b) were measured by qRT-PCR. Ct values were
normalized to miR-16 concentration.
An original phenol-free protocol for miRNA isolation from plasma has been
proposed. In pilot experiments new protocol matched the performance of
the established phenol-based protocols. Analysis of miRNA expression in
samples treated by this protocol showed that two of the selected miRNAs:
miR-19b and miR-125b exhibit noticeable trends in response to treatment.
Thus, for example, miR-19b concentration would decline (2-5 fold) after
chemotherapy, while miR-125b expression would reversely increase (2-3
fold). However, rANOVA analysis of the obtained data revealed that only
miR-19b expression differs significantly (P=0,034) across the timepoints.
Essentially a more profound analysis is required to elicit significant
predictors of treatment efficiency. Our next step would be to perform a wide
profiling of circulating miRNA to identify a panel of miRNAs that can be
used in clinical practice.
102
Cellular activity of microRNAs dysregulated in breast cancer
Carlotta ZERBINATI, Fabio Corrà, Linda MINOTTI, Marco Galasso,
Maurizio Previati, Stefano VOLINIA
Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Italy
Introduction. Breast cancer (BC) is one of the major health problems
worldwide and it is the second cause of cancer-related death in women.
Patients often develop resistance to the current therapies. For this reason,
the identification of new specific clinical molecular markers and
pharmacologic targets in cancer research is an ongoing challenge. By
regulating the expression of target genes, microRNAs can have a tumor
suppressor or oncogenic role.
Materials and methods. We studied several human BC-derived cell lines:
MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-361, SKBR3, T47D,
BT474, ZR75.1, MDA-MB-453, HBL-100, and the breast non-tumor cell
lines: 184A1, MCF10A.
All the cell lines were transfected with either one of 38 miRNAs (miR-21,
26b, 28-5p, 33b, 99a, 126, 126*, 130b, 138, 142-5p, 143, 181a, 202, 203,
206, 210, 218, 222, 145, 301a, 302a, 320c, 326, 484, let-7d*, 93, 103,
1307, 148, 328, 874, 151, 10a, 25, 30a, 615, 27a, 9), identified as important
in BC (Volinia et al. Genome Res. 2010; Volinia S. and Croce CM. PNAS
2013).
Cell proliferation was determined by means of the xCELLigence RTCA
System and the analysis of cell viability was carried out by performing MTS
and PMS. The miRNAs were further investigated for their capacity to affect
cell migration, cell invasion, and RNA profiles.
Results. The main outcome of our studies has been the identification from
such wide list of a few miRNAs involved in the regulation of cell growth and
invasion. In a first not exhaustive screening, we have identified some
genes, which negatively correlated with those key miRNAs.
Conclusions. In this work, we investigated the possible causal role of
microRNAs associated to breast cancer. We can conclude that we could
dissect and prioritize in vitro the functional role of miRNAs in breast cancer.
103
The following participants have received a grant-in-aid from:
FEBS – Federation of Biochemical Societies
Diana Alexieva, London, UK
Ross Conlon, Dublin, Ireland
Maria Inomistova, Kyiv, Ukraine
Olga Patutina, Novosibirsk, Russia
Edith Schneider, Ulm, Germany
Mario Leonardo Squadrito, Lausanne, Switzerland
Ivan Zaporozhchenko, Novosibirsk, Russia
and
EPIGEN – Progetto Bandiera Epigenomica
Laura Crisafulli, Milan, Italy
Alessandro Fiorenzano, Naples, Italy
Marija Mihailovic, Milan, Italy
Valeria Ranzani, Milan, Italy
Annamaria Salvati, Salerno, Italy
Carlotta Zerbinati, Ferrara, Italy
Printed in Naples, Italy
October 2014
by Tipografia A. TETI S.r.l.
105
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