Page 115 - ebook HCC

EASL
PROGRAMME AND ABSTRACTSAND ABSTRACTS
GENEVA, SWITZERLANDA, SWITZERLAND
112 PROGRAMME GENEV EASL HCC SUMMITHCC SUMMIT 113
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FEBRUARY 13 - 16, 2014Y 13 - 16, 2014
FEBRUAR
Poster Board Number B13 Poster Board Number B14
CIRCULATING MICRORNAS PROFILES IDENTIFY CASPASE-3 TARGETING: A FURTHER TILE
CIRRHOTIC PATIENTS WITH HCC SUSTAINING THE ANTI-APOPTOTIC ROLE OF
MIR-221 IN HEPATOCELLULAR CARCINOMA

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Francesca Fornari , Manuela Ferracin , Maddalena Milazzo , Davide Trerè ,
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Sara Marinelli , Laura Venerandi , Alberto Borghi , Clarissa Patrizi , Francesco G. Foschi , Francesca Fornari , Maddalena Milazzo , Clarissa Patrizi , Elisa Callegari ,
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Massimo Negrini , Giuseppe F. Stefanini , Luigi Bolondi , Laura Gramantieri 1 Silvia Sabbioni , Marzia Galassi , Massimo Negrini , Laura Gramantieri , Luigi Bolondi 1
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1 Dipartimento di Medicina Clinica e Centro di Ricerca Biomedica Applicata, Policlinico 1 Dipartimento di Medicina Clinica e Centro di Ricerca Biomedica Applicata, Policlinico
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Universitario S.Orsola-Malpighi, Bologna, Dipartimento di Medicina Sperimentale e Universitario S.Orsola-Malpighi, Bologna, Dipartimento di Medicina Sperimentale e
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Diagnostica e Centro Interdipartimentale per la Ricerca sul Cancro, Università di Ferrara, Diagnostica e Centro Interdipartimentale per la Ricerca sul Cancro,
Ferrara, Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Policlinico Università di Ferrara, Ferrara, Italy
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Universitario S.Orsola-Malpighi, Bologna, Dipartimento di Medicina Interna,
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Ospedale per gli Infermi, Faenza, Italy Corresponding author’s e-mail: fornarifrancesca@live.it
Corresponding author’s e-mail: fornarifrancesca@live.it Introduction: MicroRNA-221 (miR-221) is over-expressed in several cancer types
including hepatocellular carcinoma (HCC). It was previously demonstrated that miR-221
Introduction: The performance of available circulating biomarkers in the screening and reduced apoptotic cell death through the direct targeting of pro-apoptotic genes, such as
diagnostic settings of HCC is sub-optimal, therefore both EASL and AASLD guidelines BMF and PUMA. MiR-221 oncogenic role has been proved by our group in a liver-specific
identify the field of circulating non invasive biomarkers as a topic in which research efforts miR-221 transgenic model, suggesting antagomiR-221 as a possible therapeutic tool for
should be perfused. the treatment of HCC.
BASIC POSTER ABSTRACTS cirrhotic patients without liver lesions as well as in cirrhotic patients with small/unifocal and apoptotic cell death and response to treatments throught the direct regulation of caspase-3 BASIC POSTER ABSTRACTS
Aims: The aim of this study was to test the performance of circulating microRNAs in
Aims: This study aims to further characterize miR-221 involvement in the regulation of
advanced hepatocellular carcinoma (HCC).
expression.
Methodology: The study was designed as a two phase epidemiological study, with a
Methodology: An in vitro functional analysis was used to investigate miR-221 modulation
hypothesis generating step conducted by means of microarray assay, specifically aimed
of caspase-3 expression in HCC cell lines. Luciferase-reporter assay was performed to
at discovering a genome-wide aberrantly regulated circulating miRNA panel able to
differentiate cirrhotic patients (N. 11) from HCC (N. 12) patients. The validation phase was
mRNA. Chemiluminescent assays, FACS and Western blot analysis were performed to
performed by qRT-PCR specific for the selected miRNAs in an independent series of 81 evaluate the interaction between miR-221 and its complementary binding site in caspase-3
evaluate apoptotic cell death in miR-221 or anti-miR-221 transfected HCC cells following
consecutive patients with cirrhosis (N. 20), unifocal/small HCC (N. 28) and intermediate- Doxorubicin treatment. Real Time PCR and Western blot analysis were employed to
advanced HCC (N. 33). Logistic regression was used to assay circulating factors analyse miR-221 and caspase-3 expression in liver tissues of miR-221 transgenic model.
independently associated with HCC. ROC curves were constructed and the AUC was
calculated, to explore the performance of specific miRNAs as diagnostic tests for HCC. Results: Western blot analysis and luciferase reporter assay showed a direct regulation
of caspase-3 target by miR-221 in different HCC cell lines. Annexin-V/FACS analysis
Results: A signature of circulating miRNAs emerged as differentially expressed between displayed an increase of apoptotic cell death in miR-221 silenced Hep3B and SNU449
patients with LC and HCC in the training set. This miRNAs signature was confirmed in the cells following Doxorubicin challenge. In line with this data, an increase of caspase activity
validation set where it differentiated patients with cirrhosis from patients with HCC. Higher and pro-apoptotic genes expression was observed in the same settings. An ex vivo
serum levels of specific miRNAs turned out to be an independent factor for the presence analysis showed a significant decrease of caspase-3 protein levels in liver tissues of miR-
of early HCC or of HCC with macro-vascular invasion. The AUC of a panel of selected 221 transgenic mice with respect to wild type control animals.
circulating miRNAs was higher than that of AFP.
Conclusions: MiR-221 regulates apoptotic cell death of HCC cells through the simultaneous
Conclusions: Despite these results are very preliminary, this two stage study demonstrated inhibition of caspase-3 protein and other pro-apoptotic molecules. Our data provide an
that circulating microRNAs deserve much attention as potential non invasive biomarkers additional molecular rationale for miR-221 silencing as a novel therapeutic strategy, alone or
for HCC in the diagnostic setting. in combination with anti-cancer drugs, for the treatment of advanced HCCs.