P-026 - Regulation of Glypican-3 by New MicroRNAs and Implication in Hepatocellular Carcinoma

Date 04 July 2015
Event WorldGI 2015
Session Posters
Topics Hepatobiliary Cancers
Translational Research
Presenter F. Cartier
Citation Annals of Oncology (2015) 26 (suppl_4): 1-100. 10.1093/annonc/mdv233
Authors F. Cartier1, S. Lesjean1, J. Zucman-Rossi2, K. Hooks1, F. Sagliocco1, C. Grosset1
  • 1INSERM U1053, Bordeaux/FR
  • 2INSERM U674, Paris/FR

Abstract

Introduction

Glypican-3 (GPC3) is a membrane-anchored protein overexpressed in two types of liver cancer: hepatocellular carcinoma (HCC) and hepatoblastoma. GPC3 controls key signalling pathways such as Wnt/beta-catenin pathway, which is involved in liver development and carcinogenesis. This oncogenic protein is also used as a diagnostic and prognostic marker and constitutes a therapeutic target in liver cancers. In order to inhibit the expression of GPC3 and prevent its oncogenic effect, our studies focused on the post-transcriptional regulation of GPC3 by microRNAs (miRNAs) in liver tissue. We previously identified five miRNAs as well as the endoribonuclease IRE1-alpha as GPC3 regulators (Maurel M. et al, Hepatology 2013 and Maurel M. et al, RNA 2013).

Methods

To identify new GPC3-regulating miRNAs in HCC cells, we used a functional screening system named Dual-Fluorescence FunREG (Maurel M. et al, Hepatology, 2013). HuH7 cells co-expressing the Tomato and the GFP transgenes with either 3'UTR and/or 5'UTR of GPC3 were transfected by each miRNA from a library (miRBase V17.0; 1712 miRNAs; Qiagen). The miRNAs inducing a significant variation of the GFP/Tomato ratio compared to the control RNA were selected as candidates. Subsequently we evaluated the role of these miRNAs on GPC3 expression and in liver carcinogenesis using classical molecular and cellular approaches in HCC cells.

Results

The screening allowed us to identify 16 new miRNAs controlling GPC3 through the 3'UTR, 5'UTR or both. The effect of these miRNAs on endogenous-GPC3 protein expression was studied in tumoural HuH7 cells by flow cytometry and Western blotting. Among 16 miRNAs, ten can regulate the endogenous expression of GPC3 protein. The relative and absolute expression of those ten miRNAs was evaluated by RT-qPCR and showed that five miRNAs are under-expressed in HCC patients. We tested the effect of these five miRNAs on the phenotype of HuH7 cells in vitro. They significantly inhibit cell proliferation and block the cell cycle at the G0/G1 stage in vitro. Three of the miRNAs are more efficient in inducing growth arrest than a siRNA against GPC3. Additionally, miR-A can induce cell apoptosis in several tumoural liver cell lines in vitro. MiRNA target prediction programs show that mir-A has a non-conventional predicted target site in 3'UTR of GPC3 mRNA. Moreover, in silico analyses suggest that miR-A could regulate several other oncogenic genes involved in hepatocellular carcinogenesis. We are currently testing additional oncogenic targets.

Conclusion

We identified ten new miRNAs controlling GPC3 protein expression in HCC cells using a functional screening. Among these miRNAs five are significantly decreased in HCC and could participate in GPC3 overexpression in liver carcinogenesis. These five miRNAs can block tumoural cell growth and miR-A can induce tumoural cell apoptosis in vitro. MiR-A, B, C, D and E might act as tumour suppressors in HCC and constitute new potential therapeutic agents. The anti-tumoural effect of our best candidate miR-A is currently being tested in vivo in Chorioallantoic chicken membrane model (CAM) and will be soon investigated in mice.