Glypican-3 (GPC3) is an oncogene, frequently upregulated in liver malignancies such as hepatocellular carcinoma (HCC) and hepatoblastoma and constitutes a potential molecular target for therapy in liver cancer. Using a functional screening system, we identified 10 new microRNAs controlling GPC3 expression in malignant liver cells, five of them e.g. miR-4510, miR-203a-3p, miR-548aa, miR-376b-3p and miR-548v reduce GPC3 expression. These 5 microRNAs were significantly downregulated in tumoral compared to non-tumoral liver and inhibited tumor cell proliferation. Interestingly, miR-4510 inversely correlated with GPC3 mRNA and protein in HCC samples. This microRNA also induced apoptosis of hepatoma cells and blocked tumor growth in vivo in the chick chorioallantoic membrane model. We further show that the tumor suppressive effect of miR-4510 is mediated through direct targeting of GPC3 mRNA and inactivation of Wnt/β-catenin transcriptional activity and signaling pathway. Moreover, miR-4510 up-regulated the expression of several tumor suppressor genes while reducing the expression of other pro-oncogenes. In summary, we uncovered several new microRNAs targeting the oncogenic functions of GPC3. We provided strong molecular, cellular and in vivo evidences for the tumor suppressive activities of miR-4510 bringing to the fore the potential value of this microRNA in HCC therapy.

BackgroundTherapeutic outcomes using the multikinase inhibitors, sorafenib and regorafenib, remain unsatisfactory for patients with advanced hepatocellular carcinoma (HCC). Thus, new drug modalities are needed. We recently reported the remarkable capacity of miR‐4510 to impede the growth of HCC and hepatoblastoma through Glypican‐3 (GPC3) targeting and Wnt pathway inactivation.MethodsTo identify new targets of miR‐4510, we used a label‐free proteomic approach and reported down‐regulation of RAF proto‐oncogene serine/threonine‐protein kinase (RAF1) by miR‐4510. Because the tumourigenic role of RAF1 in HCC is controversial, we further studied RAF1:miR‐4510 interactions using cellular, molecular as well as functional approaches and a chicken chorioallantoic membrane (CAM) xenograft model.ResultsWe found an increase in RAF1 protein in 59.3% of HCC patients and a specific up‐regulation of its transcript in proliferative tumours. We showed that miR‐4510 inactivates the RAS/RAF/MEK/ERK pathway and reduces the expression of downstream targets (ie c‐Fos proto‐oncogene [FOS]) through RAF1 direct targeting. At a cellular level, miR‐4510 inhibited HCC cell proliferation and migration and induced senescence in part by lowering RAF1 messenger RNA (mRNA) and protein expression. Finally, we confirmed the pro‐tumoural function of RAF1 protein in HCC cells and its ability to sustain HCC tumour progression in vitro and in vivo.ConclusionsIn this work, we confirm that RAF1 acts as an oncogene in HCC and further demonstrate that miR‐4510 acts as a strong tumour suppressor in the liver by targeting many proto‐oncogenes, including GPC3 and RAF1, and subsequently controlling key biological and signalling pathways among which Wnt and RAS/RAF/MEK/ERK signals.