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.

Although hepatocellular carcinoma (HCC) is one of the most common malignancies and constitutes the third leading cause of cancer-related deaths, the underlying molecular mechanisms are not fully understood. In the present study, we demonstrate for the first time that hepatocytes express signalling lymphocytic activation molecule family member 3 (SLAMF3/CD229) but not other SLAMF members. We provide evidence to show that SLAMF3 is involved in the control of hepatocyte proliferation and in hepatocellular carcinogenesis. SLAMF3 expression is significantly lower in primary human HCC samples and HCC cell lines than in human healthy primary hepatocytes. In HCC cell lines, the restoration of high levels of SLAMF3 expression inhibited cell proliferation and migration and enhanced apoptosis. Furthermore, SLAMF3 expression was associated with inhibition of HCC xenograft progression in the nude mouse model. The restoration of SLAMF3 expression levels also decreased the phosphorylation of MAPK ERK1/2, JNK and mTOR. In samples from resected HCC patients, SLAMF3 expression levels were significantly lower in tumorous tissues than in peritumoral tissues. Our results identify SLAMF3 as a specific marker of normal hepatocytes and provide evidence for its potential role in the control of proliferation of HCC cells.

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. We recently characterized for the first time the expression of Signaling Lymphocyte Activating Molecule 3 (SLAMF3) in human hepatocytes and here, we report that SLAMF3 interacts with the HCV viral protein E2 and is implicated in HCV entry process. We found a strong correlation between SLAMF3 expression level and hepatocyte susceptibility to HCV infection. The use of specific siRNAs to down-modulate SLAMF3 expression and SLAMF3-blocking antibodies both decreased the hepatocytes susceptibility to HCV infection. Moreover, SLAMF3 over-expression significantly increased susceptibility to HCV infection. Interestingly, experiments with peptides derived from each SLAMF3 domain showed that the first N-terminal extracellular domain is essential for interaction with HCV particles. Finally, we showed that recombinant HCV envelop protein E2 can bind SLAMF3 and that anti-SLAMF3 antibodies inhibited specifically this interaction. Overall, our results revealed that SLAMF3 plays a role during HCV entry, likely by enhancing entry of viral particle within hepatocytes.

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We have previously reported that endothelin-1 (ET-1) stimulates the in vitro secretion of corticosterone and aldosterone from the adrenal gland of the frog Rana ridibunda. The aim of the present study was to investigate the pharmacological profile of the endothelin receptor subtype involved in the corticotropic effect of ET-1. The mixed ET(A)/ET(B) receptor antagonist Ro 47-0203 (10(-5) M) totally blocked the stimulatory effect of ET-1 (5 x 10(-9) M) on corticosterone and aldosterone secretion. The action of ET-1 was also inhibited by the selective ET(A) receptor antagonist BQ-485 (10(-7) M). In contrast, the selective ET(B) receptor antagonist IRL 1038 (10(-6) M) did not affect the response of the frog adrenal gland to ET-1. In addition, the selective ET(B) receptor agonist IRL 1620 (10(-6) M) did not mimic the stimulatory effect of ET-1. The high affinity ET(C) receptor agonist endothelin-3 (ET-3) stimulated corticosteroid secretion, but was 400 times less potent than ET-1. Moreover, the action of ET-3 was also blocked by BQ-485 (10(-7) M). These data indicate that the stimulatory effects of ET-1 and ET-3 on corticosteroid secretion by the frog adrenal gland are mediated by an ET(A) receptor subtype.

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The distribution of galanin-like immunoreactivity was studied in the adrenal gland of the frog Rana ridibunda using the indirect immunofluorescence technique. A dense network of varicose fibers immunoreactive to galanin was found in the adrenal tissue. A combination of HPLC analysis and RIA detection was used to characterize galanin-like immunoreactivity in frog adrenal gland extracts. The elution profile revealed the existence of a single form of galanin exhibiting the same retention time as synthetic frog galanin. The possible involvement of galanin in the regulation of corticosteroid secretion was investigated in vitro using a perifusion system for frog adrenal slices. For concentrations ranging from 10(-9) to 3 x 10(-6) M, synthetic frog galanin induced a dose-dependent inhibition of corticosterone and aldosterone release. Repeated pulses of galanin (10(-6) M), given at 90-min intervals, resulted in a reproducible inhibition of corticosteroid secretion without any apparent tachyphylaxis. During prolonged administration of galanin (10(-6) M), the steroidogenic effect of ACTH (10(-9) M) was significantly reduced. In contrast, galanin did not attenuate the stimulation of corticosteroid secretion induced by the angiotensin II analog [Sar1,Val5]angiotensin II. These results show the occurrence of galanin in fibers innervating the frog adrenal gland. The data also demonstrate that synthetic galanin inhibits spontaneous and ACTH-induced corticosteroid release. Taken together, these findings suggest that galanin, released by nerve fibers in the adrenal tissue, can act locally as a modulator of steroid secretion.