The Role Of MiR-34c In HBV Related HCC By Targeting TGIF₂

Background:Hepatocellular carcinoma (HCC), especial chronic infection with hepatitis B (HBV) related HCC, which affects hundreds of millions of people worldwide, is one of the ten top malignant tumors in the world, with high morbidity and mortality. Besides imagic examination such as B mode ultrasound and CT, the detection of alpha fetoprotein (AFP) is the most common tool for HCC early diagnosis, but is easy to be misdiagnosed. So far, there is still no statisfactory treatment method of HCC. Given this scenario, the new therapeutic strategies to combat HCC are needed.miRNAs have been identified to negatively regulate the expression of hundreds of genes involved in cell proliferation, differentiation and apoptosis. Ectopic expression of miRNAs also plays a role in the development of cancer. Several reports have shown that some miRNAs are down regulated in hepatocellular carcinoma (HCC) and the biological evidence for an involvement of miRNAs in liver tumorigenesis is increasing. Studies about miRNAs provided new ideas for elucidating the mechanism of HBV causing HCC and also new treatment targets for HCC. It has been reported that the miR-34family plays a very important role in cell proliferation, differentiation and apoptosis. The expression of miR-34c in different cancers was reported on a hot debate. As to its expression in HBV related HCC, there is no exact study up to now. In the primary work, we found that miR-34c was down regulated in the liver cancer of HBV transgenic mice compare with the control group. In this study, we mainly investigated in the function of miR-34c in the HBV related HCC.Objectives:Determine the direct target of miR-34c and detect the level of miR-34c and its target in the HBV related HCC. Then inquire into the phenotypic effects of miR-34c on the infection of HBV and the development and progress of HCC both in vitro and in vivo.Methods:Firstly, qRT-PCR on clinical tissues (human HBV-related HCC tissues and normal hepatic tissues) or hepatocellular carcinoma cell lines (HepG2.2.15and HepG2cells, BEL7402and HBV-BEL7402cells), was conducted to detect the miR-34c level. Secondly, the target gene TGIF2of miR-34c predicted by querying PicTar, TargetScan and miRanda was determined by carrying out RT-PCR on human liver cancer cell lines and luciferase assays on HepG2cells. Thirdly, after48h or72h transfecting miR-34c expression vector into HepG2.2.15cells, RT-PCR/qPCR and Western Blot were conducted to detect the mRNA and protein levels of TGIF2respectively, as well as CCK-8and FCM for assessing cell proliferation and cell apoptosis. HBV DNA qPCR and ELIS A were conducted to detect the copy of HBV DNA and the secreation of HBsAg and HBeAg separately. Finally, we established the human hepatoma cell line HepG2.2.15nude mice model to study the inhibition of miR-34c on the tumor growth.Result:In this study, we show that miR-34c is deregulated in HBV related HCC by performing qRT-PCR on clinical tissues (human HBV-related HCC tissues and adjacent normal hepatic tissues) or hepatocellular carcinoma lines (HepG2.2.15and HepG2cells, BEL7402and HBV-BEL7402cells), which is coordinated with our primary result. To study the phenotypic effects of miR-34c, we transfected miR-34c expression vector into HepG2.2.15cells and found over-expression of miR-34c not only inhibited cell proliferation but also increased cell apoptosis. Further, over-expression of miR-34c can suppress the copy of HBV DNA and the secretion of HBsAg and HBeAg. In concordance to this, the level of TGIF2expression was high in human HBV-related HCC tissues HepG2.2.15with HBV infected. MiR-34c directly targeted TGIF2and reduced both mRNA and protein levels of TGIF2which stimulates proliferation and suppresses apoptosis in HCC cells. Finally we found that the overexpression of miR-34c inhibited the tumor growth in the human hepatoma cell line HepG2.2.15nude mice model.Conclusion:In this study, it is concluded that miR-34c may play an important role as a tumor suppressive microRNA in the development and progression of HBV-related HCC by targeting TGIF2. Thus miR-34c represents a key diagnostic marker and potential therapeutic target for the prevention and treatment of HBV infection.