Silencing Glvpican-3Expression Via Rna Interference Induces Apoptosis In Human Hepatocellularcarcinoma Cells And Molecular Mechanisms

Background and purpose:Hepatocellular carcinoma (HCC) is one of the most common malignancies, rated fifth in incidence and the third in mortality worldwide. HCC, as one of the cancers seriously threatened people's health and life all over the world. Because of the complex molecular mechanisms behind its carcinogenesis. early detection is difficult, the outcome of aggressive therapies remains grave, eassily relapse and unobvious concurrent chemoradiotherapy, it is called as "The King of Cancer". Thus, there is an urgent need to identify novel molecular targets for the development of diagnostic and therapeutic approaches for HCC. Therefore, the new tumor maker has always been the hot topic among the HCC researching field. In recent years, as the rapid development of human genomics, proteomics and some other research technology, the research for detecting HCC tumor maker has gained great progress.The recent research had found that Glypican-3(GPC3, also known as MXR7), a member of the glypican family that is highly expressed in HCC cell lines and tissuesis. And it may also have the relationship with precursor lesions (namely, CH and LC) of liver cancer. In addition, as one kind of HCC oncoprotein, GPC3plays an important role in the biological behavior of HCC cells, like cell proliferation and apoptosis, but the concret research program is that the molecular mechanisms of this events still remains unknown. GPC3shows great promise as an important molecular target for the diagnosis and treatment of HCC. The precise molecular mechanism of GPC3 mediated apoptosis resistance remains unclear, therefore, deeply researching the important role of GPC3plays in HCC and providing the theoretical basis for liver cancer gene targeting therapy, this is one of very important ways to study the value of GPC3in HCC early diagnosis, cure and prognostic judgement.This study focus on using StealthTM RNAi for GPC3silencing, after immediately transfection to the HCC cell line HepG2, which with high-expression GPC3protein, then RNAi will be formed to restrain GPC3gene expression of HepG2cells, and then we used RNAi to restrain GPC3gene expression and investigated the effect of GPC3-silencing on the HCC line HepG2cells proliferation and apoptosis as well as the cspecific molecular mechanism.Methods:1. Investgated the expression of GPC3protein levels in HCC cell lines by the way of ELISA essay and Western blotting.2. Design and synthesis of the GPC3gene-targeted StealthTM RNAi sequence and then use the LipofectamineTM RNAi Max transfection Agent to immediately transfected the HCC cell line HepG2which with high-expression of GPC3protein.3. Use R-T PCR method to test the effect of the GPC3-StealthTM RNAi on HepG2cells, and then screen the StealthTM RNAi with the best silencing effect for the later gene-silencing test. The sulforhodamine B (SRB) assay was used in study cell growth rate; assessment of apoptosis with TUNEL staining and flow cytometry.4. Protein extraction and western blot analysis levels of key apoptosis related proteins at different times after GPC3is silenced, and explore the specific molecular mechanism of HCC cell apoptosis after the GPC3is silenced.Results:1. GPC3is highly expressed in the HCC lines HepG2and Huh-7, while low expressed in SMMC-7721cell line.2. Tranfection of GPC3targeted StealthTM RNAi obviously restrain the GPC3gene expression of HepG2cells, among which, HSS149and Cocktail of three lines of StealthTM RNAi has stronger restrain effect for GPC3expression. Compared with the blank group, the expression levels of GPC3gene in HepG2gene obviously gets lower (95.90%and96.25%) after HSS149tranfection for48or72hours; and the expression level of GPC3gets much lower (96.27%and96.36%) after the Cocktail transfection.3. GPC3trageted StealthTM RNAi obviously inhibited proliferation of HepG2cells. After Cocktail transfects for24,48and72hours, the inhibition rate of HepG2cell's proliferation are:40.67%,48.34%and57%.5. After GPC3targeted StealthTM RNAi transfection, the GPC3protein levels, Bcl-2and Procaspase-3and mitochondria Cytochrome c were cobviously lower in HepG2cells, while the protein levels of Bax and Cytochrome c in the cytoplasm up-regulated obviously.Conclusions:1. GPC3is highly expressed in HCC lines HepG2and Huh-7.2. GPC3targeted StealthTM RNAi can obviously restrain the expression of GPC3gene and protein in HepG2cells.3. GPC3targeted StealthTM RNAi transfection can obviously restrain proliferation of HepG2cells.4. GPC3targeted StealthTM RNAi transfection can obviously induces the apoptosis in HepG2cells.5. The apoptosis in HepG2cells caused by GPC3gene silencing may be established by dysfunction of Bax/Bcl-2/Cytochrome c/Caspase-3signaling pathway.6. GPC3gene expression plays an important role in apoptotic resistance, occurrence and development of HCC, and it also can be further investgated as a new cure method for HCC gene therapy. These results suggest that GPC3positively regulates cell proliferation by enhancing the resistance to apoptosis via promoting the dysfunction of the Bax/Bcl-2/cytochrome c/caspase-3signaling pathway in GPC3-overexpressing HCC cell lines. Therefore, Knockout of GPC3expression may cause broad-spectrum biological effects in HCC cells that merit further investigation because these effects may be widely applicable and beneficial for gene therapy in clinical settings of HCC.