| Objective: Primary hepatic carcinoma(HCC) is one of the most common fatal malignancy in the world. Chronic Hepatitis virus infection, alcohol abuse, diabetes and non-alcoholic fatty liver disease, excessive intake of aflatoxin are important causes of HCC. Hepatitis C virus(HCV) infection is one fo the most important reasons for HCC. According to the statistics of the world health organization(WHO), more than 200 million people are infected with HCV in the world. By the way, the increasing rate of the liver cancer patients developed from HCV infection is 1% to 7% each year. HCV infection has become a serious threat to human health of the public health problem. HCV virus is mainly divided into 6 kinds of genotypes, and more than 80 kinds of subtypes, which happens to 1b genotype and 6 genotype in our country. Compared with other genotypes infection, HCV 1b genotype infection has more serious liver cell damage and higher risk for the development of liver cirrhosis and liver cancer. Therefore, the research on HCV infection related to the mechanism of HCC is of great significance.HCV virus was first identified in 1989, which is the main pathogens causing hepatitis besides HBV and HAV. HCV is an single-stranded RNA virus classified within the Flaviviridae family, which viral genome is about 9.6 Kb in length. Associated with signal peptide enzyme in host cells and virus protease, HCV can produce at least 10 kinds of viral proteins, including three kinds of structural protein(Core, E1 and E2) and 7 kinds of the nonstructural protein(p7, NS2, NS3, NS4 A, NS4 B, NS5 A, NS5B). The HCV core protein(HCV core) plays a very important role in the interaction between virus and host cells and has a direct effect of cancer. Its abnormal induction of cell signal transduction and transcription factor regulation are involved in HCV related liver cancer and malignant transfer process, but the specific mechanism is unclear.Snail is one of transcription factor superfamily SNAIL members, which is a vital induction molecule involved in EMT. It is important for maintaing character of stem cells, cell live and apoptosis, and immune regulation. Snail has 264 amino acid and three function structural domains. Snail C-terminal could specifity recognize E-box 5’-CAGGTG-3’ and inhibit gene expression. This mechanism is recognized as a key factor of Snail inhibit E-cadherin and promotion of EMT.GSK-3(Glycogen synthase kinase-3) is a serine/threonine kinase, which was first discovered in 1980. It is a key enzymes involved in the regulation of Glycogen synthesis of Glycogen synthase. GSK-3 family is divided into two subtypes, ɑ and β. GSK-3β is a key component of Wnt/β-catenin and PI3K/Akt signal pathways, involved in regulating cells adhesion, differentiation, proliferation and apoptosis. It also play an important role in the signal transduction of tumor cells. Previous studies found that, in cytoplasm of human hepatocellular carcinoma cell line HepG2, levels of phosphorylated GSK-3β increased significantly while E-cadherin is down-regulated. It revealed GSK-3β might participate in regulating E-cadherin transcription. In addition, transforming growth factor β1(TGF-β1) and transcription factor Snail is considered involving regulating EMT by down-regulated the E-cadherin.Own preliminary data include:(1) In the HCV core protein over-expression of liver cancer cells, hepatic progenitor cells and HCV infection Huh7.5.1 cells, their migration and invasion ability were enhanced obviously. In the EMT molecular marker detection, we found that HCV core could induce down-regulation of E-cadherin and up-regulation of Vimentin. However exogenous expression of E-cadherin can partly reverse EMT induced by the Core.(2) The HCV core inhibit E-cadherin promoter activity, and is closely related to the binding sites of E-boxes.(3) Signaling pathway report plasmid screening experiment found that in overexpression of HCV core cell model, PI3K/Akt signal pathway is activated, the expression of p-Akt and p-GSK3β levels are rised, and GSK-3β activity has been restricted.According to the preliminary experiment found that the Core role in regulation are correlated with E-box. We hypothesis that HCV core directly inhibit GSK-3β activity, reduced the degradation of transcription factor Snail, promote the stability of Snail, and then regulate the expression of E-cadherin. This project could help to more well understand the HCV involved cell signal transduction, EMT process and the molecular mechanism of liver cancer metastasis, and to construct molecular basis for the development of HCV therapeutic drug targets.Methods: This study proposed to detect the expression of HCV Core, Snail and E-cadherin and their correlations in vitro HCV core expression model and HCV replication model, using western blot, siRNA interference technology, Ch IP technology, IP, immunofluorescence experiments and so on. We analysed whether Core and Snail or other histone modification enzyme could form compound regulating the expression of E-cadherin and whether HCV Core could promote transposition and reduction of GSK-3β enzyme activity, which involved in the EMT.Results: With the methods of Wakita, we obtained the JFH-1 RNA and transfected it into Huh7.5.1 cell and successfully detected stable expression of HCV core. Three days after transfection, we collected, enriched and filtered the cells supernatant, and transcfected HLCZ01 cells. Eight days after infection, HCV RNA obviously increased. We found the expression of HCV Core protein in immunofluorescence experiments.We observed HCV core could enhance the ability of cell migration in SK-Hep1 cell stable expression of HCV core model. EMT could be partially blocked with exogenous expression of E-cadherin. In stable expression of HCV core Huh7 cell model with silencing the endogenous E-cadheirn expression, the ability of cell migration and invasion increased. Meanwhile, we found HCV core could down-regulated E-cadherin in JFH-1 infected Huh7.5.1 cell model and JFH-1 infected HLCZ01 cell model.In JFH-1 RNA infected Huh7.5.1 cell model, HCV Core was located in the cell nucleus with Snail by immunofluorescence experiments. Indeed, HCV Core bonded with the carboxyl of Snail and down-regulated E-cadherin. Meanwhile, Snail could raise HDACs, which could promote HCV Core bond to the promoter of CDH1, and coregulate the expression of E-cadherin. In JFH-1 RNA infected Huh7.5.1 cell model, we found HCV Core could promote transposition and phosphorylation of GSK-3β, resulting in reduction of GSK-3β enzyme activity and EMT finally.Conclusion: We found HCV Core could promote transposition of GSK-3β and reduction of GSK-3β enzyme activity. HCV Core was found to bind to Snail C-terminal 153-264 amio acid, Recruited HDACs formatting an inhibitor complex to inhibit the expression of E-cadherin, promoting EMT. Those reveal that HCV core promot hepatoma associating with Snail. These findings increase understanding of factors associated with occurance and progression in hepatoma and may ultimately lead to the development of novel treatment strategies for HCV-associated hepatocellular carcinoma. |
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