The Study On The Mechanism Of HCV C Protein Suppressing P14-p53-p21 Pathway And Promoting Hepatocarcinoma Cell Proliferation

Hepatitis C virus (HCV) has been identified as the major causative agent of non-A, non-B hepatitis, which is a significant public health problem with an international scope, for the virus has infected approximately 170 million people, representing nearly 3% of the world's population. The majority remains chronically infected, leading to chronic hepatitis, steatosis, cirrhosis and hepatocellular carcinoma. In some country, the HCV is the leading cause of hepatocellular carcinoma and liver transplantation. In our country, the incidence of HCV-infected patients is not higher than that of HBV-infected patients, or not as high as in other countries, but the HCV infection often leading chronic hepatitis, cirrhosis and hepatocellular carcinoma also causes seriously health problem. And approved therapy for HCV infection shows not much efficient, and to the day no effective vaccine has been developed. HCV, as a RNA virus, only replicates in the cytosol and fails to integrate with the host genome to activate oncogene, mainly acting with its proteins. HCV core protein is one of HCV structural proteins. Many researches indicate that the core protein plays a key role in HCV pathogenesis. In addition to roles of the C protein in binding viral RNA, regulating HCV RNA translation, making homotypic interactions for particle assembly, Cprotein is involoved in cell signalling, apoptosis, carcinoma, and lipid metabolism, which plays a major role in promoting cellular transformation, immortalization and hepatocarcinoma progression. p14^ARF-p53-p21^WAF1 pathway has been shown to be a key element of the cell's antiproliferation machinery, capable of inducing either cell cycle arrest or apoptosis in response to numerous types of stress. The C protein effects on the well-known pathway, but there were controversial results about the effects.So we want to explore the mechanism of C protein effecting on p14^ARF-p53-p21^WAF1 pathway and hepatocarcinoma cellular proliferation.Aims: To investigate the mechanism of C protein effecting on p14^ARF-p53-p21^WAF1 pathway, and promoting human hepatocellular and hepatocarcinoma cell proliferation.Methods:1. Collect liver tissues of 23 cases with HCV infected and 42 cases with HCC, and determine the expression of HCV core, mutant p53, Mdm2, p14^ARF, p21^waf1 proteins with immunohistochemical method, using En Vision method; analyze the correlation of these proteins by statistics methods.2. Constructions of pEGFPCl-C191 and pEGFPCl-C173 plasmid were made and the plasmids were transfected using Lipofectamine 2000 regent into HepG2 cells, then the subcellular localization of core fluorescent proteins (the innate form of core protein l-191aa. and the mature form of core protein l-173aa.) were observed under inverted fluorescorpe.3. Constructions of pcDNA3.1-C191 and pcDNA3.1-C173 plasmids were carried out, and the plasmids were transfected using Lipofectamine 2000 regent into HepG2 cells for making C191-HepG2 and C173-HepG2 models constitutionally expressing C proteins. Then EnVisionimmunohistochemical staining, RT-PCR, Flow cytometry (FCM), and measuring the cell growth curve were utilized to detect proteins expression(including C protein, p53, p21, pl4 protein), their mRNA transcription, cell cycle and cell growth in HepG2 before and after transfection.4. The pcDNA3 ARF expressing pl4 protein, and pcDNA3 wt p53 expression wild type p53 were contransfected into C191-HepG2 and C173-HepG2 which express innate or mature form of C protein using Lipofect amine 2000 regent. En Vision immunohistochemical staining, confocal immunofluorescence method, RT-PCR, Western blot method were made to detect the proteins expression (including C protein,p53, p21,p14 protein), the protein colocalization, their mRNA transcription.Results:1 .The expression incidences of mutant p53 and Bcl-2 proteins were 87.0%, 95.75% respectively in the liver tissues of HCV C protein positive staining and were 47.6%,83.3% respectively in HCC; the test of positive intensity expression between HCV core and mutant p53 proteins was correlationship; the test of positive intensity expression between HCV core and Bcl-2 proteins in HCV hepatitis and cirrhosis was correlationship, but wasn't correlationship in HCC. The expression incidence of Mdm2 was very high in HCV hepatitis and in HCC, and there was no correlation between C protein and Mdm2, but there was correlation between mutant p53 and Mdm2 protein expression. The expression of p21 was very low both in HCV hepatitis and in HCC, and the expression of p21 wasn't correlated with C protein or mutant p53. Though the expression of pl4 was higher both in HCV hepatitis and in HCC, there was no correlation with C protein.2. The C191 protein was predominantly localized in cytoplasm and in the perinucleoplasmic region of HepG2 24h after transfection withpEGFPCl-C191, while C173 protein accumulated predominantly in the nucleus and perinucleoplasmic region of HepG2. But both of C191 and C173 proteins were all localized predominantly in nucleus and perinucleoplasmic region of HepG2 6 day after transfection.3. The HepG2 cells consistutionaly expressed a little C protein, p53 protein and pl4 protein.4. C proteins in C191-HepG2 and C173-HepG2 cells inhibited transcription of p53 mRNA and pl4 mRNA. And the expressions of p53, pi4 proteins were decreased from immunocytochemical staining and Wester blot. The cell cycle showed that the S phase was increased, while the Gl phase was decreased in C191-HepG2 and C173-HepG2 cells compared with HepG2's.5. The p53 protein colocalized with C protein mainly in nucleus and perinucleoplasmic region of cells. It seamed that the C protein also colocalized with pi4 or p21 protein.6. Though the mRNA and protein of p53 or pl4 were increased after C191-HepG2 and C173-HepG2 cells transfected with pcDNA3- ARF or pcDNA3-wt p53,but the expression of C protein wasn't affected.Conclusions:1. Mutant p53's expression was very common in the tissues of HCV core positive staining, the core protein probably induce out the mutant p53 and promote its expression. The core protein and mutant p53 may enhance Bcl-2 expression indirectly. The incidence of Mdm2's expression was very high, which may be due to the pl4 and mutant p53's increase. Though the incidence of p14^ARF protein was not low being due to C protein's effect, p14^ARF protein wouldn't exert its tumor suppress because of abrogation of wild-type p53, mutant p53 or p14^ARF protein itself mutation. The absence of p21^WAF1 protein expression was universal in the tissues of HCV core positive staining and in HCC, which is probably...