Effect Of Hepatitis C Virus NS5A Protein On TLR3,TLR4

BackgroundHepatitis C virus (HCV) infection is world-widely distributed, it is one of the main reasons of the end-stage liver diseases in Europe, America and Japan. According to the World Health Organization, the global rate of HCV infection is about 3%.The number of infections is more than 170 million, there are about 35,000 new cases every year. The hcv infection rate is in the range of 1.5～3.5% in China, and totally more than 30 million people in China have been infected. There are 80% of the infected persons progress to chronic hepatitis, Some of which developed to liver cirrhosis or hepatocellular carcinoma. HCVNS5A as an important non-structural protein has become the focus of research because of its multi-functions in HCV replication, pathogenesis. Currently, the mechanism of interaction between TLRs and the host immnunity becomes more and more interested. TLR3 was mainly expressed in dendritic cells, it can identify the virus dsRNA and play an important role in the process of anti-virus. TLR4 is the first protein found in mammalian TLRs. The surface of much cells such as monocytes, plasma cells, neutrophils, endothelial cells can express TLR4, it is considered to be relevant to gram-negative bacteria and endotoxin recognition and activation, and also found to play an important role in alcoholic liver disease, viral hepatitis, liver fibrosis. In this study, plasmid pcNS5A was transfected to QSG7701 cells. We detect the impact of HCV NS5A on the expression of TLR3 mRNA, TLR4 mRNA and protein in the liver cells. It can afford the experimental evidence for the pathogenesis of the hepatitis C.Methods1. Plasmid transfection:We planted QSG7701 cells in six-well plates before transfection, until 60%-70% cells merging, put pcNS5A or pRc/ CMV plasmids into cells according to the procedure of Lipofectin2000, changed for 10% of fetal bovine serum medium after 12 hours, harvested after 48 hours.2.Immuno- cytochemistry:We detected the expression of TLR4 and NS5A protein by immunocytochemistry method.3. RT-PCR:transfected cells were extracted total RNA in accordance with the procedure of RNA extraction kit. We measured the concentration and purity of RNA by UV spectrophotometer, took 2μgRNA to make cDNA on the basis of reverse transcription, the products were augmented by PCR and separated by 2% agarose gel electrophoresis.4. Indirect immunofluorescence method:We used indirect immunofluorescence method to detect TLR4 protein expression.5. Western blot:We detected TLR4 protein expression by Western blot method, usingβ-actin as a reference.Results 1. The cells transfected with plasmid pcNS5A expressed HCV NS5A protein which stained as brown particle. It is distributed in the cytoplasm, obviously around the nucleus. Non-transfection cells and that of transfected pRc/CMV plasmid did not express HCV NS5A protein.2. By the immunohistochemistry method, it is found the cells transfected with plasmid pcNS5 A expressed TLR4 protein. It is mainly distributed on the cell membrane. Non-transfection cells and that of transfected pRc /CMV plasmid did not express TLR4 protein.3. TLR4 mRNA expression in pcNS5A transfected cells is higher than in the untransfected or pRc/CMV plasmid transfected cells. The level of TLR4 mRNA expression between the pRc/CMV plasmid transfected cells and the untransfected cells is similar. This shows that HCV NS5A can stimulate the transcription of TLR4 mRNA. TLR3mRNA expression between pcNS5A transfected cells, blank group and the group of transfected pRc/CMV plasmid is similar. This shows that HCV NS5A doesn't stimulate the transcription of TLR3 mRNA.4. By the indirect immunofluorescence method, It is found the cells transfected with plasmid pcNS5A expressed higher level of TLR4 protein than non-transfection group and the group of transfected pRc/CMV plasmid. There is no difference inTLR3 protein expression between the cells of transfected with plasmid pcNS5A, in non-transfection cells and that of transfected pRc/CMV plasmid. This shows that HCV NS5A can't stimulate the expression of TLR3 protein.5. By western blot method, we found that the TLR4 protein expression in pcNS5A transfected cells was much higher than in the untransfected cells or in the cells transfected with pRc/CMV plasmid. TLR4 protein expression level between the cells transfected with pRc/CMV plasmid and the untransfected cells is similar. This shows HCV NS5A up-regulated the expression of TLR4 protein.Conclusion:1. HCV NS5A expression plasmid was transfected to QSG7701 cells successfully, and HCVNS5 A protein expression was detected.2. HCV NS5A stimulates the transcription of TLR4 mRNA.3. HCV NS5A promote the expression of TLR4 protein.4. HCV NS5A doesn't stimulate the transcription of TLR3 mRNA and the expression of TLR3 protein.