Primary Research: Hepatistis C Virus NS5A Protein Inhibit Hepcidin Gene Expression

Purpose:Hepatitis C virus(HCV) infection is the global distribution.The number of infections is more than 170 million,it is 1.5～3.5%in China, more than 30 million people infected.There are 80%infections that became the patients of chronic hepatitis,cirrhosis and primary liver cancer.HCV is closely related to the liver cancer.HCV-induced liver disease is increasing.All of these may clarify that the HCV and other cofactors have the important impact in the progress of hepatitis C-related liver diseases.There are also many studies prove that iron plays an important role in the progress on chronic liver disease of HCV infection. HCVNS5A is very active non-structural protein of the HCV,it plays an important role in forecasting in response to interferon therapy,viral replication,antiviral resistance,the occurrence of hepatocellular carcinoma.Hepcidin(hepatic bactericidal protein) the small molecule antimicrobial peptide is discovered recently and secreted by the liver.It regulats the body iron homeostasis and plays the central role in the process of the iron-regulatory hormone.HCVNS5A can generate peroxide to make liver cells in a state of oxidative stress,so that the levels of intracellular ROS increased.Because ROS can regulate several cell signaling pathways to inhibit or reduce hepcidin gene expression,we speculate that HCVNS5A-induced ROS can also reduce or inhibit hepcidin gene expression.In this study,plasmid pcNS5A was transfected to QSG7701 cells transiently and stablely.We use the technique of indirect immunofluorescence method to make sure the success of transfection, and adop the method of RT-PCR to detect the impact of HCV NS5A on the hepcidin gene.Methods:1.HCV NS5A expression plasmid's restriction enzyme digestion: pcNS5A was digested by the restriction enzyme method.Product was separated by 1%agarose gel electrophoresis.2.Plasmid transfection:1) transient transfection:We planted 5×105 QSG7701 cells in 35mm Petri dishes before transfection,put pcNS5A or pRc/ CMV plasmids into cells according to the procedure of Lipofectin2000, changed for 10%of fetal bovine serum medium after 24 hours, harvested after72 hours.2) stable transfection:We planted 5×105 QSG7701 cells in 35mm Petri dishes before transfection,put pcNS5A or pRc/CMV plasmids into cells according to the procedure of Lipofectin2000,changed for 10%of fetal bovine serum medium which is added in the appropriate concentration of G418 for screening after 24 hours.Cell clones occur around two weeks.3.Add LPS:pcNS5A or p Rc / cmv plasmid transfected into QSG7701 cells transiently in accordance with the above-mentioned procedure.We put LPS(200μg/ml)in culture medium after 48 hours,then harvested the cells after 24 hours.4.Indirect immunofluorescence method:We use indirect immunofluorescence method to detect HCVNS5A protein expression and identify the success of transfection.5.RT-PCR:transfected cells were extracted total RNA in accordance with the procedure of RNA extraction kit.We took 10μgRNA to make cDNA on the basis of reverse transcription,the products were amplificated by PCR and separated by 2%agarose gel electrophoresis.Results:1.We get the expected size of the HCV NS5A fragment after restriction enzyme digestion.2.The cells transfected with plasmid pcNS5A expressed HCV NS5A protein,a kind of green fluorescent particle.It is distributed in the cytoplasm.Non-transfection group and the group of transfected pRc /CMV plasmid did not express HCV NS5A protein。3.Plasmid p CNS5A or p Rc/CMV was tranfected to QSG7701 cells in accordance with gene transfer technology.The groups were detected the expression of hepcidin mRNA by RT-PCR.Hepcidin mRNA expression in the experimental group is lower than the blank group and the group of transfected pRc/CMV plasmid,hepcidin mRNA expression of the transfected pRc /CMV plasmid group and the blank group is similar,This shows that the pRc /CMV plasmid had no effect in the expression of hepcidin mRNA,and HCV NS5A reduced expression of hepcidin mRNA.4.The use of gene transfer technology,pcNS5A plasmids was transfected into QSG7701 cells transiently.Expression of hepcidin mRNA was detected by RT-PCR in each group of cells.Hepcidin mRNA expression of LPS+the blank control group is higher than the others.Conclusion:1.HCV NS5A expression plasmid was transfected to QSG7701 cells successfully,and QSG7701 express the HCVNS5A protein.2.HCV NS5A inhibit the expression of hepcidin mRNA.3.Effect of enhancing hepcidin mRNA expression about LPS is weaken by HCV NS5A.