Hepatitis C Virus (HCV) Core Protein Influence The Expression Of Tissue Inhibitor Of Metalloproteinases-1 (TIMP-1)

AimLiver cirrhosis is one of the major complications of hepatitis C virus (HCV) infection, but the mechanisms underlying HCV-related fibrogenesis are still not clear. Although the roles of HCV core protein remain poorly understood, it is supposed that HCV core protein play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to investigate the influence of HCV core protein on tissue inhibitor of metalloproteinases-1 (TIMP-1) expression via molecular methods, providing a potential pathogenesis for hepatic fibrosis during chronic HCV infection. Methods1.The TIMP-1 promoter was amplified by polymerase chain reaction(PCR) from human genomic DNA template by using primers . PCR product was subcloned into Pcat3-basic vector to construct report vector Pcat3-TIMP-1P. after Pcat3-TIMP-lp was transfected into HepG2 cells by using transient transfection, the expression levels of Chloramphenycol acethyltransferase (CAT) were measured by enzyme-linked immunoassay( ELISA). Pcat3-basic was transfected into cells served as negative-control, positive-control cells transfected with Pcat3- control.2. Chloramphenycol acethyltransferase (CAT) analysis was performed on LX-2 cells cotransfected with PcDNA3.1(-)-HCVcore and Pcat3-TIMP-lp, while negative-control cells transfected with Pcat3-basic, positive-control cells transfected with Pcat3-control. Results1.TIMP-1 promoter DNA fragment was amplified successfully and confirmed by restriction enzyme digestion and sequencing.The report vector Pcat3-TIMP-lpwas constructed successfully. ELISA analysis showed the optical density(OD) of HepG2 cells transfected with Pcat3-basic was 0.004±0.002, the OD of Pcat3-TIMP-lp was 2.329±0.685. Statistics was performed by ANOVA, and differences were considered to be significant at F =26.075 (P<0.05 ), TIMP-1 promoter stimulated expression of CAT.2. After cotransfected into LX-2 cell with PcDNA3.1(-)-HCVcore and Pcat3-TIMP-lp, the OD of Pcat3-TIMP-lp was 0.833±0.040. In contrast, the OD of Pcat3-TIMP-lp transfected alone was 0.677±0.049. Statistics was performed by ANOVA, and differences were considered to be significant at F =13.401 (P<0.05 ). After cotransfected with PcDNA3.1(-)-HCVcore and Pcat3-TIMP-lp, the expression of CAT was elevated.Conclusion1. The report vector Pcat3-TIMP-lp was constructed successfully, and transfection experiment in vitro confirmed the activity of TIMP1 promoter.2. This result suggested that through enhancing TIMP-1 promoter activity, HCV core protein facilitated the expression of TIMP-1. We further demonstrated that HCV play a pathogenic role in liver fibrogenesis during chronic HCV infection.