Inhibitory Effect Of Antisense RNA On HCV Gene Expression

Background and Objective:Hepatitis C virus (HCV), an enveloped and positive-sense singlestranded RNA virus belonging to the genus Hepacivirus within the Flaviviridae family, is the main causative agent of non-A, non-B hepatitis. Chronic infection often leads to liver cirrhosis and hepatocellular carcinoma. No prophylactic or therapeutic vaccine is available, and current treatment for HCV infection is interferon in combination with ribavirin, but only about half of the treated patients maintain a sustained antiviral response. Thus, the development of safe and effective therapies for HCV infection is of great significance. Gene treatment for HCV has become hot research point in recent years. Specialist has used molecule technique including antisensenucleic acids and RNA interference to procure much development in the primary and clinic research. In order to investigate the contribution of antisensenucleic acids to HCV, our study is to construct the recombinant plasmid pHCV-EGFP which is expressed and replicated in Huh-7 cells, then observe effect of antisense EGFP on HCV replication. It provides effective pathway and experiment evidence for exploring new device against HCV infection.Methods:(1) The HCV replicon plasmid was reconstructed in vitro to be the plasmid HCV-EGFP, in which the EGFP gene replaced the HCV E1 and E2 gene. The resultant recombinant plasmid was confirmed by restriction endonuclease and sequencing, and then was designated as HCV-EGFP. (2) The recombinant plasmid HCV-EGFP was transfected into human hepatoma cell Huh-7 by lipofectamine 2000 mediation. The reporter gene expression in the transfected cells was examined by detecting green fluorescence and semi-quantitative RT-PCR were used to certify whether the HCV gene was effectively replicated in Huh-7 cells. (3) Human hepatoma cell Huh-7 as the research object, the plasmid HCV-EGFP and antisense EGFP were transiently cotransfected into Huh-7 via lipofectamine 2000 mediation, at equal pace pHCV-EGFP/ plus sense EGFP, pHCV-EGFP, blank control were set up, EGFP protein was examined by detecting green fluorescence and western blot, and HCV RNA replication was determined by semi-quantitative RT-PCR.Results:(1) The recombinant pHCV-EGFP was successfully constructed and could be correctly expressed and replicated effectively in Huh-7 cells. (2) The expression of EGFP protein was down-adjusted extremely and replication of HCV RNA decreased substantially (p<0.05) in pHCV-EGFP/antisense EGFP compared with control groups.Conclusion:(1) This recombinant plasmid pHCV-EGFP and the resultant Huh-7 cell model provide an effective pathway and basis for further study on HCV replication and pathogenic mechanism. (2) Antisense EGFP could inhibit HCV replication and protein expression, which offers feasibility and experiment evidence for research of HCV therapy.