Research Of HBV Replication Inhibitied By SiRNA

Chronic infection with hepatitis B virus (HBV) is endemic to sub-Saharan Africa and parts of Asia where persistence of the virus is commonly associated with complicating cirrhosis and hepatocellular carcinoma (HCC).Licensed therapies that have been employed to eliminate HBV from chronic carriers of the virus include interferon-a, and the nucleoside analogues, lamivudine. Interferon-a are partially effective only in HBV e antigen(HBeAg)-positive carriers. Moreover, prolonged therapy with lamivudine is associated with an increased incidence of viral resistance. The limitations of HBV therapy, together with an improved understanding of HBV molecular biology,have prompted exploration of novel approaches to inhibiting HBV replication.RNA interference (RNAi) ,also called post transcriptional gene silencing (PTGS)is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. Short double-stranded RNAs, known as small interfering RNAs (siRNA), are incorporated into an RNA-induced silencing complex that directs degradation of RNA containing a homologous sequence.RNAi and related RNA silencing mechanisms are believed to act as a natural defence against incoming viruses and the expression of transposable elements.In mammalian systems, siRNAs are incorporated into a nuclease complex known as the RNA-induced silencing complex (RISC). RISC is directed by the unwound antisense siRNA strand to homologous target RNAs, which undergo endonucleolytic cleavage. Within RISC, mRNA cutting is affected by Slicer.The genome of HBV, as with other hepadnaviruses, is composed of circular partly doublestranded DNA . The long (minus) strand is approximately 3200 bases in length, and contains four open reading frames (ORFs) that encode precore/core, polymerase, surface and HBVx (HBx) proteins. After infecting hepatocytes, the partly double-stranded HBV genome is converted to covalently closed circular DNA, which is the template that is transcribed by cellular RNA polymerase II. RNAi could inhibit HBV protein expression and HBV DNA replication. RNAi generated by the siRNA might represent an alternative approach for the treatment of chronic HBV infection.In this study, we set up HBV-adr cell model(HepG 2.2.15 cell), and designed siRNA and used it as a tool to inhibit HBV replication in 2.2.15 cells. Three specific siRNA, siRNA1, siRNA2 and siRNA3 were confirmed effective in attenuating HBV activity at the mRNA and protein level in HepG2.2.15 cells, but the transfection with nonspecific siRNA as the negative control had no such effects.The inhibitions were sequence-specific, effective.1、Establish HBV-adr cell modelMETHODS AND RESUILS1) The HBV—adr gene segment was separated and depurated.2) The HBV double—copy gene segment was inserted to enzyme incise site in plasmid pcDNA3.3) The recombinant plasmid pHBV2 was screened.4 ) The recombinant plasmid pHBV2 was transfected into HepG2 by lipid body. 5 ) The cell line was screened by G418.CONCLUSIONSWe established HBV-adr cell model.2、Design and construction of siRNAMETHODS AND RESUILS1) Three X-protein of HBV genome sequences from GenBank were aligned and analyzed to identify the conservative regions containing at least 19 continuous nucleotides.2) These siRNA were further analyzed by BLAST to ensure that they did not have significant sequence homology with human genome sequences.The NControl-05815 was chosed to nonspecific siRNA.3) These siRNA were transfected into HepG 2.2.15 cells with Lipofectamine 2000 reagent according to the manufacturer’s instructions.CONCLUSIONS1) We designed three siRNAs based on the X-ptorein of HBV sequences2) Three siRNA were transfected into HepG 2.2.15 cells with Lipofectamine 2000 reagent.3、Test interfering effectMETHODS AND RESUILS1) We dedive the all HepG 2.2.15 cells into five groups:the empty control(which has not any siRNA), the negative control(which has nonspecific siRNA), siRNA1, siRNA2, and siRNA3.2) Quantitative assay of HBV mRNA: The amount of HBV DNA in the culture medium was quantitated by real time quantitative reverse transcription polymerase chain reaction(RT-PCR). The degree of inhibition by siRNA of HBV at the mRNA in HepG2.2.15 cells on hours 24,48 and 72 posttransfection: the negative control: 0.98 %±0.13%,—2.86%±0.30%, 034%±0.08%; siRNA1: 40.41%±4.35%, 47.82 %±3.46%, 23.37%±2.46%; siRNA2: 51.25%±3.51%, 55.33%±2.39%, 41.90 %±3.37%; siRNA3: 43.84%±5.32%, 45.18%±3.26%, 34.42%±3.42%.3) Inhibition of HBV proteins by RNAi: The viral proteins of hepatitis B surface antigen (HBsAg) in culture medium from transfected cells at various times were measured using western blot. There were a decrease in the levels HBsAg from the transfected cells. However, the transfection with nonspecific siRNA as the negative control had no decrease.CONCLUSIONS1) Three specific siRNA, siRNA1, siRNA2 and siRNA3, were confirmed effective in attenuating HBV activity at the mRNA and protein level in HepG2.2.15 cells.2) The transfection with nonspecific siRNA as the negative control had no interfering effects.3) The inhibitions by RNAi were sequence-specific...