Inhibition Of HBV Replication And Expression By RNA Interference

Hepatitis B virus (HBV) is a 3.2-kb DNA virus, replicating almostexclusively in the liver.Hepatitis B virus (HBV) infection affects nearly 400million people worldwide. Hepatitis B virus infection is very common in ourcountry. Chronic hepatitis B can develop hepatitis, cirrhosis, liver failure andhepatocellμlar carcinoma. Current therapies includes recombinant interferon alfaand nucleoside analogs, t the therapeutic effect is not satisfied. herefore, manyalternative strategies are being explored to find more safe and effective method. ecan find the reports about technique of antisense oligonucleotides, ribozyme andDNAzyme frequently.But each of them exists its defect. RNA interference (RNAi) is a cellμlar mechanism of post-transcriptionalgene silencing (PTGS) and the process whereby double-stranded RNA (dsRNA)induces the sequence-specific degradation of homologous messenger RNA(mRNA). This process is mediated by 21 to 23 nucleotides,called smallinterfering RNAs (siRNA), cleaved from dsRNA. With the development of RNAitechnology, there has been much research into the use of RNA interference for thetreatment of human diseases. Researchers have done some experiments aboutantiviral therapy with RNAi technology. In our experiment we use specific RNAiproducing vector to inhibit HBV replication and expression.The depressant effectis more permanent than PCR producing siRNA expression cassettes. The kind of cell line we used is the HepG2.2.15 cell.The HepG2 2.2.15human hepatoblastoma cell line was used as a model system. A head-to-tail dimerof HBV DNA (strain ayw) had transfected into this cell line stably. The HepG22.2.15 is a very important tool for the anti-HBV therapy research.The aims of thisstudy were to develop an RNAi approach that specifically targets the precore andcore regions and S regions of hepatitis B virus (HBV) by short interfering RNA(siRNA) expressing vector in vivo, and to explore the inhibitory effect of siRNAson HBV replication and expression, and to provide some evidences for anti-HBVtherapy by RNAi.We choose different targets from HBV S region and C region and we desireand synthesize the insert fragment. Then we ligate the annealed insert fragmentand the plasmid with U6 promoter. We transfect the right plasmids to theHepG2.2.15 cell, which are identified by enzyme incision and sequencing. Wemeasured different transfection concentration,different transfection time, whichaffected replication and expression of HBV. At the same time, we desire thecontrol plasmid with sequence unrelated to HBV. We use ELISA assay to test theeffect to HBV protein pression level by RNAi. The role of HBV siRNA producingsystems in the inhibition of HBV DNA levels was measured with dothybridization in HepG2.2.15 cells by RNAi. The resμlts are as follows:1,Two pairs of DNA fragments that can transcribe shRNAs are cloned intoU6 promoter containing plasmid. which targeted sequences within S and C geneof HBV.The targeting site in the S gene locates from 460nt to 478nt. HBV.Thetargeting site in the C gene locates from 2134nt to 2152nt And we also desire apair of DNA fragments which are unrelated to HBV genome and clone them intoU6 promoter containing plasmid. We determine if each plasmid is the right one wewanted through being digested by Stu I and sequencing.We named the plasmid asSC-S,SC-C and SC-N .2 , The depressant effect of shRNA producting plasmids SC-S isdose-dependent in the range from 0.5μg to 2.0μg per 5×104 cells.Moreconcentration,the inhibitory effects are higher, whereas SECs-CON had no effect.The inhibition ratios of HBeAg and HBsAg are 90.51% and 100% 6 days aftertransfection with SC-S at the concentrations of 2.0μg per 5×104 cells.We find thesame result through dot blot assays.The amount of DNA reduces when theconstration of plasmid increases.We find SC-S at the constration of 1.0μg per5×104 cells can inhibit HBeAg and HBsAg significantly and the inhibition ratiosare 51.2% and 68.53%。We also notice that the HBsAg depressant effect of SC-Sis better than the HBeAg depressant effect.3,Both shRNA producting plasmids successfully reduced the expression ofHBsAg and HBeAg , even the presence of HBV replication.Different shRNAproducting plasmids had different inhibitory ratio.The depressant effect of shRNAproducting plasmids targeted at surface gene (SC-S)is better than the shRNAproducting plasmids targeted at core gene (SC-C).4,The depressant effect of HBV expression is manifest on the 3rd day aftertransfection by SC-S. In our experiment the inhibition ratio is the highest on the6th day after transfection by SC-S.And the depressant effect of HBV expression isstill significant on the 9th day after transfection by SC-S.In conclusions, In conclusions, We construct two shRNA productingplasmids inhibiting HBV replication and expression specifically with U6 promoterwhich targeted at two distinct 21nt sequences ,one of which in the HBV surfacegene,the other in the HBV core gene.Both shRNA producting plasmidssuccessfully reduced the expression of HBsAg and HBeAg , even the presence ofHBV replication.Different shRNA producting plasmids had different inhibitoryratio.The depressant effect of shRNA producting plasmids targeted at surface gene(SC-S)is better than the shRNA producting plasmids targeted at core gene(SC-C).The depressant effect of shRNA producting plasmids is dose-dependent inthe range from 0.5μg to 2.0μg.The depressant effect of shRNA productingplasmids is more persistent than the other method.In a word, shRNA productingplasmids that transcript HBV-specific siRNAs with RNA polymerase Ⅲ is anefficient approach in reducing the level of HBV proteins and in suppression ofHBV replication.The resμlts in this study sμggest that RNAi-based anti-HBVstrategy may open a path toward using siRNAs to improve HBV therapy, andopen a new avenue for treating chronic HBV infection, which remains a commonand serious disease by siRNAs only or as an adjuvant therapy to other antiviraltherapy.In brief, we use shRNA producing plasmid system to transcribe HBVspecifically siRNA in vivo and the shRNA producing plasmid can be highlyeffective to inhibit the replication and expression of HBV in HepG2.2.15 cell.Ourstudy confirmed expressing siRNA truly has the anti-HBV effect in vivo.