Identification Of Genes Involved In HBV Replication Using RNAi Library

Objective: To develop a method to identify genes involved in HBV replication by RNAi library.Approaching for the mechanism of HBV replication to provide some new targets for anti-HBV drugs.Methods:(1) Set up and validate the screening strategy: Construting HBx-siRNA and HBs-siRNA, transfecting them into HepG2.2.15 cells respectivly, MEIA(microparticle enzyme immunoassay) revealed the levels of HBsAg and HBeAg in supernatant of HepG2.2.15 cells, the levels of HBV DNA and HBV cccDNA were quantified using real time RT-PCR. FACS was used to show the levels of HBsAg on surface of HepG2.2.15 cells. (2) HepG2.2.15 cells were infected with RNAi library, cells expressing less HBsAg were isolated using immunomagnetic beads. After PCR amplification and sequencing of the siRNA conding sequences, corresponding siRNA was used to verify the screening results. The levels of corresponding protein were detected by Western blot. The replication of HBV was detected by MEIA and real time RT-PCR . FACS was also used to show the levels of HBsAg on surface of HepG2.2.15 cells .Results: (1) the levels of HBsAg,HBeAg and HBV DNA in supernatant of HepG2.2.15 cells by HBx-siRNA and HBs-siRNA were all decreased. HBV cccDNA and surface HBsAg were inhibited. (2) By random sequencing, there were 20 clones to be sequenced. We found that DDB1 might be involved in HBV replication. After transfecting DDB1-siRNA into HepG2.2.15 cells , DDB1 protein expression was inhibited. MEIA and real time RT-PCR revealed that the levels of HBsAg,HBeAg and HBV DNA in supernatant of HepG2.2.15 cells with DDB1-siRNA were decreased. The relative cccDNA expression of cells was reduced. FACS indicated that the surface HBsAg was siginificantly inhibited.Conclusion: we developed a method that can be used to identify genes involved in HBV replication and DDB1 can enhance the replication of HBV.