The inhibitory effecacy of siRNAs targeting nuclear localization signal region on hepatitis B virus covalently closed circular DNA

Objective To evaluate the inhibitory efficacy of constructed nuclear localization signal(NLS) region of hepatitis B virus(HBV) targeted siRNAs on HBV covalently closed circular DNA(cccDNA) in vitro.Methods The NLS and C regions of HBV genome targeted siRNAs were constructed and transfected into HepG2.2.15 cells,which were constitutive HBV producing human hepatoblastoma cells.Enzyme-linked immunosorbent assay was used to measure the HBsAg and HBeAg levels in culture medium,while HBV DNA and HBV cccDNA in cultural supernatant and cells were determined by real-time fluorescence quantitative PCR 72 hours after transfection.The inhibitory efficay on the mRNA of HBV NLS gene was assessed by RT-PCR.Results The both 2 constructed siRNAs targeting NLS significantly down-regulated the HBV cccDNA level,inhibited DNA replication and HBeAg expression;with inhibitory rates on HBV cccDNA of 93.3% and 85.0%,on HBV NLS mRNA of 62.8% and 48.9%,on HBV DNA replication of 76.56 and 66.41%,and on HBeAg of 57.25% and 43.48%(P0.01).These effects were not revealed in the other irrelevant siRNA.Conclusion These results demonstrate that siRNA targeting NLS can consistently inhibit the HBV cccDNA amplification,HBV DNA replication and expression of HBeAg with great potency and specificity in vitro,suggesting that RNAi strategy may represent a potentially efficacious approach to the clinical management of HBV infection.