Inhibitory effect of siRNA targeting hepatitis B viral X gene on hepatitis B viral replication and HBsAg and HBeAg expression in vitro

Objectives To establish an RNAi approach that can specifically target the X gene sequence of hepatitis B virus ( HBV ) by synthesizing short interfering RNA ( siRNA ) in vivo, and to assess the inhibitory effect of this siRNA on HBV replication and its antigen expression. Methods A recombinant plasmid pSIHBV/X which can transcribe the siRNA targeting HBV X gene was constructed by cloning the annealed synthesized sequences into linearized pTZU6+1 vector and named as pSIHBV/X. Secondly, the eukaryotic expression plasmid pHBV1.3, which contains 1.3-fold-overlength genome of HBV, were cotransfected into HepG2 cells using GenePORTER2 transfection reagent with either the RNAi plasmid pSIHBV/X or unrelated control plasmid pSIGFP. At 24, 48 and 72 hours after transfection, the levels of HBsAg and HBeAg in the cell culture supernatant were determined with ELISA kits. The HBV DNA in the supernatant was also determined by fluorogenic quantitative PCR ( FQ-PCR ). Results A siRNA transcribing vector, pSIHBV/X, which targeted the X gene of hepatitis B virus was constructed successfully. The introduction of RNAi plasmid had shown to efficiently and specifically inhibit the synthesis of surface antigen and e antigen of HBV, with inhibitory rates of 64% and 61%, peaking at 72 hours after transfection. FQ-PCR also showed that replication of HBV DNA was reduced to lower levels, when the ratio of pSIHBV1.3 and pSIHBV/X was at 1: 20, whereas the control vector did not exhibit any inhibitory effect on the replication and expression of HBV. Conclusions Results in this experiment demonstrates that the short interfering RNA targeting HBV X gene exerts robust inhibition on HBV replication and antigen expression.