Inhibition of hepatitis B virus expression and replication by vector-based siRNAs targeting X gene

Objective To construct pSUPER vectors expressing short interfering RNAs (siRNA) targeting HBV X gene and to evaluate inhibitory effect of these siRNAs on HBV gene expression and replication in HepG2 2.2.15 cells.Methods Based on the sequence of HBV in HepG2 2.2.15 cells, double sequences targeting HBV X gene were cloned into pSUPER vector. The recombinant plasmids were cotransfected and monoclonal cell strains were screened in hygromycin media. Next- the HBsAg and HBeAg in supernatant of the screened cell strains were assayed with Abbott MEIA Kits, HBV DNA was determined by fluorescent quantitative PCR(FQ-PCR), and HBV mRNA were detected by RT-PCR.Results The pSUPER-X1 and pSUPER-X2 siRNA expression vectors were successfully constructed. The siRNAs could effectively inhibit the secretion of surface antigen and e antigen of HBV in HepG2 2.2.15 cells by 97% and 88% respectively, FQ-PCR results revealed that copy of HBV DNA in supernatant was significantly reduced and RT-PCR results showed that the HBV mRNA was markedly decreased. Whereas the irrelevant siRNA expression vector and control did not show any inhibitory effect on the expression and replication of HBV.Conclusion These results demonstrate that vector-based siRNAs targeting HBV X gene can stably inhibit HBV replication with great potency and specificity in vitro.