Long hairpin RNA expression vector targeting HBV X gene inhibits replication of hepatitis B virus / 中华临床感染病杂志

ABSTRACT

Objective To investigate the effect of long hairpin RNA ( lhRNA) expression vector targeting HBV X gene ( HBx) on replication of hepatitis B virus ( HBV) and gene expression.Methods Four kinds of small interference RNAs ( siRNAs) were synthesized and lhRNA expression vectors targeting HBx were constructed.Four siRNA oligonucleotides and two lhRNA expression vectors were transfected into HepG2.2.15 cells.HBsAg, HBV DNA in culture supernatants and HBx mRNA in HepG2.2.15 cells were detected by time-resolved immunofluorometric assay, real-time quantitative PCR, and reverse transcription PCR, respectively.Negative sequence group or empty vector group was taken as the control.Independent-samples t test was performed to evaluate the inhibition effect on replication of HBV and gene expression. Results Compared with the negative control, HBsAg, HBV DNA level in culture supernatants and HBx mRNA in HepG2.2.15 cells were significantly decreased after siRNA-1 and siRNA-4 transfected at high concentrations (60 nmol/L or 90 nmol/L) (P<0.05), especially the HBsAg and HBV DNA levels in the siRNA-1 transfection group, which were significantly decreased at 24, 48 and 72 h after transfection ( P<0.05 or P <0.01 ) . Two lhRNA expression vectors ( pMD-HBxlh1 and pMD-HBxlh4 ) were successfully constructed and transfected into HepG2.2.15 cells, HBsAg and HBV DNA level in transfected cells was significantly lower than those in negative control (P<0.05).Conclusion The novel siRNA-1 is confirmed to target HBx gene and lhRNA expression vector targeting HBx can effectively inhibit the replication of HBV and expression of HBV gene.