Research On RNA Interference's Inhibition Of Hepatitis B Virus Gene Expression And Replication In HepG2-N10 Cells

AIM: RNA interference refers to the phenomenon of sequence-specific degradation of homologous mRNA induced by double-stranded RNA. It has been successfully utilized to down-regulate the endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study,we evaluated the effect of vector-based small interfering RNAs promoted by U6 (pSilencer2.0-U6) inhibit HBV replication in cell culture.METHODS: Several sequences targeting on different regions of HBV genome were inserted into pSilencer vectors. These expression plasmids were transfected into HepG2-N10 cells, a cell line which stably express HBsAg,HBeAg and adw2 subtype Dane Particles. Viral antigens were measured by ELISA. Viral mRNA was analyzed by RT-PCR. The cccDNAs secreted into the culture media were measured by quantitative real-time PCR.RESULTS: Vector-based RNA interference could potently reduce HBsAg and HBeAg expression in cell culture. Furthermore,RT-PCR analysis showed that viral mRNAs were effectively degraded,thus eliminating the messengers for protein expression as well as templates for reverse transcription. Quantitative Real-time PCR analysis of cccDNAs revealed that vector-based RNA interference can inhibit HBV replication efficiently.CONCLUSION: Our results indicated that RNA interference can inhibit HBV gene expression and replication, and it may have therapeutic effect of HBV.