siRNA-mediated inhibition of HBV replication and expression

AIM: RNA interference (RNAi) is a newly discovered phenomenon provoked by dsRNA. The dsRNA is initially cleaved by Dicer into 21-23 nt small interfering RNA (siRNA) and can then specifically target homologous mRNA for degradation by cellular ribonucleases. RNAi 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 investigated whether vector-based siRNA promoted by U6 (pSilencer1.0-U6) could efficiently inhibit HBV replication in cell culture. METHODS: pSilencer vectors with inserts targeting on different regions of HBV genome were constructed. These plasmids were co-transfected with pHBV3.8 into Huh-7 cells via lipofection and viral antigens were measured by ELISA. Viral RNA was analyzed by Northern blot. The mRNA of MxA and 2'-5'OAS was reverse transcribed and quantified by real-time PCR.RESULTS: Vector-based siRNA could potently reduce hepatitis 13 virus antigen expression in transient replicative cell culture. Furthermore, Northern blot analysis showed that viral RNA was effectively degraded, thus eliminating the messengers for protein expression as well as template for reverse transcription. Real-time PCR analysis of cellular MxA and 2'-5'OAS gene expression revealed that vectorbased siRNA did not provoke the interferon pathway which reassured the specificity of the vector-based RNA interference technique. CONCLUSION: Our results indicate that RNA interference may be a potential tool to control HBV infection.

AIM:RNA interference(RNAi)is a newly discovered phenomenon provoked by dsRNA.The dsRNA is initially cleaved by Dicer into 21-23 nt small interfering RNA(siRNA) and can then specifically target homologous mRNA for degradation by cellular ribonucleases.RNAi 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 investigated whether vector-based siRNA promoted by U6(pSilencer1.0-U6) could efficiently inhibit HBV replication in cell culture. METHODS:pSilencer vectors with inserts targeting on different regions of HBV genome were constructed.These plasmids were co-transfected with pHBV3.8 into Huh-7 cells via lipofection and viral antigens were measured by ELISA. Viral RNA was analyzed by Northern blot.The mRNA of MxA and 2'-5'OAS was reverse transcribed and quantified by real-time PCR. RESULTS:Vector-based siRNA could potently reduce hepatitis B virus antigen expression in transient replicative cell culture.Furthermore,Northern blot analysis showed that viral RNA was effectively degraded,thus eliminating the messengers for protein expression as well as template for reverse transcription.Real-time PCR analysis of cellular MxA and 2'-5'OAS gene expression revealed that vector- based siRNA did not provoke the interferon pathway which reassured the specificity of the vector-based RNA interference technique. CONCLUSION:Our results indicate that RNA interference may be a potential tool to control HBV infection.