Establishment of a screening system for selection of siRNA target sites directed against hepatitis B virus surface gene.
To study the inhibitory effects of plasmid-derived small interfering RNA (siRNA) and synthetic siRNA on the expression of the hepatitis B virus surface (HBs) gene, three plasmid-derived siRNAs and one synthetic siRNA that complement the coding region of the HBs gene were prepared. The HBs expression plasmid pHBs-EGFP was also constructed. HeLa cells were co-transfected with pHBs-EGFP and the above siRNAs. The HBs mRNA quantities were measured by reverse-transcription PCR, and the level of HBs-EGFP fusion protein was quantified by fluorescent microscope. The concentrations of the hepatitis B virus surface antigen (HBsAg) derived from the culture supernatant of transfected HepG2.2.15 cells were measured by an enzyme-linked immunosorbent assay (ELISA) kit. The results showed that the three plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the quantities of HBs mRNA and protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBs expression. It can inhibit HBs-EGFP expression by 63.3% and suppress HBs mRNA by 75.6%. To further substantiate the above observations, psiRNA1 was transfected into HepG2.2.15 cells (an HBV secreting cell line). The transfections resulted in almost complete blockage of HBsAg production, whereas control vector-transfected cells secreted high levels of HBsAg 7 days post-transfection. In conclusion, our data suggests that RNA interference (RNAi) is an efficient approach for reducing the level of HBs transcripts and proteins and for suppressing HBsAg production.
Zhou
Xiu-Min
XM
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Lin
Ju-Sheng
JS
Shi
Yi
Y
Tian
De-An
DA
Huang
Huan-Jun
HJ
Zhou
He-Jun
HJ
Jin
You-Xin
YX
D001483:Base Sequence; D005240:Feasibility Studies; D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D006367:HeLa Cells; D006509:Hepatitis B / Q000235:genetics / Q000628:therapy; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics*; D016415:Sequence Alignment / Q000379:methods; D017423:Sequence Analysis, RNA / Q000379:methods*
D006514:Hepatitis B Surface Antigens; D034741:RNA, Small Interfering