siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection.
The main objective was to determine whether a pool of small interfering RNAs (siRNAs) targeting different regions of hepatitis B virus surface antigen (HBsAg) efficiently inhibits hepatitis B virus (HBV) infection. siRNAs targeting different regions of HBsAg were transfected into HBV-producing HepG2.2.15 cells and at 72 h post-transfection, the culture medium was collected for ELISA to determine HBsAg, while total RNA was isolated from the cells for real-time PCR. Three siRNA sequences that efficiently inhibited HBV infection were converted into small hairpin RNAs (shRNAs) and then cloned into a single plasmid psiSTRIKE driven by a single U6 promoter. These shRNA expressing plasmids were tested for HBsAg gene silencing in HepG2.2.15 cells. A pool of siRNAs targeting HBsAg efficiently inhibited HBV replication and antigen expression when transfected into HepG2.2.15 cells, compared with the use of single siRNA. Similarly, the plasmid encoding three different shRNAs driven by a single U6 promoter was more effective in silencing HBsAg at DNA, mRNA and protein levels compared with the plasmid encoding single shRNA. No apoptotic change was observed in the cells when the plasmid was transfected at a dose of 0.5-2 microg/1 x 10(6) cells after complex formation with Lipofectamine LTX. Furthermore, transfection with siRNA or shRNA did not increase interferon-gamma (IFNs-gamma) release, suggesting no induction of IFN response. In conclusion, a pool of chemically synthesised siRNAs as well as the shRNA expression plasmid encoding multiple shRNAs targeting different regions of HBsAg showed high gene silencing in HepG2.2.15 cells.
Chen
Yong
Y
Huaian 4th People's Hospital, Jiangsu, PR China.
Mahato
Ram I
RI
D017209:Apoptosis; D045744:Cell Line, Tumor; D004279:DNA, Viral; D004797:Enzyme-Linked Immunosorbent Assay; D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D006509:Hepatitis B / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics; D006801:Humans; D010957:Plasmids; D034741:RNA, Small Interfering / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D014162:Transfection / Q000379:methods; D014779:Virus Replication / Q000235:genetics*
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D034741:RNA, Small Interfering