Targeted screening of SiRNA directed HBV polymerase gene for effective inhibition of HBV expression.
In order to screen potential mRNA locations of P gene in which targeting siRNAs can effectively inhibit HBV expression, 5 recombinant plasmids containing 4 targeting-specific siRNA fragments and a control were prepared and transfected into 2.2.15 cells respectively. The expression levels of HBx mRNA, HBs mRNA and HBc mRNA were detected by RT-PCR. The concentrations of the hepatitis B virus antigens, including HBsAg and HBeAg harvested from the culture supernatant of transfected 2.2.15 cells, were measured by ELISA. X protein was tested by Western blot. The results showed that four siRNAs against distinct mRNA locations of HBV polymerase gene had different inhibitory effects on their targeted mRNA. The plasmid-derived psiRNA1 and psiRNA2 could effectively inhibit the transcription and translation of HBs gene, whereas the inhibitory efficiency of psiRNA3, psiRNA4 for HBe gene was much higher than that of psiRNA1 and psiRNA2. In comparison to the rest of psiRNAs in this study, psiRNA4 was the most effective to suppress the transcription and translation of HBx. It is suggested that siRNA can be considered as a powerful therapeutic agent for reducing HBV expression. The siRNAs against HBV polymerase are effective largely depending on the location of targeted sites. To enhance inhibitory efficiency, hunting for high effective target in polymerase gene is necessary and feasible.
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Jinjian
J
Yao
Weiling
W
Yu
Ying
Y
Chang
Jinghua
J
Ren
Dong
D
Xu
Siyuan
S
Han
Jusheng
J
Lin
D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D015967:Gene Expression Regulation, Viral; D015684:Gene Products, pol / Q000096:biosynthesis / Q000235:genetics*; D016679:Genome, Viral; D006509:Hepatitis B / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection
D000998:Antiviral Agents; D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; D012333:RNA, Messenger; D034741:RNA, Small Interfering