Selective Silencing of Viral Gene Expression in HPV6bE6-positive Cell Line Treated with Specific siRNA
- VernacularTitle:小干预RNA对人乳头瘤病毒6bE6基因的沉默作用
- Author:
Kejia ZHAO
;
Hao CHENG
;
Jiayi DING
;
Xing ZHANG
- Publication Type:Journal Article
- Keywords:
Papillomavirus,Human;
Gene silencing;
Small interfering RNA
- From:
Chinese Journal of Dermatology
2003;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the inhibition of pathogenic viral gene expression in HPV6bE6-positive cell line by specific siRNA, which might have great potential for clinical use. Methods B16 cells were transfected with recombinant plasmid pcDNA3.1(+)-GFP/HPV6bE6, and the positive cell clones were selected by fluorescence protein observation and RT-PCR. Four specific siRNAs, none of which shares homology with exons of known human genes, were designed and synthesized to target HPV6bE6 mRNA. Quantitative real-time PCR was performed to measure the inhibition rates of target gene expression by comparing HPV6bE6 mRNA concentrations before siRNA transfection with those after transfection. The inhibition rates of target gene expression with different siRNA concentrations of 0.2 nmol/L, 1 nmol/L, 10 nmol/L, 50 nmol/L, 150 nmol/L and with different treatment time at 24 h, 48 h, 72 h and 96 h after transfection were measured respectively. Results More than 90% reduction of HPV6bE6 mRNA was observed following treatment with HPV6bE6-siRNA, and HPV-negative cells were apparently unaffected by HPV6bE6-siRNA. The decrease of HPV6bE6 mRNA was maximal at 24 h after siRNA treatment and sustained for at least 4 days. The minimal level of siRNA to efficiently silence the homogeneous target gene HPV6bE6 was 1 nmol/L. Conclusion HPV6bE6-siRNA can efficiently and specifically silence target genes and may be developed as a potential therapeutic approach for HPV infection.
