小干扰RNA对人乳头状瘤病毒6bE7基因表达的沉默作用

Interference of HPV-6bE7 gene expression induced by smail interfering RNA

目的研究靶向人乳头状瘤病毒HPV-6b型E7基因的二聚体小干扰RNA（siRNA-HPV-66E7）对靶基因表达的沉默作用。方法建立并筛选稳定表达HPV-6bE7基因的B16和293T转染细胞株，用脂质体转染法将体外合成的siRNA-HPV-6bE7转染上述细胞株，采用实时荧光定量PCR分析靶基因HPV-6bE7的mRNA表达情况。结果用不同浓度的siRNA-HPV-6bE7转染细胞48h，50nmol／L浓度对B16细胞中靶基因表达的抑制作用最强（抑制率87．05％），1nmol／L抑制作用较低（9．14％）；而在293T细胞，10nmol／L的siRNA对靶基因表达的抑制效应最大（78．87％），1nmol／L仍有一定抑制作用（46．92％）。50nmol／LsiRNA-HPV-6bE7转染B16细胞后，靶基因的mRNA表达在24h内开始受抑制（32．47％），48h作用最强（74．72％），96h作用很低（8．91％）；25nmol／L和10nmol／L的siRNA转染293T细胞后，均在24h内起效（26．66％、20．31％），抑制作用至少能维持72h（65．93％、35．23％）。结论siRNA-HPV-6bE7对B16和293T细胞外源性靶基因表达均有较强的特异性沉默作用，在不同细胞株达到最大抑制效应的siRNA浓度不同，但时效曲线的变化趋势基本一致，siRNA均在24h内起效，48—72h达到高峰，抑制作用至少能维持72h。本研究结果为下一步在动物或临床进行siRNA干扰试验提供了实验依据。

Objective To investigate the specific interference of HPV-6bE7 gene expression induced by RNAi technique in B16 and 293T cell lines transfected with HPV-6bE7 gene. Methods B16 and 293T cells were transfected with recombinant plasmids pcDNA3.1 （ ＋ ）-GFP/HPV-6bE7, and the positive cell clones were selected by fluorescence protein observation and RT-PCR. One specific dicer siRNA targeted to HPV-6bE7 mRNA was designed and synthesized, sharing no homology with exons of known human genes. Quantitative real-time PCR was performed to measure the inhibitory rate of target gene expression by comparing HPV-6bE7 mRNA concentrations before and after siRNA transfection. Results 50 nmol/L siRNA-HPV-6bE7 elicited the highest level of gene silence in B16 which were transfected with siRNA after 48 h（87.05%）, Comparing to 293T, the highest gene silence was caused by 10 nmol/L（78.87% ）. The inhibition of HPV-6bE7 expression induced by 50 nmol/L siRNA in B16 was maximal at 48 h（74.72%）. While in 293T, the time of maximal inhibitory effect was at 48-72 h （65.93%, 41.16% ）. Conclusion The exogenous HPV-6bE7 expression could be significantly inhibited by treatment with specific siRNA in a dose and time-dependent manner in B16 and 293T cells which may provide a useful profile for further investigation of inhibition of HPV viral protein, and a promising treatment approach for HPV infection.