小干扰RNA对人乳头状瘤病毒6bE7基因表达的沉默作用

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-6bE7)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48 h,50 nmol/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%)。50 nmol/L siRNA-HPV-6bE7转染B16细胞后,靶基因的mRNA表达在24 h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。

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

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-PER.One specific dicer siRNA targeted to HPV-6bE7 mRNA was de- signed 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 transfecfion.Results 50 nmol/L siRNA-HPV-6bE7 elicited the highest level of gene si- lence in B16 which were transfected with siRNA after 48 h(87.05%),Comparing to 293T,the highest gene si- lence 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.