RNA干扰技术抑制HPV16 E6基因表达对宫颈癌细胞株CaSki增殖的影响

Effect of siRNA Inhibiting HPV16 E6 Gene Expression on Proliferation and Cell Cycle of Cervical Cancer Cell Line CaSki

目的观察HPV16 E6小干扰RNA(siRNA)能否抑制宫颈癌细胞生长,并探讨其作用机理。方法化学合成针对HPV16 E6的siRNA,借脂质体转染宫颈癌CaSki细胞,应用细胞计数的方法测定细胞生长曲线、活细胞率及细胞生长抑制率。运用实时荧光定量RT-PCR、流式细胞术检测转染后不同时间点细胞周期、HPV16 E6、p53、p21 mRNA及蛋白表达的变化。结果转染HPV16 E6 siRNA后,细胞生长明显受到抑制。流式细胞术结果显示并未将细胞阻滞在G1期。实时荧光定量RT-PCR显示,转染24h,E6 mRNA的表达比空白组降低了20.11倍(P<0.05),而p53、p21 mRNA的表达无明显变化。转染48h,E6蛋白表达明显下调,Pp53、P21蛋白表达相应地升高。结论HPV16 E6 siRNA可通过特异、高效地沉默宫颈癌细胞E6mRNA的表达,减少对野生型p53的降解,恢复P53蛋白的功能活性而发挥抑制宫颈癌细胞生长的作用。

Objective To observe whether human papillomavirus 16 （HPV16） E6-specific small interfering RNAs （siRNAs） can be employed to inhibit the growth of cervical cancer cell line, and to investigate the associated mechanism. Methods RNAi was performed using synthetic small interfering RNAs transferred into CaSki cell line by lipofectamine. The cell growth curves ,live cell ratio and inhibition ratio of cells were measured by using cell counting. At various time points of post-transfection, the distributions of cell cycle, the expression levels of HPV16 E6, p53, p21 mRNA and proteins were detected by using flow cytometry （FCM） and real-time quantitative reverse transcription-polymerase chain reaction （real-time RT-PCR）. Results The growth inhibition of E6 siRNA to CaSki cells was demonstrated after cells treated with E6 siRNA. No substantial G1 arrest was observed by FCM analysis. For 24 hours after cell transfection, the level of E6 mRNA was decreased by 20. 11 folds compared with control （P〈0.05）. However, p53 and p21 mRNA levels appeared unaffected. 48 hours after cell transfection, the expression level of E6 protein was efficiently decreased, but the P53 and P21 protein levels increased in comparison. Conclusions The inhibitory effect of HPV16 E6 siRNA to CaSki cell maybe due to specially and efficiently silence E6 mRNA expression, decrease the degradation of wild type P53 protein, and then recover the function activity of P53 protein.