RNA干扰对宫颈癌细胞中E6AP基因的影响及其意义

SiRNA inhibition of E6AP expression in cervical cancer cells

目的探讨RNA干扰（RNAi）对宫颈癌细胞系HeLa细胞E6AP基因表达及细胞增殖和凋亡的影响。方法实验分为3组：空白对照组（未经转染的HeLa细胞）、转染阴性对照的小干扰RNA（siRNA）组及转染特异性E6AP siRNA组。采用半定量RT-PCR技术、Western blot方法检测E6AP mRNA、蛋白表达水平，用四甲基偶氮唑蓝比色（MTT）法检测细胞增殖状况，用流式细胞术检测细胞凋亡。结果转染E6AP siRNA24、48、72h后，E6AP siRNA组E6AP mRNA表达水平与对照siRNA组比较下降33％、72％、70％。Western blot结果显示，在转染48及72h，E6AP蛋白表达下降38％、59％。MTT法检测显示，转染HeLa细胞24、48、72、96h后细胞生长速度明显降低，E6AP siRNA组与空白对照组（F=101．38，P〈0．05）、对照siRNA组（F=38．64，P〈0．05）比较，差异均有统计学意义。E6AP siRNA作用24、48、72h后E6AP siRNA组凋亡率明显高于对照siRNA组（F=41．48，P〈0．05）和空白对照组（F=86．36，P〈0．05），差异有统计学意义。结论体外合成的siRNA能有效封闭宫颈癌细胞中E6AP基因的表达，抑制细胞增殖，促进细胞的凋亡，为宫颈癌的基因治疗提供理论依据。

Objective To study the effect of RNA interference （RNAi） targeting E6AP on the proliferation and apoptosis of HeLa cells. Methods HeLa cells were cultured and divided into 3 groups： blank control group, cells transfected with nonsense siRNA（ small interference RNA） , and cells transfected with specific E6AP siRNA. The expressions of E6AP mRNA and protein were detected by RT-PCR and Western blot before and after the transfection respectively. Cell proliferation was determined by methylthiazolyl tetrazolium （MTT）. The cell apoptosis index was assessed by flow cytometry. Results Upon treatment with E6AP siRNA for 24,48 and 72 h, the expression level of E6AP mRNA decreased 33% ,72% and 70% than siRNA treated group. The protein expression levels in 48 h and 72 h E6AP siRNA groups decreased 38% , 59% comparing with those of the nonsense siRNA treated group （P 〈0. 05）. The proliferative capacity of cells transfectd with E6AP siRNA was significantly lower than blank control group （ F = 101.38, P 〈0. 05 ） and siRNA treated group （ F = 38. 64, P 〈 0. 05 ）. The apoptosis index of HeLa cells treated with E6AP siRNA was significantly higher than that of the nonsense siRNA （ F = 41.48, P 〈0. 05） and the blank control group （F = 86. 36, P 〈 0. 05 ）. Conclusion SiRNA targeting can effectively suppress the expression levels of E6AP mRNA, corresponding with a proliferation inhibition and an enhanced apoptosis of HeLa cells.