靶向抑制Survivin基因对子宫内膜癌细胞增殖与凋亡影响

Effect of targeted Survivin gene inhibition on proliferation and apoptosis of endometrial cancer cells

目的肿瘤形成与细胞凋亡异常密切相关，Survivin基因是凋亡抑制蛋白家族中最强细胞凋亡抑制因子之一。本研究利用特异性小干扰RNA（small interfering RNA，siRNA）技术抑制人RL95-2子宫内膜癌细胞Survivin基因表达，探讨沉默Survivin基因对人子宫内膜癌细胞增殖和凋亡影响。方法合成靶向Survivin基因siRNA特异性序列，脂质体转染人RL95—2子宫内膜癌细胞，转染6h后，荧光显微镜和流式细胞仪评估细胞转染效率。转染48h后，应用qPCR检测SurvivinmRNA水平的变化，蛋白质印迹法检测Survivin蛋白的表达。细胞转染72h后，CCK-8检测细胞增殖情况，流式细胞仪检测细胞凋亡变化。结果脂质体介导siRNA转染效率达到61．3％（6130／10000）。转染48h后，qPCR检测结果显示，实验组SurvivinmRNA表达量为0．240±0．023，对照组为0．998±0．030，空白组为1．006±0．022。转染48h后，蛋白质印迹法检测结果显示，实验组Survivin蛋白相对表达量为0．371±0．089，对照组为0．639±0．127，空白组为0．619±0．094，各分组Survivin蛋白相对表达量比较，差异有统计学意义，F=6．100，P=0．036。细胞转染72h后，实验组吸光度值为0．558±0．088，对照组为1．047±0．128，空白组为1．116±0．159，各分组吸光度值比较，差异有统计学意义，F=17．075，P=0．003。转染72h后，实验组细胞早期凋亡率为（18．9±3．44）％，对照组为（3．7±1．09）％，空白组为（3．1±1．22）％，各分组细胞早期凋亡率比较，差异有统计学意义，F=49．728，P=0．018。结论应用siRNA技术沉默Survivin基因表达，可降低RL95—2子宫内膜癌细胞生长速度，抑制肿瘤细胞增殖，诱导细胞凋亡。Survivin基因可作为抗子宫内膜癌治疗潜在靶点。

OBJECTIVE Tumor formation is closely related to cell apoptosis,and Survivin gene is one of the stron gest apoptotic inhibitors in apoptotic inhibitor family,the specific small interfering RNA（siRNA） was used to silence the expression of Survivin gene of RL95-2 endometrial cancer cells to investigate the effect of Survivin gene inhibition on pro liferation and apoptosis of endometrial cancer cells in the study. METHODS According to the related foreign literatures, the Survivin gene targeted specific siRNA sequences was synthesized and transfected into endometrial cancer cells by lipidosome. Six hours after transfection, transfection efficiency was detected and analyzed by fluorescence microscope and flowcytometry. Forty-eight hours after transfection,the expression of Survivin mRNA was measured by using qPCR and the expression of Survivin protein was measured by using Western Blot. Seventy two hours after transfection,cell proliferation were detected by CCK-8;72 hours after transfection,the cell apoptosis was tested by flowcytometry. RESULTS The transfection rate reached 61.3 % （6 130/10 000）. Forty-eight hours after transfection, the results of qPCR showed that the expression quantity of Survivin mRNA in the experimental group was 0. 240i0. 023 ,the expression of Survivin mRNA in the control group was 0. 998!0. 030,and the expression of Survivin mRNA in the blank group was 1. 006±0. 022. Fortyeight hours after transfection, the results of western bolt showed that the relative Survivin protein expression in the experimental group was 0. 371±0. 089, the relative Survivin protein expression in the control group was 0. 639 ± 0. 127, and the relative Survivin protein expression in the blank group was 0. 619±0. 094, the difference was statistically significant, F= 6. 100,P= 0. 036. Seventy-two hours after transfection, the results of CCK-8 assay showed that the absorbance in the experimental group was 0. 558 ±0. 088, the absorbance in the control group was 1. 047± 0. 128, and the absorbance in the blank group was 1.116± 0. 159, the difference was statistically significant, F = 17. 075, P = 0. 003. Seventy-two hours after transfection, the early apoptosis rate in the experimental group was （18.9 ± 3.44）%, the early apoptosis rate in the control group was （3.7±1.09） % ,and the early apoptosis rate in the blank group was （3.1±1.22） % ,the difference was statistically significant,F= 49. 728,P=0. 018. CONCLUSIONS Utilizing the siRNA technology can decrease the expression of Survivin gene, reduce the speed of cell proliferation,inhibit the tumor cell proliferation, and induce apoptosis of endometrial cancer cells. Sunrivin may be a potential target for antitumor therapy.