RNAi抑制MCF-7细胞survivin基因及对多西他赛敏感性影响

Study of the RNA interference silencing of survivin gene and chemosensitivity to docetaxel in MCF-7 cells

目的利用RNAi技术抑制乳腺癌细胞株MCF-7sur-vivin基因,观察MCF-7细胞株的细胞凋亡率、survivin蛋白表达及对多西他赛(docetaxel)药物敏感性的变化。方法以培养的乳腺癌MCF-7细胞株为体外研究模型,设对照组、空载体组及RNAi组进行实验。(1)半定量RT-PCR、Western blot法检测survivin mRNA、蛋白在各组细胞中的表达;(2)用流式细胞术分析各组细胞周期及细胞凋亡率的影响;(3)用MTT法分析多西他赛对各组细胞存活率作用的浓度。结果(1)RT-PCR检测结果显示,RNAi组survivin扩增条带(134bp)明显弱于空载体组及对照组,mRNA相对含量较空载体组明显下降(75.4%),差异有统计学意义(P<0.05);Western blot检测空载体组与对照组survivin蛋白条带明显存在,且基本一致,而RNAi组同样位置的条带基本消失(79.8%);(2)RNAi组细胞阻滞在G0/G1期,与空载体组和对照组分别比较,差异有统计学意义(P<0.05);(3)MTT比色法检测结果显示,不同浓度多西他赛处理后对照组、空载体组、RNAi组的IC50分别为(124.7±0.21)、(116.9±0.25)、(15.2±2.8)μg.L-1,RNAi组MCF-7细胞对多西他赛的药物敏感性增高(P=0.000)。结论RNAi明显抑制了survivin在转录及翻译水平的表达;RNAi抑制survivin基因表达,能明显提高乳腺癌MCF-7细胞对多西他赛的药物敏感性。

Aim Through interfering the expression of survivin with short hairpin RNA （shRNA） technology, to investigate the effect of downregulation of survivin on cell apoptosis and chemosensitivity to docetaxel in breast cancer MCF-7 ceils. Methods （ I ） Using MCF-7 ceils as a model system, three groups were set up transfected with lipofectamine, RNAi control plasmid and survivin RNAi plasmid, respectively. The expression of survivin in MCF-7 ceils was measured at transcriptional and translational level by using RT-PCR and Western blot methods. （2） The effect on the cell cycle and apoptosis was analyzed with flow cytometry. （3） The viability of ceils applied with different doses of ducetaxel was determined by using the method of 3- [ 4, 5-Dimethyhhiazol-2-yl ]-2, 5-diphenyhetrazolium bromide reduction（ MTT method）. Results （ 1 ） RT- PCR and Western blot demonstrated that survivin ex- pression was significantly decreased by transfection with RNAi targeting plasmid; the expression proportion was reduced by nearly 75.4% and 79.8% （P 〈 0. 05）. （2） RNAi-treated ceils were arrested at G0/ G1 phase. It was satistifically significant from lipo- fectamin control and control at a level of P 〈 0. 05. （3） The ICs0 was calculated as 124. 7±0. 21.116. 9 ± 0. 25.15.2 ± 2. 8 μg · L^-1 respectively by MTT assay after dealing with different concentrations of docetaxel. Docetaxel significantly strengthened the chemosensitivity of the ceils in a dose-dependent manner （P = 0. 000）. Conclusions These results showed that by inducing RNAi-targeting plasmid, survivin expression could be effectively decreased at both transcriptional and translational level. The reduction of surviving expression exerted significant effects on chemosensitivity of MCF-7 ceils to docetaxel.