沉默miR-21对子宫内膜癌顺铂耐药细胞株Ishikawa/DDP的影响

Effect of silencing miR-21 on Ishikawa/DDP cisplatin resistant cell in endometrial carcinoma

目的:观察沉默miR-21对子宫内膜癌顺铂耐药细胞株Ishikawa/DDP的影响。方法:以Lipofectamine 2000介导miR-21抑制剂转染Ishikawa/DDP细胞株,同时设置阴性组和耐药组。采用反转录PCR检测miR-21、多药耐药基因MDR1、促凋亡基因Bax和抗凋亡基因Bcl-2的表达。采用蛋白印迹法检测多药耐药蛋白P-gp、促凋亡蛋白Bax和抗凋亡蛋白Bcl-2的表达。采用噻唑蓝比色法检测细胞对顺铂的敏感性。采用流式细胞术检测细胞凋亡情况。结果:与耐药组和阴性组比较,抑制剂组miR-21,MDR1和Bcl-2mRNA表达显著下调(P<0.01),而Bax mRAN表达显著上调(P<0.001);抑制剂组P-gp和Bcl-2蛋白表达显著低下调(P<0.05),而Bax蛋白表达显著上调(P<0.001)。与耐药组和阴性组比较,顺铂对抑制剂组的IC50值显著(P<0.001);顺铂对抑制剂组细胞的诱导凋亡率显著增加(P<0.001)。结论:沉默miR-21可显著提高Ishikawa/DDP细胞株对顺铂的敏感性,并促进细胞凋亡,其具体机制可能与下调MDR1,P-gp和Bcl-2表达,以及上调Bax表达有关。

Objective： To observe the effects of silencing miR-21 on Ishikawa/DDP cisplatin resistant cell in endometrial carcinoma. Methods： Ishikawa/DDP cisplatin resistant cell was transfected with miR-21 inhibitor by Lipofectamine 2000. At the same time, control group and drug resistant group were set up. The expression of miR-21, MDR1, Bax and Bcl-2 was detected by reverse transcription PCR. The protein expression of P-gp, Bax and Bcl-2 was detected by Western blot. The sensitivity of Ishikawa/DDP cells to cisplatin was detected by MTT. The cell apoptosis was detected by flow cytometry. Results： Compared with the drug resistant group and the control group, the mRNA expression of miR-21, MDR1, Bcl-2 in the inhibitor group significantly decreased （P〈0.01）. The mRNA expression of Bax in the inhibitor group significantly increased （P〈0.001）. The protein expression of P-gp, Bcl-2 in the inhibitor group significantly decreased （P〈0.05）. The protein expression of Bax in the inhibitor group significantly increased （P〈 0.001）. The IC50 value in the inhibitor group significantly decreased （P〈0.001）. The apoptosis rate in the inhibitor group significantly increased （P〈0.001）. Conclusion： Silencing miR-21 can significantly increase the sensitivity of Ishikawa/DDP cells to cisplatin and promote apoptosis. The specific mechanism may be related to down regulation of MDR1, P-gp, Bcl-2, as well as up regulation of Bax.