MiR433靶向调控Notch1逆转人乳腺癌细胞对多西他赛的耐药性

MiR-433 reverses chemoresistance to docetaxel by targeting Notch1 in breast cancer cells

目的探讨miR-433在多西他赛对乳腺癌耐药机制中的作用。方法将MCF-7乳腺癌细胞暴露于逐渐增强浓度的多西他赛中建立对多西他赛耐药的乳腺癌细胞(MCF-7/DOX),并用miR-433抑制剂及对照剂转染MCF-7/DOX细胞,采用酶标仪(Bio Tek)测量吸光度和FACS流式细胞仪检测来评估miR-433的功能和作用;通过生物信息学分析、荧光素酶报告基因测定法等技术确定miR-433的下游靶标。结果实时定量PCR结果显示miR-433在MCF-7/DOX细胞中的表达下调;细胞活力测定结果表明miR-433抑制剂能促进产生多西他赛耐药,并减弱MCF-7细胞的凋亡,而miR-433过表达能增强多西他赛的敏感性并且诱导MCF-7/DOX细胞凋亡。荧光素酶报告基因测定结果显示在miR-433表达显著下调的MCF-7细胞中,Notch1的m RNA和蛋白质表达水平明显增强;miR-433mimics转染的MCF-7/DOX细胞与对照组(mimics-Ctrl)相比,Notch1的m RNA和蛋白表达显着降低。结论 miR-433可通过抑制Notch1的表达来逆转乳腺癌对多西他赛的耐药性,继续发挥药物的有效性。

Objective To investigate the role of miR-433 in chemoresistance to docetaxel in breast cancer cells. Methods A docetaxel-resistant MCF-7 breast cancer cell line（MCF-7/DOX） was established by exposure of parental MCF-7 cells to progressively increased docetaxel concentrations. The functional role of miR-433 was investigated by assessing the changes in viability and apoptosis of the cells transfected with a miR-433 inhibitor or a miR-433 mimics. The downstream targets of miR-433 were determined by bioinformatics analysis, cell transfection and luciferase reporter assay. Results Quantitative real-time PCR analysis showed that miR-433 was down-regulated in MCF-7/DOX cells. Transfection of the cells with the miR-433 inhibitor obviously enhanced chemoresistance to docetaxel and attenuated cell apoptosis in MCF-7 cells; miR-433 overexpression significantly increased the sensitivity to docetaxel and promoted apoptosis in MCF-7/DOX cells. Luciferase reporter assay showed that the down-regulation miR-433 expression was associated with significantly increased expressions of Notch1 at both m RNA and protein levels in MCF-7 cells. Compared with the control cells, Mc F-7/DOX cells transfected with miR-433 mimics exhibited significantly decreased m RNA and protein expressions of Notch1. Conclusions miR-433 may reverse chemoresistance to docetaxel by targeting Notch1 in breast cancer cells.