microRNA-21对乳腺癌MCF-7/ADR细胞株多柔比星耐药性影响的研究

目的:探讨microRNA-21(miR-21)基因表达改变对乳腺癌细胞多柔比星化疗耐药的影响.方法:人工合成miR-21模拟序列或干扰序列,以脂质体为载体,转染乳腺癌细胞MCF-7及其多柔比星耐药细胞株MCF-7/ADR;应用荧光定量RT-PCR检测miR-21的表达；应用MTT法检测细胞转染前后对多柔比星的耐药性；应用流式细胞仪检测细胞凋亡；应用蛋白质印迹法检测PTEN、BAX及Bcl-2蛋白的表达.结果:MTT检测结果示,MCF-7及其耐药株MCF-7/ADR细胞多柔比星半数抑制浓度(IC50)分别为(0.28±0.03)和(17.5±0.12) μmol/L.miR-21表达上调,MCF-7细胞对多柔比星的IC50明显增高为(3.65±0.12) μmol/L;miR-21表达下调,MCF-7/ADR细胞对多柔比星的IC50明显降低为(7.53±0.11) μmol/L.流式细胞分析显示,miR-21下调后MCF-7/ADR细胞凋亡率明显增加.同时,细胞内PTEN、BAX蛋白表达水平增加,Bcl-2表达降低.结论:miR-21在乳腺癌化疗耐药中具有重要作用,抑制miR-21的表达可以逆转乳腺癌多柔比星耐药细胞株对多柔比星的耐药性.

Dysregulation effect of microRNA-21 gene on drug resistance in ADM-resistant MCF-7 breast cancer cells

OBJECTIVE: To explore the effects of miR-21 gene expression changes on doxorubicin(ADM) resistance in breast cancer cells.METHODS:Synthetic miR-21 sequence or analog interference sequences were transfected into MCF-7 cells and its doxorubicin(ADM) resistant cell line——MCF-7/ADR with lipofectamine 2000 vector.miR-21 level was determined by real time RT-PCR.Drug resistance of cells was detected by MTT assay.Cell apoptosis were assayed by flow cytometry.The expression of PTEN,BAX and Bcl-2 protein were analyzed by western blot.RESULTS: The IC50 of ADM in MCF-7 and MCF-7/ADR cells was(0.28±0.03) and(17.5±0.12) μmol/ L,respectively.Upregulation of miR-21,the IC50 of ADM in MCF-7 cells increased(3.65±0.12) μmol/L and downregulation of miR-21,the IC50 of ADM in MCF-7/ADR cells decreased to(7.53±0.11) μmol/L.Moreover,miR-21 inhibitor could enhance apoptosis in MCF-7/ADR cells,increase PTEN and BAX expression and decrease Bcl-2 protein expression.CONCLUSIONS: Dysregulation of miR-21 plays critical roles in the ADM resistance of breast cancer.The resistance on ADM of human ADM-resistant breast cancer cells is reversed by miR-21 gene down-regulating.