微小RNA-21对人乳腺癌细胞株紫杉醇耐药性的影响及其机制

Effects of miRNA-21 on paclitaxel-resistance in human breast cancer cells

目的：探讨微小RNA-21（miR-21）对人乳腺癌细胞MCF-7和SKBR-3及其紫杉醇耐药株细胞生物学活性的影响。方法：体外研究采用紫杉醇逐步加量诱导法建立人乳腺癌细胞紫杉醇耐药株；通过观察细胞形态的变化和检测耐药基因MDRl、BCRP、MRPl的表达以鉴定耐药细胞株；检测MCF-7和SKBR-3亲本及耐药细胞中miR-21和凋亡相关基因Bax、Bcl-2的表达水平；用miR-21抑制剂和模拟物分别转染MCF-7和SKBR-3耐药和亲本细胞株，MrIT法和流式细胞术检测转染miR-21抑制剂后细胞对紫杉醇的敏感程度和细胞周期、凋亡的变化，并检测耐药基因和凋亡相关基因的表达。结果：成功建立人乳腺癌细胞紫杉醇耐药株MCF-7／PR和SKBR-3／PR，耐药细胞株中耐药基因MDRl、BCRP、MRPl表达均升高，抗凋亡基因Bcl-2升高，凋亡基因Bax下降，miR-21表达升高（P〈0．05）；与对照组比较，miR-21抑制剂可提高耐药细胞株对紫杉醇的药物敏感性，增加细胞凋亡水平，增加细胞周期中G0／G1期细胞（P〈0．05），并可抑制耐药细胞株耐药基因的表达，降低Bcl-2／Bax比值（P〈0．05），而miR-21模拟物可刺激亲本细胞出现耐药细胞形态改变，并上调耐药基因的表达及Bcl-2／Bax比值（P〈0．05）。结论：乳腺癌紫杉醇耐药细胞株MCF-7／PR和SKBR．3／PR中miR-21表达上调；抑制miR-21表达可逆转人乳腺癌MCF-7／PR、SKBR-3／PR细胞株对紫杉醇化疗的耐药性。

Objective： To investigate the effects of miR-21 on paclitaxel-resistance in human breast cancer MCF-7/PR and SKBR-3/PR cells. Methods： Paclitaxel- resistant human breast cancer cell lines MCF-7/PR and SKBR-3/PR were established by stepwise selection in increasing concentration of paelitaxel. Cellular morphology, mRNA and protein level of MDR1, BCRP and MRP1 in MCF-7/PR and SKBR-3/PR ceils were determined. The expression of Bax, Bcl-2 and miR-21 in parental and paelitaxel-resistant cells was detected by RT-PCR and Western blotting. The synthetic miR-21 inhibitor or miR-21 mimic were transfected into MCF-7/PR, SKBR-3/PR and MCF-7, SKBR-3 ceils with Lipofectamine 2000. The miR-21 levels were determined by RT-PCR, and P-gp, Bcl-2 and Bax protein levels were examined by Western blotting. MTI＂ assay was used to measure the cell viability, and flow cytometry was performed to analyze the cell cycle and apoptosis. Results： The levels of MDR1, BCRP, MRP1, Bcl-2/Bax and miR-21 in MCF-7/PR and SKBR-3/PR cells were significantly higher than those in MCF-7 and SKBR-3 cells. The protein levels of P-gp, Bcl-2 were up- regulated, and Bax was down-regulated compared with parental cells. MiR-21 was significantly down-regulated after miR-21 inhibitor was transfected; and the levels of MDR1, BCRP, MRP1 and Bcl-2/Bax （P 〈 0.05） were also down-regulated. MiR-21 inhibitors significantly suppressed G0/G1 transition of the cell cycle, and induced cell apoptosis in MCF-7/PR and SKBR-3/PR ceils. MTF results showed that miR-21 inhibitors induced sensitivity of MCF-7/PR and SKBR-3/PR cells to paclitaxel. And miR-21 mimic can increase the expression of MDR1, Bcl-2/Bax and change cell morphology from parental ceils to resistant cells. Results： The established MCF-7/PR and SKBR-3/PR breast cancer cells show typical muhidrug resistance characteristics, which can be used as the model for drug resistance study. Down-regulated miR-21 expression in MCF-7/PR and SKBR-3/PR breast cancer cells can enhance cell sensitivity to paclitaxel.