miR-130a靶向调节PTEN基因对人乳腺癌细胞多柔比星敏感性影响的观察

Effects of miR-130a on chemosensitivity of human breast cancer cells to adriamycin by targeting PTEN

目的:探讨miR-130a对PTEN基因表达的调控,及其与乳腺癌MCF-7细胞株多柔比星耐药的关系。方法:利用miRNA芯片结合RT-qPCR的方法筛选到miR-130a在亲代敏感MCF-7/S细胞与多柔比星耐药细胞(MCF-7/Adr)中表达差异;通过靶基因预测软件预测PTEN与miR-130a基因的关系;通过miR-130a模拟物和抑制物转染实验结合四甲基偶氮唑蓝(MTT)法、RT-qPCR和蛋白质印迹法观察miR-130a的表达变化对乳腺癌细胞多柔比星耐药性的影响及其与PTEN基因表达间的关系。结果:与MCF-7/S相比,miR-130a在MCF-7/Adr中的表达水平明显增高(116 834.70±4 728.32)倍,t=19.035,P=0.000;但在多西紫杉醇耐药株MCF-7/Doc中的表达差异无统计学意义,t=0.703,P=0.521。MCF-7/S细胞转染miR-130amimics后,与阴性对照组相比,其miR-130a表达水平明显增高(17.686±1.057)倍,t=8.360,P=0.001;MCF-7/S空白对照组、阴性对照组和实验组对Adr的IC50分别为(0.240±0.099)、(0.181±0.060)和(0.606±0.164)mg/L;与阴性对照相比,转染miR-130amimics后细胞的IC50显著增高,t=5.200,P=0.007。同时PTEN mRNA和蛋白表达水平明显下调,PTEN mRNA水平是阴性对照组的(0.362±0.076)倍,t=2.927,P=0.043;蛋白表达水平是阴性对照组的(0.386±0.020)倍,t=20.713,P=0.000 3;而MCF-7/Adr转染miR-130ainhibi-tors后,其miR-130a表达水平是阴性对照组的(0.169±0.035)倍,t=12.036,P=0.000 2;MCF-7/Adr空白对照组、阴性对照组和实验组对Adr的IC50分别为(50.793±3.970)、(46.206±1.963)和(23.366±1.304)mg/L;与阴性对照相比,转染miR-130ainhibitors后细胞的IC50显著降低,t=16.784,P=0.000 7;同时PTEN mRNA和蛋白表达水平均明显上调,PTEN mRNA水平是阴性对照组的(3.564±0.336)倍,t=4.122,P=0.015;蛋白表达水平是阴性对照组的(2.019±0.268)倍,t=8.999,P=0.000 8。结论:miR130a可能通过靶定PTEN基因来增强乳腺癌细胞对多柔比星的耐药性。

OBJECTIVE：To investigate the effect of miR-130a on expression of PTEN,and their association with the chemosensitivity of MCF-7 breast cancer cells to adriamycin. METHODS： MicroRNA microarray and RT-qPCR were used to screen and identify the differential expression of miR-130a between MCF-7/Adr and MCF-7/S. The potential target genes of miR-130a were predicted by online bioinformatic softwares,and PTEN could be a specific target, miR-130a mim- ics were transfected into MCF-7/S cells,and miR-130a inhibitors were transfected into MCF-7/Adr, respectively. TheIC50 of these transfected cells to Adr was evaluated by MTT assay and the expression levels of PTEN mRNA and protein were analyzed by RT-qPCR and Western blot. RESULTS： Compared with the parental MCF-7/S, the relative expression level of miR-130a in MCF-7/Adr was significantly up-regulated to （116 834. 70±4 728.32） fold（t= 19. 035,P=0. 000）. There were no statistical differences in the level of miR-130a between MCF-7/S and MCF-7/Doc（t=0. 703,P= 0. 521）. Compared with control MCF-7/S, the relative expression level of miR-130a in miR-130a mimics-transfected MCF-7/S was significantly increased to （17. 686±1. 057） folds （t = 8. 360, P = 0. 001）, and drug resistance of mimics-transfected MCF-7/S cells to Adr became significantly increased[IC50 ：（0. 606±0. 164） mg/L vs （0. 181±0. 060） mg/L;t= 5. 200, P=0. 007]. The expression levels of PTEN mRNA and protein in mimics group were down-regulated to （0. 362±0. 076） folds （t=2.927,P=0.043） and to （0.386±0.020） folds （t=20.713,P=0.000 3）. Compared with control MCF-7/ Adr, the relative expression level of miR-130a in miR-130a inhibitors-transfected MCF-7/S was significantly decreased to （0. 169±0. 035） folds （t= 12. 036,P=0. 000 2）, and drug resistance of inhibitors-transfected MCF-7/Adr cells to Adr became significantly decreased[IC50 ： （23. 366±1. 304） mg/L vs （46. 206±1. 963） mg/L;t = 16. 784, P =0. 007]. The expression levels of PTEN mRNA and protein in inhibitors group were up-regulated to （3. 564±0. 336） folds （t=4. 122, P=0. 015） and to （2. 019±0. 268） folds （t=8. 999,P=0. 000 8）. CONCLUSION： miR-130a may confer breast cancer cell resistance to adriamycin by targeting PTEN.