微RNA-99b靶向调节mTOR基因表达可逆转人乳腺癌细胞对多柔比星的耐药性

MicroRNA-99b reverses chemosensitivity of breast cancer cells to doxorubicin by targeting and regulating the expression of mTOR gene

目的:探讨微RNA-99b(microRNA-99b,miR-99b)通过靶向调节哺乳动物雷帕霉素靶分子(mammalian target of rapamycin,mTOR)基因的表达,在人乳腺癌细胞对多柔比星(doxorubicin,DOX)耐药过程中的作用及其可能的分子机制。方法:采用实时荧光定量PCR法检测miR-99b在亲本敏感细胞株MCF-7/S和DOX耐药细胞株MCF-7/DOX中的表达水平。应用生物信息学软件预测miR-99b的潜在靶基因,选取mTOR作为研究靶基因。将miR-99b模拟物和miR-99b抑制子分别转染MCF-7/S和MCF-7/DOX细胞(以转染阴性对照序列作为阴性对照组),然后采用实时荧光定量PCR和蛋白质印迹法检测备组细胞中miR-99b和mTOR的表达变化,并采用MTT法和FCM法分别检测miR-99b抑制表达或过表达后MCF-7/S和MCF-7/DOX细胞对DOX的敏感性以及细胞凋亡变化。同时,MTT法检测mTOR抑制剂雷帕霉素处理后MCF-7/DOX细胞对DOX的敏感性变化。结果:耐药细胞MCF-7/DOX中miR-99b的相对表达水平比敏感细胞MCF-7/S明显降低(P<0.001)。与阴性对照组或未转染对照组相比,miR-99b模拟物转染MCF-7/DOX细胞后,miR-99b的相对表达水平明显升高(P<0.001),DOX对细胞的半数抑制浓度(half maximal inhibitory concentration,IC_(50))明显降低(P<0.001),细胞凋亡率明显升高(P<0.01),而且mTOR的mRNA和蛋白表达水平均明显降低(P<0.01,P<0.001)。当MCF-7/S细胞转染miR-99b抑制子后,miR-99b的相对表达水平比阴性对照组明显降低(P<0.05),DOX的IC_(50)值明显升高(P<0.001),mTOR的mRNA和蛋白表达水平明显升高(P值均<0.001),而细胞凋亡率比未转染对照组明显降低(P=0.001)。在MCF-7/DOX细胞中分别加入100、400和1 600 nmol/L的mTOR抑制剂雷帕霉素处理后,DOX对细胞的IC_(50)值均明显降低(P值均<0.001)。结论:miR-99b可以靶向下调mTOR基因的表达,从而增加人乳腺癌细胞对DOX作用的敏感性。

Objective： To explore the effect of microRNA-99b （miR-99b） on drug-resistance of human breast cancer cells to doxorubicin （DOX） through targeting and regulating the expression of mammalian target of rapamycin （roTOR） gene, and to investigate its potential mechanism. Methods： The real-time fluorescent quantitative PCR （RFQ-PCR） were used to detect the expression level of miR-99b in human breast cancer MCF-7/S cells （as a parental sensitive cell line） and MCF-7/DOX cells （as a DOX-resistant cell line）. The potential target genes of miR-99b were predicted by bioinformatics software, and roTOR as a target gene was selected. MiR-99b mimics and inhibitors were transfected into MCF-7/DOX and MCF-7/S cells, respectively. The negative control sequences were transfected into these two cells as the negative controls. The expressions of miR-99b and mTOR after transfection were detected by RFQ-PCR and Westen blotting, respectively. The influences of miR-99b or mTOR inhibitor rapamycin on the sensitivity of breast cancer cells to DOX as well as the cell apoptosis were detected by MTT method and FCM, respectively. Results： The expression level of miR-99b in MCF-7/DOX cells was lower than that in MCF-7/S cells （P 〈 0.001）. As compared with the negative control group or the untransfection group, in MCF-7/DOX cells transfected with miR-99b mimics, the expression level of miR-99b was increased （P 〈 0.001）, the half maximal inhibitory concentration （ICso） value of DOX was significantly decreased （P 〈 0.001）, the cell apoptotic rate was increased （P 〈 0.01）, and the levels of mTOR mRNA and protein were down-regulated （P 〈 0.01, P 〈 0.001）. On the contrary, as compared with the negative control group or the untransfection group, in MCF-7/S cells transfected with miR-99b inhibitors, the expression of miR-99b was down-regulated （P 〈 0.05）, the ICs0value of DOX was significantly increased （P 〈 0.001）, the cell apoptotic rate was decreased （P = 0.001）, and the expression levels of mTOR mRNA and protein were up-regulated （both P 〈 0.001）. After treatment with three different doses of rapamycin （100, 400 and 1 600 nmol/L, respectively）, the ICso value of DOX for MCF-7/DOX cells was significantly reduced （P 〈 0.001） as compared with that of the negative control group （without rapamycin treatment）. Conclusion： MiR-99b can down-regulate sensitivity of human breast cancer cells to the expression of mTOR, thereby increasing the DOX.