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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 investigate the effect of microRNA-99b (miR-99b) on the expression of mammalian target of rapamycin (mTOR) gene in human breast cancer cells. (DOX) -mediated resistance and its possible molecular mechanism. Methods: The expression of miR-99b in parental MCF-7 / S cell line and DOX resistant cell line MCF-7 / DOX was detected by real-time fluorescence quantitative PCR. Bioinformatics software was used to predict potential target genes of miR-99b and mTOR was selected as target gene. The miR-99b mimics and miR-99b inhibitors were transfected into MCF-7 / S and MCF-7 / DOX cells (transfected with a negative control sequence as a negative control), and then using real-time fluorescence quantitative PCR and Western blot Detect the changes of miR-99b and mTOR in the prepared cells, and detect the sensitivity of MCF-7 / S and MCF-7 / DOX cells to DOX by MTT and FCM respectively Apoptosis changes. At the same time, the sensitivity of MCF-7 / DOX cells to DOX was detected by MTT assay after mTOR inhibitor rapamycin treatment. Results: The relative expression level of miR-99b in MCF-7 / DOX cells was significantly lower than that in MCF-7 / S cells (P <0.001). Compared with the negative control group or untransfected control group, the relative expression level of miR-99b was significantly increased (P <0.001) in MCF-7 / DOX cells transfected with miR-99b mimics, (P <0.01), and the apoptosis rate was significantly increased (P <0.01), and the mRNA and protein expression of mTOR were significantly decreased (P <0.01, P <0.001). The relative expression level of miR-99b in MCF-7 / S cells transfected with miR-99b inhibitor was significantly lower than that in the negative control group (P <0.05), and the IC 50 value of DOX was significantly increased (P <0.001) , mTOR mRNA and protein expression levels were significantly increased (P values were <0.001), while the apoptosis rate was significantly lower than the untransfected control group (P = 0.001). IC50 values of DOX were significantly decreased in MCF-7 / DOX cells treated with 100,400 and 1 600 nmol / L of rapamycin (all P <0.001). Conclusion: miR-99b can down-regulate the expression of mTOR gene and thus increase the sensitivity of human breast cancer cells to DOX.