摘要
目的探讨miR-34a对人乳腺癌细胞系MCF-7多柔比星(Adr)耐药性的影响及其潜在分子机制。方法用低浓度逐步加量诱导法,建立MCF-7的Adr耐药细胞系(MCF-7/Adr),用miRNA芯片结合RT-qPCR筛选并验证miRNA在MCF-7/Adr及MCF-7细胞中的表达差异;用生物信息学软件对miR-34a进行基因靶向预测;通过miR-34a模拟物(mimic)和抑制物(inhibitor)转染实验结合MTT、实时荧光定量PCR(RT-qPCR)观察miR-34a的表达变化对细胞耐药性的影响,western blot检测转染后Notch1蛋白表达水平。结果成功构建MCF-7/Adr耐药亚系;与MCF-7细胞相比,MCF-7/Adr中有156个差异表达miRNA;其中miR-34a的表达水平明显降低(t=11.597,P=0.000)。与阴性对照组相比,转染miR-34a mimic后MCF-7/Adr细胞miR-34a表达水平增高(t=8.013,P=0.001),且增加了对Adr的敏感性(t=18.160,P=0.000);转染miR-34a inhibitor后MCF-7细胞miR-34a的表达水平降低(t=9.979,P=0.000),且降低了对Adr的敏感性(t=4.130,P=0.009)。靶基因预测结果显示,Notch1为miR-34a的特异性靶基因。western blot结果表明,与空白对照组和阴性对照组相比,MCF-7/Adr细胞转染miR-34a mimic后,Notch1蛋白的表达水平下降(F=64.949,P=0.000)。MCF-7细胞转染miR-34a inhibitor后,Notch1蛋白的表达水平升高(F=10.938,P=0.010)。结论 MCF-7/Adr及MCF-7细胞miRNA表达谱存在差异;miR-34a参与调节细胞对Adr的耐药过程,Notch1基因可能是调控靶基因之一。
Objective：To explore the effect of miR-34a on the resistance of human breast cancer cell line MCF-7 to adriamycin （Adr） and its potential molecular mechanism. Methods：An Adr-resistant subline （MCF-7/Adr） was developed by exposing human breast cancer parental cell （MCF-7） to gradually increasing concentrations of Adr in vitro. MicroRNAs microarray and RT-qPCR were conducted to analyze the differentially expressed miRNAs in MCF-7/Adr compared with its parental MCF-7 cells. The potential target gene of miR-34a was predicted by online bioinformatic software. Both the MiR-34a mimic and inhibitor were transfected into MCF-7/Adr and MCF-7 cells, respectively, and the effects of the changes of miR-34a expression levels on the drug resistance were observed in the transfected cells by MTT and RT-qPCR assays. The level of Notch1 protein was determined by western blot. Results：MCF-7/Adr subline was successfully established. Compared to MCF-7, there were 156 differentially expressed miRNAs in MCF-7/Adr cell, among which miR-34a showed significantly low expression （t=11.597, P=0.000）. The expression level of miR-34a in miR-34a mimic transfected MCF-7/Adr elevated compared with negative control, and the sensitivity of the cells to Adr increased （t=8.013,P=0.001 and t=18.160, P=0.000）, while the expression level of miR 34a reduced in miR 34a inhibitor transfected MCF 7 and the sensitivity of the cells to Adr decreased （t=9.979, P=0.000 and t=4.130, P=0.009）. Bioinformatics prediction indicated Notch1 gene should be the specific target gene of miR-34a. Western blot analysis showed that the level of Notch1 protein decreased in miR-34a mimic-transfected MCF-7/Adr （F=64.949, P=0.000） and increased in miR-34a inhibitor-transfected MCF-7 （F=10.938, P=0.010）, compared with blank control and negative control. Conclusion：MCF-7/Adr resistant subline shows different miRNA expression profile as compared with its parental cell line. miR-34a is involved in the resistance formation of breast cancer cells to Adr and Notch1 may be one of the target genes in regulation of miR-34a.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2014年第1期15-19,共5页
Chinese Journal of Clinical Laboratory Science
基金
国家自然科学基金面上项目(81272470)
