摘要
目的:探讨miRNA-138-5p过表达对人乳腺癌MCF-7细胞顺铂(DDP)耐药的影响,并阐明其可能机制。方法:采用浓度梯度递增法诱导MCF-7细胞,建立DDP耐药细胞株MCF-7/DDP,再将miR-138-5p mimics或HIF-1α过表达质粒分别或同时转染至MCF-7/DDP细胞中。将细胞分为阴性对照组(NC组,转染miR-138-5p mimics阴性对照)、miR-138-5p mimics组(转染miR-138-5p mimics)、miR-138-5p mimics+Vector组(共转染miR-138-5p mimics和空载质粒)和miR-138-5p mimics+HIF-1α组(共转染miR-138-5p mimics和HIF-1α过表达质粒),另选未经任何转染的MCF-7/DDP细胞作为空白对照组。采用不同浓度(0、10、20、40、80和100μmol·L~(-1))DDP处理细胞24 h,MTT法检测细胞增殖活性,并计算半数抑制浓度(IC50)和耐药指数(RI);实时荧光定量聚合酶链式反应(qRT-PCR)法检测细胞中miR-138-5p和HIF-1αmRNA表达水平;流式细胞术检测细胞凋亡率;Western blotting法检测细胞中HIF-1α、P-gp和MRP1蛋白表达水平;采用TargetScan软件预测miR-138-5p与HIF-1α靶向结合位点,并采用双荧光素酶报告系统验证两者靶向关系。结果:与亲本MCF-7细胞比较,耐药细胞株MCF-7/DDP对DDP的IC50值明显升高(P<0.01),且RI值为5.72。与亲本MCF-7细胞比较,耐药细胞株MCF-7/DDP中miR-138-5p表达水平明显降低(P<0.01),而HIF-1αmRNA和蛋白表达水平明显升高(P<0.01)。与空白对照组或NC组比较,miR-138-5p mimics组细胞中miR-138-5p表达水平和细胞凋亡率明显升高(P<0.01),HIF-1α蛋白表达水平和细胞增殖活性明显降低(P<0.01)。双荧光素酶报告系统实验,与NC组比较,miR-138-5p mimics组野生型HIF-1α细胞中荧光素酶活性明显降低(P<0.01)。与miR-138-5p mimics+Vector组比较,miR-138-5p mimics+HIF-1α组细胞增殖活性明显升高(P<0.05),细胞凋亡率明显降低(P<0.01),细胞中HIF-1α、P-gp和MRP1蛋白表达水平明显升高(P<0.05)。结论:miRNA-138-5p通过靶向抑制HIF-1α表达和下调耐药相关蛋白P-gp和MRP1表达水平,增强MCF-7/DDP细胞对DDP的敏感性。
Objective:To investigate the effect of miRNA-138-5p overexpression on cisplatin(DDP)resistance of the human breast cancer MCF-7 cells,and to elucidate its possible mechanism.Methods:The MCF-7 cells were induced by concentration gradient increasing method to establish the DDP cell strain MCF-7/DDP.Then miR-138-5p mimics or hypoxia inducible factor-1α(HIF-1α)overexpression plasmid were transfected into the MCF-7/DDP cells separately or simultaneously.The MCF/DDP cells were divided into negative control group(NC group,transfected with miR-138-5p mimics negative control),miR-138-5p mimics group(transfected with mir-138-4p mimics),miR-138-5p mimics+Vector group(transfected with miR-138-5p mimics and empty plasmid)and miR-138-5p mimics+HIF-1αgroup(tranfected with miR-138-5p mimics and HIF-1αoverexpression plasmid);and the other MCF-7/DDP cells without transfection were used as blank control group.The cells were treated with different concentrations(0,10,20,40,80 and 100μmol·L-1)of DDP for 24 h and the proliferation activity of cells was detected by MTT,and half inhibitory concentration(IC50)and drug resistance index(RI)were calculated.The expression levels of miR-138-5p and HIF-1αmRNA in the cells were detected by Realtime fluorescence quantitative polymerase chain reaction(qRT-PCR);flow cytometry was used to detect the apoptotic rate of cells;Western blotting method was used to detect the expression levels of HIF-1α,P-gp and MRP1 proteins in the cells.TargetScan software was used to predict the target binding sites of miR-138-5p and HIF-1α,and the dual luciferase reporter system was used to verify the targeting relationship between miR-138-5p and HIF-1α.Results:The IC50 of drug-resistant cell strain MCF-7/DDP for DDP was significantly higher than that of its parent MCF-7 cells(P<0.01),and the RI value was5.72.Compared with parental MCF-7 cells,the miR-138-5p expression level in drug-resistant cell strain MCF-7/DDP was markedly reduced(P<0.01),while the HIF-1αmRNA and protein expression levels were significantly increased(P<0.01).Compared with blank control group and NC group,the miR-138-5p expression level and apoptotic rate in miR-138-5p mimics group were significantly increased(P<0.01),while the HIF-1αprotein expression level and the proliferation activity of cells were significantly decreased(P<0.01).Compared with NC group,the luciferase activity of wild-type HIF-1αcells in miR-138-5p mimics group was markedly decreased(P<0.01).Compared with miR-138-5p mimics+Vector group,the proliferation activity of cells in miR-138-5p mimics+HIF-1αgroup was significantly increased(P<0.05),the apoptotic rate of cells was significantly decreased(P<0.01),and the expression levels of HIF-1α,P-gp and MRP1 proteins in the cells were significantly increased(P<0.05).Conclusion:miRNA-138-5p inhibits the expression of HIF-1αand down-regulates the expression levels of resistance-related proteins P-gp and MRP1,thereby enhances the sensitivity of MCF-7/DDP cells to DDP.
作者
黄果
王佑权
陈娟
HUANG Guo;WANG Youquan;CHEN Juan(Departmet of Breast and Thyroid Surgery,Second Affiliated Hospital,South China University,Hengyang 421001,China;Departmet of Radiotherapy,Second Affiliated Hospital,South China University,Hengyang 421001,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2021年第2期360-368,共9页
Journal of Jilin University：Medicine Edition
基金
湖南省科技厅临床医疗技术创新引导项目(2018SK51513)
湖南省科技厅自然科学基金面上项目(2018JJ2354)
湖南省科技厅、湖南省卫健委乳甲疾病防治临床医学研究中心科研项目(2018SK4001)。
