吉林大学学报(医学版) ›› 2021, Vol. 47 ›› Issue (2): 360-368.doi: 10.13481/j.1671-587X.20210215

• 基础研究 • 上一篇    下一篇

miRNA-138-5p靶向抑制HIF-1α表达对乳腺癌MCF-7细胞顺铂耐药的逆转作用及其机制

黄果1,王佑权1(),陈娟2   

  1. 1.南华大学附属第二医院乳腺甲状腺外科,湖南 衡阳 421001
    2.南华大学附属第二医院放疗科,湖南 衡阳 421001
  • 收稿日期:2020-08-15 出版日期:2021-03-28 发布日期:2021-03-25
  • 通讯作者: 王佑权 E-mail:gfzq1410@163.com
  • 作者简介:黄 果(1988-),男,湖南省祁阳县人,主治医师,医学硕士,主要从事乳腺癌基础和临床方面的研究。
  • 基金资助:
    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)

Reverse effect of miR-138-5p targeted inhibition of HIF-1α expression on cisplatin resistance of breast cancer MCF-7 cells and its mechanism

Guo HUANG1,Youquan WANG1(),Juan CHEN2   

  1. 1.Departmet of Breast and Thyroid Surgery,Second Affiliated Hospital,South China University,Hengyang 421001,China
    2.Departmet of Radiotherapy,Second Affiliated Hospital,South China University,Hengyang 421001,China
  • Received:2020-08-15 Online:2021-03-28 Published:2021-03-25
  • Contact: Youquan WANG E-mail:gfzq1410@163.com

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100 μmol·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-1α mRNA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-1α mRNA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。

关键词: miR-138-5p, 缺氧诱导因子1α, 乳腺肿瘤, 细胞耐药

Abstract: 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-1α group(tranfected with miR-138-5p mimics and HIF-1α overexpression 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 100 μmol·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-1α mRNA in the cells were detected by Real-time 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 was 5.72. 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-1α mRNA 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-1α protein 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-1α cells 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-1α group 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-1α and down-regulates the expression levels of resistance-related proteins P-gp and MRP1, thereby enhances the sensitivity of MCF-7/DDP cells to DDP.

Key words: miR-138-5p, hypoxia inducible factor-1α, breast neoplamsms, drug resistance

中图分类号: 

  • R737.9