摘要
目的:探讨miR-186-5p对乳腺癌MCF-7细胞化疗敏感性的影响及分子机制。方法:采用梯度5-fluorouridine(5-Fu)处理MCF-7细胞建立耐药细胞株MCF-7/5Fu,实时荧光定量PCR(qPCR)检测细胞中miR-186-5p表达;脂质体介导法分别将miR-186-5p inhibitor、miR-186-5p mimic及阴性对照转染至MCF-7细胞或MCF-7/5Fu细胞;qPCR检测细胞中miR-186-5p表达,CCK-8法检测细胞活性,流式细胞术检测细胞凋亡,双荧光素酶报告基因实验检测miR-186-5p和线粒体转录因子A(TFAM)的靶向关系,qPCR检测TFAM表达变化。结果:与MCF-7细胞比较,miR-186-5p在MCF-7/5Fu细胞中的表达水平显著下降(P<0.05)。与NC mimic组比较,MCF-7/5Fu细胞转染miR-186-5p mimic后,细胞中miR-186-5p表达水平升高(P<0.05),不同浓度5-Fu处理后细胞活性降低,IC50下降(P<0.05),细胞凋亡增加(P<0.05)。TFAM-WT中miR-186-5p inhibitor组MCF-7细胞荧光素酶活性较NC inhibitor组增强(P<0.05),而miR-186-5p mimic组MCF-7/5Fu细胞荧光素酶活性较NC mimic组下降(P<0.05),且MCF-7细胞中miR-186-5p inhibitor组TFAM的相对表达量较NC inhibitor组升高(P<0.05);MCF-7/5Fu细胞中miR-186-5p mimic组TFAM的相对表达量较NC mimic组显著下降(P<0.05)。结论:miR-186-5p通过靶向调控TFAM表达增强乳腺癌细胞对5-Fu化疗敏感性。
Objective:To investigate the effect of miR-186-5p on the chemotherapy sensitivity of breast cancer MCF-7 cells and its molecular mechanism.Methods:Drug-resistant cell line MCF-7/5Fu was established by culturing human breast cancer MCF-7 cells with increasing concentrations of 5-Fu.MiR-186-5p inhibitor,miR-186-5p mimic and their negative control(NC)miRNAs were transfected into MCF-7 or MCF-7/5Fu cells using Lipofectamine 2000.Cell viability was assessed using a Cell Counting Kit-8(CCK-8).Cell apoptosis analysis was performed using flow cytometry.Dual luciferase reporter assay was used to validate whether miR-186-5p targets mitochondrial transcription factor A(TFAM).Real-time fluorescence quantitative PCR(qPCR)was used to detect miR-186-5p and TFAM expression.Results:The miR-186-5p level in MCF-7/5Fu cells was decreased significantly compared with that in MCF-7 cells(P<0.05).The miR-186-5p level in MCF-7/5Fu cells was prominently increased after transfected with miR-186-5p mimic compared with the miR-NC mimic group(P<0.05).The cell activity and IC50 decreased while the apoptotic rate increased after treatment with different concentrations of 5-Fu(P<0.05).The relative luciferaseactivity was significantly higher in MCF-7cellscotransfected with miR-186-5p inhibitor and TFAM-WT than in cells transfected with NC inhibitorand TFAM-WT.However,the relative luciferaseactivity was lower in MCF-7/5Fucellscotransfected with miR-186-5p mimic and TFAM-WT than in cells transfected with NC mimic and TFAM-WT.Compared with the NC inhibitor group,miR-186-5p inhibitor elevated the mRNA expression of TFAM in MCF-7 cells(P<0.05);compared with the NC mimic group,miR-186-5p mimic reduced the relative expression of TFAM mRNA in MCF-7/5Fu cells(P<0.05).Conclusion:MiR-186-5p enhances the sensitivity of breast cancer cells to 5-Fu chemotherapy through targeted regulation of TFAM expression.
作者
陈曦
王亚莉
Chen Xi;Wang Yali(Department of Thoracic Oncology,E’dong Medical Group Central Hospital,Huangshi 435000,China;Personnel Division,E’dong Medical Group Central Hospital,Huangshi 435000,China)
出处
《广西医科大学学报》
CAS
2020年第11期1973-1979,共7页
Journal of Guangxi Medical University
基金
湖北省卫生健康委员会联合基金资助项目(No.WJ2019H020)。
