POLG抑制剂阻碍线粒体生物合成抑制三阴性乳腺癌细胞迁移和侵袭  

POLG inhibitor suppresses migration and invasion of triple-negative breast cancer cells via blocking mitochondrial biogenesis

作　　者：刘行 樊双琴 晏小敏 赵仕杰 王荣[1] 沈祥春[1] 周雪[1] 张玥[1] 陈妍[1] Liu Xing;Fan Shuangqin;Yan Xiaomin;Zhao Shijie;Wang Rong;Shen Xiangchun;Zhou Xue;Zhang Yue;Chen Yan(Key Laboratory of Optimal Utilization of Natural Medicinal Resources,Guizhou Medical University,Anshun 561113)

机构地区：[1]贵州医科大学天然药物资源优效利用重点实验室,安顺561113

出　　处：《安徽医科大学学报》2024年第10期1720-1728,共9页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金项目(编号:82273957);贵州省科技计划项目(编号:黔科合平台人才[2021]5632号、黔科合基础-ZK[2023]重点034);贵州省中医药、民族医药科学技术研究课题(编号:QZYY-2022-025);贵州省卫生健康委科学技术基金项目(编号:gzwkj2021-519);贵州医科大学大学生创新创业训练计划项目(编号:S202210660139)。

摘　　要：目的探究线粒体DNA聚合酶γ(POLG)抑制剂扎西他滨(ddC)对三阴性乳腺癌MDA-MB-231细胞迁移和侵袭的作用,并初步探讨其对线粒体生物合成的影响。方法MTT法测定ddC对细胞活力的影响;细胞划痕实验和Transwell侵袭实验检测细胞的迁移和侵袭能力;应用流式细胞术和V-FITC/PI细胞凋亡检测试剂盒检测细胞的凋亡水平;Western blot检测POLG、NADH脱氢酶亚基Ⅰ(NADH1)、NADH脱氢酶亚基Ⅱ(NADH2)、ATP合成酶亚基6(ATPase6)、细胞色素C氧化酶亚基Ⅰ(COX-1)和细胞色素C氧化酶亚基Ⅲ(COX-3)的蛋白表达水平;qPCR实验检测POLG的mRNA水平和mtDNA拷贝数;应用MitoTracker Green荧光探针和ATP定量试剂盒测定细胞的线粒体含量和ATP水平;将POLG的过表达载体转染到MDA-MB-231细胞中,用Western blot、细胞划痕实验和Transwell侵袭实验验证POLG抑制剂对细胞的迁移和侵袭能力的影响。结果POLG在MDA-MB-231细胞中的表达高于人正常乳腺上皮MCF-10A细胞(P<0.01)。ddC剂量依赖性地抑制细胞活力,降低了MDA-MB-231细胞的迁移(P<0.01)和侵袭能力(P<0.01),且在实验剂量下对正常乳腺上皮MCF-10A细胞无明显毒性。ddC可下调MDA-MB-231细胞内POLG蛋白(P<0.01)和mRNA水平(P<0.01),并降低mtDNA拷贝数(P<0.01)以及mtDNA编码的NADH1、NADH2、ATPase6、COX-1和COX-3蛋白表达(P<0.01)。ddC可抑制MDA-MB-231细胞的线粒体含量(P<0.01)和ATP水平(P<0.01)。在MDA-MB-231细胞内过表达POLG增强了细胞的迁移(P<0.05)和侵袭能力(P<0.05),而ddC对POLG过表达的MDA-MB-231细胞的迁移、侵袭能力未表现出明显抑制。结论ddC可下调MDA-MB-231中POLG的表达,阻碍线粒体生物合成和降低ATP水平,进而抑制MDA-MB-231细胞的迁移和侵袭。Objective To investigate the effects of zalcitabine(ddC),a mitochondrial DNA polymeraseγ(POLG)inhibitor,on the migration,invasion,and to preliminarily explore mitochondrial biogenesis of human triple-negative breast cancer MDA-MB-231 cells.Methods The effect of ddC on cell viability was detected using the MTT assay.The migration and invasion abilities of the cells were evaluated using the cell scratch and Transwell invasion assays.Cell apoptosis was determined using flow cytometry and a V-FITC/PI cell apoptosis detection kit.The protein expression of POLG,NADH dehydrogenase subunitⅠ(NADH1),NADH dehydrogenase subunitⅡ(NADH2),ATP synthase subunit 6(ATPase6),cytochrome c oxidase subunitⅠ(COX-1)and cytochrome c oxidase subunitⅢ(COX-3)were determined using Western blot.The POLG mRNA level and mtDNA copy number were determined using qPCR.The mitochondrial content and ATP levels were determined using MitoTracker Green fluorescent probe staining and an ATP determination kit.MDA-MB-231 cells were transfected with pcDNA3.1-EGFP-POLG plasmids to overexpress POLG.The inhibitory effects of ddC on cell migration and invasion were detected in POLG-overexpressed MDA-MB-231 cells.Results POLG expression was higher in MDA-MB-231 cells than in normal mammary epithelial cells(MCF-10A)(P<0.01).ddC inhibited cell viability in a dose-dependent manner.ddC inhibited the migration(P<0.01)and invasion(P<0.01)of MDA-MB-231 cells;however,it displayed no significant inhibitory effects on cell viability in normal mammary epithelial cells(MCF-10A)at the same concentration.ddC downregulated the protein(P<0.01)and mRNA(P<0.01)levels of POLG,reduced mtDNA copy number(P<0.01)and downregulated mtDNA-coded NADH1,NADH2,ATPase6,COX-1 and COX-3 protein expression(P<0.01)in MDA-MB-231 cells.Furthermore ddC inhibited mitochondrial content(P<0.01)and ATP(P<0.01)levels in MDA-MB-231 cells.POLG overexpression increased the migration(P<0.05)and invasion(P<0.05)abilities of MDA-MB-231 cells,while ddC did not significantly inhibit the migration and inv

关 键 词：三阴性乳腺癌 迁移 侵袭 POLG抑制剂 线粒体生物合成 ATP合成 

分 类 号：R737.9[医药卫生—肿瘤]