miR-146b-5p通过抑制ATR通路诱导肺癌A594细胞凋亡  

miR-146 b-5 p induced apoptosis of lung cancer A594 cells by inhibiting the ATR pathway

作　　者：陈晓[1] 魏立[1] 陈重[1] 李基伟[1] 

机构地区：[1]河南省人民医院(郑州大学人民医院)胸外二科,河南郑州450003

出　　处：《现代肿瘤医学》2017年第20期3238-3241,共4页Journal of Modern Oncology

基　　金：河南省科技攻关项目(编号:162102310029)

摘　　要：目的:研究miR-146b-5p对ATR的靶向抑制、诱导A594细胞凋亡的作用。方法:CCK8实验、克隆形成实验检测对照组、miR-146b-5p类似物转染组和阴性转染组,观察A594细胞增殖和存活能力;Target Scan7.1与miRanda软件预测miR-146b-5p靶向ATR。实时定量PCR检测对照组、miR-146b-5p组和Negative mimics组ATR mRNA表达变化。设计ATR 3'UTR突变序列(ATR-3'UTR-mut)和荧光报告载体实验验证miR-146b-5p对ATR的靶向作用;Western Blot实验检测各组癌细胞中ATR通路蛋白ATR、Chk1、p53表达变化,流式细胞仪检测各组细胞凋亡。结果:CCK8实验和克隆形成实验结果:与对照组比较,miR-146b-5p组细胞数降低,克隆形成率从0.98±0.01降低到0.57±0.03,差异显著(P<0.05);Negative mimics组细胞数和克隆形成率0.92±0.03均无显著差异(P>0.05)。实时定量PCR结果与对照组比较,miR-146b-5p组ATR mRNA表达从0.96±0.02降低到0.38±0.03,差异显著(P<0.05);无义序列组ATR mRNA表达为0.94±0.02,差异不显著(P>0.05);荧光报告载体实验结果:ATR 3'UTR miR-1 4 6 b-5p组ATR mRNA降低到0.36±0.03,差异显著(P<0.05);ATR 3'UTR-mut miR-146b-5p组ATR mRNA表达量为0.96±0.02,差异不显著(P>0.05)。Western Blot结果显示miR-146b-5p组ATR、Chk1、p53蛋白表达降低。流式细胞仪结果显示miR-146b-5p组细胞凋亡率增加(26±2.18)%,差异显著(P<0.05)。结论:miR-146b-5p通过抑制ATR信号通路诱导肺癌A594细胞凋亡。Objective：To investigate the effect of miR - 146b -5 p induced lung cancer cell apoptosis by inhibiting ATR signaling pathways by on ATR mRNA. nethods：CCK8 assay and clonogenic survival assay were performed to study the cell survival ability. Combined with quantitative real -time PCR, the luciferase reporter assay and transfec- tion assay were carried out to evaluate the targeting effect of miR - 146b - 5p mimics on ATR mRNA. Western blot- ting and flow eytometry were performed to study the underlying signal pathway. Results. The clonogenic survival rate （0.57 ± 0.03 ）and the number of live ceils decreased significantly in the miR - 146b -5p mimics transfected group （P 〈0.05 ）. The qRT- PCR results showed that transfected with miR- 146b- 5p mimics could remarkably inhibit the expression of ATR mRNA （0.38 ± 0.03 ） （ P 〈 0.05 ）. Besides, the luciferase activity in the miR - 146b - 5p transfection of ATR - 3＂ UTR reduced sharply （0.36 ± 0.03 ） （ P 〈 0.05 ）. Western blot assay showed that protein level of ATR, Chkl and p53 reduced in the miR - 146b - 5p transfected group. Cell apoptosis rate of miR - 146b - 5p transfection group increased obviously to （26 ± 2.18 ） % （ P 〈 0.05 ）. Conclusion ： miR - 146b - 5 p could effectively induce the cell apoptosis of lung cancer cell by inhibiting ATR pathway.

关 键 词：miR-146b-5p ATR 肺癌 细胞凋亡 

分 类 号：R734.2[医药卫生—肿瘤]