靶向沉默ATM基因对人肺腺癌A549细胞放射敏感性影响  

Effect of silencing of ATM expression by siRNA on radiosensitivity of human lung adenocarcinoma A549 cells

作　　者：刘小群[1] 乔田奎[1] 

机构地区：[1]复旦大学附属金山医院肿瘤科,上海201508

出　　处：《中华放射肿瘤学杂志》2014年第1期73-77,共5页Chinese Journal of Radiation Oncology

基　　金：上海市金山区科委基金项目（2012-3-15）

摘　　要：目的探讨质粒介导RNA干扰抑制ATM基因表达对人肺腺癌A549细胞放射敏感性影响。方法构建ATM基因小分子干扰RNA（siRNA）真核表达质粒pSilencer2．1-ATM并转染A。细胞（阳性组），转染pSilencer2．1-nonspecific质粒为阴性组，未转染为对照组。RT—PCR和蛋白印迹法分别检测ATM基因mRNA及蛋白表达，细胞克隆形成实验观察细胞放射敏感性变化，流式细胞仪检测细胞周期和细胞凋亡。结果成功构建了ATM基因siRNA真核表达质粒。RT—PCR和蛋白印迹法证实阳性组细胞ATM基因表达下调，阳性组、阴性组的放射增敏比（D0值比）分别为1．50、1．01，流式细胞仪检测显示阳性组G1、G2＋M期细胞比例减少[51．27％：61．85％（P=0．012）、6．34％：10．91％（P=0．008）]，细胞凋亡率则高于对照组、阴性组[49．31％：13．58％（P=0．000）、49．31％：13．17％（P=0．000）]。结论ATM基因沉默可增加A549细胞的放射敏感性，其机制可能与细胞周期变化及凋亡有关。Objective To investigate the effect of silencing of ataxia-telangiectasia mutated （ATM） expression by plasmid-mediated RNA interference on the radiosensitivity of human lung adenocarcinoma A549 cells. Methods Eukaryotic expression plasmid containing ATM small interfering RNA （siRNA） （pSilencer2. 1-ATM ） , as well as pSilencer2. 1-nonspecifie, was constructed. Lung adenoearcinoma A549 cells were divided into positive group, negative group, and control group to be transfected with pSilencer2. 1- ATM, pSilencer2. 1-nonspecific, and no plasmid, respectively. The mRNA and protein expression of ATM was measured by RT-PCR and Western blot, respectively. The change in cell radiosensitivity was observed by colony-forming assay. Cell cycle and cell apoptosis were analyzed by flow cytometry. Results The eukaryotic expression plasmid containing ATM siRNA was successfully constructed. The RT-PCR and Western blot demonstrated that the expression of ATM was down-regulated in the positive group. The sensitization enhancement ratios （Do ratios） for the positive group and negative group were 1.50 and 1. 01, respectively. The flow cytometry revealed that the proportions of A549 cells in G1 and G2/M phases were significantly lower in the positive group than in the control group （51.27% vs 61.85% , P = 0. 012 ;6. 34% vs 10. 91% , P = 0. 008 ） and that the apoptosis rate was significantly higher in the positive group than in the control group and negative group （49.31% vs 13.58%0 , P = 0. 000;49.31% vs 13.17% , P = 0. 000）. Conclusions Silencing of ATM expression may increase the radiosensitivity of human lung adenocarcinoma A549 cells, probably by affecting the cell cycle and promoting cell apoptosis.

关 键 词：毛细血管扩张共济失调突变基因 RNA干扰 细胞系 肺腺癌 放射敏感性 

分 类 号：R734.2[医药卫生—肿瘤]