靶向沉默ATM基因对人肺腺癌A549细胞放射敏感性影响

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A₅₄₉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。

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

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-nonspecific, was constructed. Lung adenocarcinoma 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 (D₀ 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%, 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.