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目的:研究siRNA沉默毛细血管扩张—共济失调突变(atxia-telangiectasia mutated,ATM)基因的表达增强胞嘧啶鸟嘌呤二核苷酸寡脱氧核苷酸(cytosine-phophate-guanine oligodeoxynucleotide,CpG ODN)7909对人非小细胞肺癌A549细胞的放射增敏作用。方法:将ATM-siRNA转染至A549细胞中,Western blotting检测A549细胞中ATM蛋白的表达。A549细胞随机分为6组:对照组、CpG组、X射线(IR)组、CpG+IR组、ATM-siRNA+CpG+IR组和NC-siRNA+CpG+IR组,克隆形成分析法检测各组细胞克隆形成率,Graphpad prism 5.0软件进行单击多靶模型和L-Q线性模型拟合辐射后A549细胞的生存曲线,以D0、Dq、N、α/β及SF2等参数分析A549细胞辐射损伤修复能力,流式细胞术检测A549细胞的凋亡。结果:ATM-siRNA转染可明显抑制A549细胞中ATM蛋白的表达(P<0.01)。X射线可剂量依赖性抑制A549细胞的克隆形成能力(P<0.05);且CpG+IR组A549细胞的克隆形成能力进一步降低(P<0.01);ATM-siRNA转染后,CpG处理的A549细胞克隆形成能力再度降低[10 Gy时,(0.05±0.00)%vs(0.71±0.00)%,P<0.01]。辐射损伤剂量生存曲线结果显示,ATM-siRNA转染后,ATM-siRNA+CpG+IR组较CpG+IR组A549细胞的α/β值明显增大(1.48 vs 0.97,P<0.05),对放射损伤修复能力明显减弱。CpG+IR组较IR组细胞凋亡率显著升高[(9.18±0.16)%vs(6.56±0.33)%,P<0.01];ATM-siRNA+CpG+IR组A549细胞凋亡率进一步升高[(10.45±0.40)%vs(9.18±0.16)%,P<0.05]。结论:siRNA沉默ATM的表达可增强CpGODN 7909对A549细胞的放射增敏作用,ATM可作为肺癌治疗的潜在靶点。
OBJECTIVE: To study the effect of siRNA silencing of atxia-telangiectasia mutated (ATM) gene expression on cytosine-phophate-guanine oligodeoxynucleotide (CpG ODN) 7909 Radiosensitization effect on human non-small cell lung cancer A549 cells. Methods: ATM-siRNA was transfected into A549 cells and the expression of ATM protein in A549 cells was detected by Western blotting. A549 cells were randomly divided into 6 groups: control group, CpG group, X-ray group, CpG + IR group, ATM-siRNA + CpG + IR group and NC-siRNA + CpG + IR group. The cell clonality rate of A549 cells was determined by Graphpad prism 5.0 software. The survival curves of A549 cells after single-target and LQ linear models were fitted were analyzed. The radiation injury repair of A549 cells was analyzed by D0, Dq, N, α / β and SF2 parameters Capacity, flow cytometry A549 cell apoptosis. Results: ATM-siRNA transfection significantly inhibited the expression of ATM protein in A549 cells (P <0.01). X-ray could inhibit the colony-forming ability of A549 cells in a dose-dependent manner (P <0.05), and the colony formation ability of A549 cells was further decreased in CpG + IR group (P <0.01) The clonality was reduced again [(0.05 ± 0.00)% vs (0.71 ± 0.00)% at 10 Gy, P <0.01]. The results of radiation dose-survival curve showed that after ATM-siRNA transfection, the α / β of ATM-siRNA + CpG + IR group was significantly higher than that of CpG + IR group (1.48 vs 0.97, P <0.05) Damage repair ability was significantly weakened. The apoptosis rate of A549 cells in CpG + IR group was significantly higher than that in IR group [(9.18 ± 0.16)% vs (6.56 ± 0.33)%, P <0.01] [(10.45 ± 0.40)% vs (9.18 ± 0.16)%, P <0.05]. Conclusion: The silencing of ATM by siRNA can enhance the radiosensitization effect of CpG ODN 7909 on A549 cells. ATM can be a potential target for the treatment of lung cancer.