siRNA抑制肺腺癌细胞DNA—DPKCS表达与放射敏感性关系研究  

siRNA in silencing the expression of DNA-dependent protein kinase and its effect on radiosensitivity of lung adenocarcinoma cells

作　　者：潘燚[1] 李伟雄[1] 杨素清[2] 曾子君[1] 林映如[1] 郭爱林[2] 

机构地区：[1]广东省人民医院广东省医学科学院肿瘤中心放疗科,广州510080 [2]广东省人民医院广东省医学科学院医学研究中心

出　　处：《中华放射肿瘤学杂志》2010年第1期66-69,共4页Chinese Journal of Radiation Oncology

基　　金：广东省自然科学基金资助（06020905）

摘　　要：目的探讨稳定表达的小干扰RNA（siRNA）抑制DNA依赖蛋白激酶催化亚基（DNA—DPKCS）表达对非小细胞肺癌细胞增殖、细胞周期及放射敏感性的影响。方法构建DNA—DPKCS—siRNA重组质粒转染肺腺癌细胞A549。流式细胞仪检测细胞周期和凋亡，成克隆实验测定细胞存活曲线。结果建立了与A549细胞同源的DNA—DPKCS低表达细胞系549pRNA—DNA—DPKCS、阴性对照549pRNA—C细胞、空白549pSUPER细胞。与A549细胞相比549pRNA—DNA—DPKCS细胞的DNA—DPKCS mRNA（0．110：1．000；F=80．55，P〈0．01）、蛋白表达水平（0．870：2．967；F=63．96，P〈0．01）以及DNA—DPKCS活性（0．004：0．266；F=51．62，P〈0．01）均显著降低。549pRNA—DNA—DPKCS细胞2Gy存活分数、平均致死剂量、亚致死性损伤剂量均较A549细胞明显降低（0．25：0．76；F=996．86，P〈0．01：1．42：1．62：F=17．41，P〈0．05；0．06：1．00；F=68．92，P〈0．01）。DNA—DPKCS表达受抑后549pRNA—DNA—DPKCS细胞较A549细胞s期和G2期细胞比例明显减少（24．5％：35．5％；F=4．83，P〈0．05和10．7％：11．0％：F=32．04，P〈0．01）。结论抑制DNA—DPKCS表达可提高肺腺癌细胞A549的放射敏感性，同时调控细胞周期时相分布。Objective To discuss the role of DNA-dependent protein kinase catalytic subunit （DNA-DPKCS） in human lung adenocarcinoma cell line （A549） by using small interfering RNA （siRNA） to specifically knockdown DNA-DPKCS expression and its effects on cell proliferation, cell cycle and radiosensitivity. Methods The DNA-DPKCS-siRNA expression vector was constructed and transfected into A549 cell line. The transformed clones were randomly selected and isolated. The cell cycle distribution and apoptosis were analyzed by flowcytometry analysis. Cell survival was detected by using clonogenic formation assay. Results With specific inhibition of DNA-DPKCS expression, stable transfected cell line 549pRNA- DNA-DPKCS was constructed by RNA interference technique. The 549pRNA-C and 549pSUPER cell lines were the control cell lines tansfected with control and blank plasmids, respectively. Compared with A549 cells, the expression levels of DNA-DPKCS mRNA （0.110： 1. 000）, protein （0. 870： 2. 967 ） and activity of DNA-DPKCS （0.004： 0.266） in 549pRNA-DNA-DPKCS cells were significantly lower （ F = 80.55 ,P 〈 0.01 ; F = 63.96, P 〈 0.01 ; F = 51.62, P 〈 0.01, respectively ）. The analysis of SF2 （ 0.25 ： 0.76 ）, DO （ 1. 42： 1.62） and Dq （0.06： 1.00） showed significant difference between 549pRNA-DNA-DPKCS and A549 cells （ F = 996.86, P 〈 0.01 ; F = 17.41, P 〈 0.05 ; F = 68.92, P 〈 0.01 ）. The number of 549pRNA-DNA- DPKCS cells in S （24.5% ： 35.5% ） and G2 （ 10.7% ： 11.0% ） phases was significantly decreased （ F = 4.83, P〈0.05 and F=32.04, P〈0.01, respectively）. Conclusions In A549 cells, inhibit of DNA- DPKCS gene expression can enhance the radiosensitivity and affect cell cycle distribution.

关 键 词：脱氧核糖核酸依赖蛋白激酶催化亚基 核糖核酸干扰 细胞系 肺肿瘤 放射敏感性 

分 类 号：R686[医药卫生—骨科学]