CAJ | 학술논문

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莺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2 Gy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G_2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.
목적 탐토은정표체적소간우RNA(siRNA)억제DNA의뢰단백격매최화아기(DNA-DPKCS)표체대비소세포폐암세포증식、세포주기급방사민감성적영향.방법 구건DNA-DPKCS-siRNA앵조질립전염폐선암세포A549.류식세포의검측세포주기화조망,성극륭실험측정세포존활곡선.결과 건립료여A549세포동원적DNA-DPKCS저표체세포계549pRNA-DNA-DPKCS、음성대조549pRNA-C세포、공백549pSUPER세포.여A549세포상비549pRNA-DNA-DPKCS세포적DNA-DPKCS mRNA(0.110:1.000;F=80.55,P<0.01)、단백표체수평(0.870:2.967;F=63.96,P<0.01)이급DNA-DPKCS활성(0.004:0.266;F=51.62,P<0.01)균현저강저.549pRNA-DNA-DPKCS세포2 Gy존활분수、평균치사제량、아치사성손상제량균교A549세포명현강저(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-DPKCS표체수억후549pRNA-DNA-DPKCS세포교A549세포S기화G_2기세포비례명현감소(24.5%:35.5%;F=4.83,P<0.05화10.7%:11.0%;F=32.04,P<0.01).결론 억제DNA-DPKCS표체가제고폐선암세포A549적방사민감성,동시조공세포주기시상분포.
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 radio-sensitivity. 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 apop-tesis were analyzed by flowcytometry analysis. Cell survival was detected by using clonogenic formation as-say. 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 SF_2(0.25:0.76), D_0 (1.42:1.62) and D_q (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 G_2 (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.