DNA-PKcs在石英诱导的DNA双链断裂修复中的作用

目的 探讨DNA依赖性蛋白激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)在石英致人胚肺成纤维细胞(HELF)DNA双链断裂修复中的作用.方法 脂质体转染法建立DNA-PKcs的小干扰RNA(siRNA)及其阴性对照重组质粒稳定转染HELF系(简称HELF-PKcs和HELF-NC).3种方式分组及对细胞的处理:(1)25、50、100、200、300、400μg/ml浓度的石英刺激HELF 12 h;(2)200μg/ml的石英刺激HELF 0、1、2、6、12、24 h;(3)200μg/ml的石英刺激HELF-PKcs和HELF-NC 0、12、24 h.用免疫印迹法检测DNA-PKcs表达、磷酸化H2AX(H2AX)的水平.用Image-Pro plus6.0软件对条带光强度进行半定量分析;用中性彗星实验(彗尾DNA百分含量值)判断石英诱导的DNA舣链断裂损伤强度.结果 不同浓度的石英刺激HELF 12 h,γH2AX水平及彗尾DNA百分含量随着石英浓度的增加逐渐升高.200 μg/ml的石英刺激HELF6 h时,彗尾DNA百分含量(38.7±6.9)%]与对照组相比明显增加,并且在12h达峰值,24h相对12h时点明显降低,差异均有统计学意义(P<0.05).在抑制DNA-PKcs的表达的细胞,12 h时,石英刺激的HELF-PKcs的石英诱导的γH2AX水平增加受抑制,彗尾DNA百分含量为(43.09±3.68)%,与石英刺激的HELF-NC相比,差异无统计学意义(P>0.05);24 h时,石英刺激的HELF-PKcs的石英诱导的γH2AX水平与石英刺激的HELF-NC相比无明显筹异,差异无统计学意义(P>0.05).石英刺激的HELF-PKcs的彗尾DNA百分含量(35.79±4.26)%]明显高于石英刺激的HELF-NC,差异有统计学意义(P<0.05).结论 石英可诱导DNA双链断裂损伤,DNA-PKcs是石英诱导的DNA双链断裂损伤的感受器,通过磷酸化H2AX,促进石英诱导的DNA双链断裂损伤的修复.

Role of DNA-dependent protein kinase catalytic subunit in silica-induced DNA double-strand break repair in human embryo lung fibroblasts

Objective To study the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in silica-induced DNA double-strand break repair in human embryo lung fibrobtasts (HELF).Methods Two stable transfectants,HELF transfectcd with DNA-PKcs siRNA (HELF-PKcs) and with negative control siRNA (HELF-NC),were established.HELF cells were treated with 0,25,50,100,200,300 anti 400 μg/ml silica for 12 h and with 200 μg/ml silica for different times (0,1,2,6,12 and 24 h ).HELF-PKcs and HELF-NC were treated with 200 μg/ml silica for 0,12 and 24 h.The expression levels of DNA-PKcs and phosphor-H2AX(H2AX) were determined by Western blot.DNA double strand breaks were measured by neutral comet assay.Results After treatment with difterent doses of silica for 12 h,the levels of H2AX and the percentages of tail DNA increased in concentration-dependent manner.After treatment with 200 μg/ml silica for different times,the levels of H2AX increased in a time-dependent manner.The percentages of tail DNA increased sig-nificantly at 6 h,and reaching maximum at 12 h and then decreasing at 24 h.The expression level of DNA-PKcs was suppressed in HELF-PKcs.After treatment with silica at 12 h,the level of H2AX was lower in HELF-PKcs than in HELF-NC,and the percentages of tail DNA increased obviously in both HELF-PKcs and HELF-NC compared with non-treated cells,but no significant difference was found in the percentages of tail DNA between them.The percentages of tail DNA decreased markedly in silica-treated HEI,F-NC and was sig-nificantly lower than in HELF-PKcs at 24 h (P<0.05).Conclusion Silica can induce DNA double strand breaks in human embryo lung fibroblasts.DNA-PKcs might play a major role in silica-induced DNA double strand break repair.Silica-induced histone H2AX phosphorylation was dependent on DNA-PKcs.