辐射对沉默ATRX的H460细胞增殖以及DNA损伤修复的影响

The effects of radiation on proliferation and DNA damage repair in H460 cells silenced ATRX

目的探讨辐射对沉默ATRX的肺癌H460细胞增殖和DNA损伤修复的影响及二者的关系。方法靶向ATRX的3个慢病毒载体转染293T细胞后,慢病毒感染H460细胞,获得ATRX低/无表达的细胞株shATRX1-H460、shATRX2-H460和shATRX3-H460,并以shControl-H460作为对照,利用Western blot检测沉默效率。分别以克隆形成实验检测细胞增殖,免疫荧光技术检测γH2AX和Rad51焦点数,同时以Western blot检测PARP1、γH2AX和Rad51蛋白的表达。结果shControl-H460细胞中可见ATRX表达,而shATRX1-H460、shATRX2-H460和shATRX3-H460细胞中ATRX表达均出现不同程度的降低。克隆形成实验显示,shATRX2-H460和shATRX3-H460细胞的存活分数(survival fraction,SF)均较shControl-H460细胞降低。shControl-H460和shATRX3-H460细胞经4 Gy照射后1 h,γH2AX焦点最多,而3 h时Rad51焦点最多,而后均降低,与shControl-H460细胞比较,在1和6 h时shATRX3-H460细胞γH2AX焦点,以及1、3和6 h时Rad51焦点显著增加(P<0.05,P<0.001)。而且shATRX3-H460细胞中PARP1、γH2AX和Rad51蛋白在3和6 h时均较shControl-H460细胞表达增加。结论成功地获得靶向沉默ATRX的细胞模型,辐射后细胞增殖能力降低,可能与DNA损伤修复能力降低有关。

Objective To explore the effects of radiation on proliferation and DNA damage repair,and to clarify the relationship between them.Methods Lentivirus plasmids of shATRX1,shATRX2 and shATRX3 were transfected into 293T cells,H460 cells were infected by lentivirus,shATRX1-H460,shATRX2-H460 and shATRX3-H460 cell lines with low/no ATRX expression were obtained,shControl-H460 cells was used as the control.The silencing efficiency was detected by Western blot.Cell proliferation was measured by colony formation assay,γH2AX and Rad51 foci were detected by immunofluores-cence,and the numbers were counted,PARP1,γH2AX and Rad51 expressions were measured by Western blot,respectively.Results ATRX expressed in H460 and shControl-H460 cells,while reduced in shATRX1-H460,shATRX2-H460 and shA-TRX3-H460 cells.The colony formation assay showed that the SFs of shATRX2-H460 and shATRX3-H460 cells was lower than that of shControl-H460 cells.After shControl-H460 and shATRX3-H460 cells were irradiated by 4 Gy at 1 h,γH2AX foci reached a maximum,while Rad51 foci reached a maximum,at 3 h,and then decreased.Compared with shControl-H460 cells,γH2AX foci at 1 and 6 h,Rad51 foci at 1,3 and 6 h were significantly increased(P<0.05,P<0.001).And PARP1,γH2AX and Rad51 protein expressions in shATRX3-H460 cells increased at 3 and 6 h compared with that in shContral-H460 cells.Conclusion The cell models of targeted silencing of ATRX in H460 cells were successfully obtained,and the cell proliferation ability was reduced after radiation,which may be related to the reduction of DNA damage repair ability.