摘要
目的:以RNA干扰(RNAi)沉默肺癌H460细胞ATRX基因,探讨电离辐射对肺癌H460细胞凋亡的影响及其作用机制。方法:以靶向ATRX基因的慢病毒表达载体转染293T细胞,所得慢病毒2次感染肺癌细胞H460,通过puromycin阳性筛选获得ATRX沉默的细胞模型,命名为sh-ATRX1-H460、sh-ATRX2-H460和sh-ATRX3-H460细胞,以sh-control-H460细胞作为对照。根据沉默结果分为sh-control-H460组和shATRX3-H460组,分别给予0、2和8Gy X射线照射。Western blotting法检测细胞中ATRX、多聚腺苷酸二磷酸核糖基聚合酶1(PARP1)和caspase-3蛋白表达量;AnnexinⅤ-FITC/PI试剂盒和流式细胞术检测各组细胞凋亡率。结果:成功获得ATRX沉默的肺癌sh-ATRX3-H460细胞模型。与照射前比较,2和8Gy X射线照射后sh-control-H460组细胞中ATRX蛋白表达量增加,sh-ATRX3-H460组细胞中无ATRX蛋白表达;与照射前比较,照射后sh-control-H460组和sh-ATRX3-H460组细胞凋亡率均明显升高(P<0.05或P<0.01);8Gy X线照射后sh-ATRX3-H460组细胞凋亡率明显高于sh-control-H460组(P<0.01);2组细胞中cleaved PARP1蛋白表达量均增加且规律相似;sh-control-H460组细胞中procaspase-3蛋白表达量无明显变化,8Gy X射线照射后sh-ATRX3-H460组细胞中procaspase-3蛋白表达量明显增加。结论:RNAi可以实现ATRX沉默,ATRX沉默能够增加辐射诱导的细胞凋亡,其机制可能与PARP1-caspase-3通路有关联。
Objective： To explore the effect of ionizing radiation on apoptosis of lung cancer H460 cells after ATRX was silenced by RNAi and its mechanism. Methods： The lentivirus expression vectors targeting ATRX were transfeeted into the 293T cells, and the lung cancer H460 cells were infected with lentivirus twice, and the ATRX silenced cell model was obtained after puromycin positive screening, then they were named as sh-ATRX1-H460, sh-ATRX2-H460, and sh-ATRX3-H460 cells; the sh-eontrol-H460 cells were regarded as control cells. The cells were divided into sh-control-H460 group and sh-ATRX3-H460 group, accroding to the silencing results and were irradiated by 0, 2 and 8 Gy X-rays. The expression levels of ATRX, poly （ADP-ribose） polymerase 1 （PARP1）, and caspase-3 proteins were measured by Western blotting method; the apoptotic rate was measured by flow cytometry and Annexin V-FITC/PI kits. Results： The lung cancer cell model of sh-ATRX3-H460 silenced by ATRX was obtained successfully. After 2 and 8 Gy X-ray irradiation, compared with before irradiation, the expression level of ATRX protein in sh-control-H460 group was increased, while there was no expression of ATRX protein in sh-control-H460 group; compared with before irradiation, the apoptotic rates of cells in two groups were increased （P〈0. 05 or P〈0. 01）; the apoptotic rate in sh-ATRX3-H460 group was significantly higher than that in sh-control-H460 group after 8 Gy X-ray irradiation （P〉0.01）. The expressions of cleaved PARP1 in the cells in both two groups after 2 Gy and 8 Gy X-ray irradiation were increased and showed similar rule. The expression level of procaspase-3 protein in sh-control-H460 group had little change, and it was increased significantly in sh-ATRX3- H460 group after 8 Gy X-ray irradiation. Gonclusion.. ATRX silencing can be achived by RNAi, then the silencing could increase the apoptosis induced by irradiation and its mechanism may be related to the PARPl-caspase-3 pathway.
作者
张晶
王志成
赵大力
卢晓倩
沈智渊
齐亚莉
ZHANG Jing WANG Zhicheng ZHAO Dali LU Xiaoqian SHEN Zhiyuan QI Yali(Department of Epidemiology, School of Public Health, Beihua University, Jilin 132011, China Key Laboratory of Radiobiology, Ministry of Health, School of Public Health, Jilin University, Changchun 130021, China Cancer Institute, State University of New Jersey, New Jersey 08903-2681, USA Jilin Province Entry and Exit Inspection and Quarantine Bureau, Changchun 130013, China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2017年第3期522-526,共5页
Journal of Jilin University：Medicine Edition
基金
吉林省教育厅"十二五"科研规划项目资助课题(2014-192)
