靶向沉默ATRX对辐射诱导宫颈癌HeLa细胞周期阻滞和凋亡的作用

Effects of targeting-silenced ATRX on cell cycle arrest and apoptosis induced by radiation in cervical cancer HeLa cells

目的:靶向沉默HeLa细胞中α-地中海贫血/精神发育迟滞综合征X染色相关蛋白(ATRX),探讨其对辐射诱导的细胞周期阻滞和细胞凋亡的作用。方法:利用RNA干扰(RNAi)技术建立稳定沉默ATRX的HeLa细胞模型,命名为shATRX1-HeLa,以shCon-HeLa为阴性对照。分别给予0、2和8 Gy X射线照射后,采用CCK-8法检测各组细胞增殖活性,采用PI单染和流式细胞术检测各组细胞中G0/G1、S和G2/M期细胞百分率,采用AnnexinⅤ-PE/7-AAD双染和流式细胞术检测各组细胞凋亡率,采用Western blotting法检测各组细胞中多聚(腺苷酸二磷酸核糖基)聚合酶1(PARP1)、裂解的半胱氨酸天冬氨酸蛋白酶9(cleaved caspase-9)和裂解的半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3)蛋白表达情况。结果:0、2和8 Gy X射线照射后0、6、10、24和48 h,与shCon-HeLa组比较,不同照射剂量shATRX1-HeLa组细胞增殖活性均明显降低(P<0.05或P<0.01)。0和2 Gy X射线照射后,与shCon-HeLa组比较,shATRX1-HeLa组G0/G1期细胞百分率明显降低(P<0.05),G2/M期细胞百分率和细胞凋亡率则明显升高(P<0.05);8 Gy X射线照射后,与shCon-HeLa组比较,shATRX1-HeLa组G0/G1期细胞百分率明显升高(P<0.05),而G2/M期细胞百分率明显降低(P<0.05),细胞凋亡率明显升高(P<0.01)。Western blotting法检测,与shCon-HeLa组比较,经2和8 Gy照射后24和48 h,shATRX1-HeLa组细胞中PARP1、cleaved caspase-9和cleaved caspase-3蛋白表达均明显增加。结论:靶向沉默HeLa细胞ATRX可降低辐射诱导的细胞G2/M期阻滞、抑制细胞增殖并促进细胞凋亡,本研究结果为肿瘤放疗提供了新的分子靶点。

Objective:To targetedly silence the α-thalassemia/mental retardation syndrome X-linked(ATRX) in the cervical cancer HeLa cells,and to explore the roles of targeting-silenced ATRX in radiation-induced cell cycle arrest and apoptosis. Methods:The HeLa cell models stably silenced ATRX were established by RNA interference(RNAi)technique,which was named shATRX1-HeLa,and shConHeLa was used as negative control. After 0,2 and 8 Gy X-ray irradiation,the cell proliferation activities in various groups were detected by CCK-8 method,and the percentages of HeLa cell in the G0/G1,S and G2/M phases in various groups were measured by PI staining and flow cytometry. In addition,the apoptotic rates were measured by Annexin Ⅴ-PE/7-AAD double staining and flow cytometry,and the expression levels of poly[ADP-ribose]polymerase 1(PARP1),cleaved caspase-9 and cleaved caspase-3 proteins in the cells in various groups were detected by Western blotting method. Results:At 0,6,10,24 and 48 h after0,2 and 8 G y X-ray irradiation,compared with shCon-HeLa group,the proliferation activities of the cells in shATRX1-HeLa group were decreased significantly(P<0. 05 or P<0. 01). Compared with shConHeLa group,after 0 and 2 Gy X-ray irradiation,the percentages of cells in G0/G1 phase in shATRX1-HeLa group were decreased significantly(P<0. 05),and the percentages of cells in G2/M phase and the apoptotic rates were increased significantly(P<0. 05 or P<0. 01). Compared with shCon-HeLa group,after 8 Gy X-ray irradiation,the percentage of cells in G0/G1 phase and the apoptotic rate were increased significantly(P<0. 05),and the percentage of cells in G2/M phase was decreased significantly(P<0. 05). The Western blotting results showed that compared with shCon-HeLa group,at 24 and 48 h after 2 and 8 Gy X-ray irradiation,the expression amounts of PARP1,cleaved caspase-9 and cleaved caspase-3 proteins in the cells in shATRX1-HeLa group were increased. Conclusion:Targeting-silenced ATRX can decrease the G2/M phase arrest of the cells induced by radiaton in the HeLa cells,and inhibit the cell proliferation and promote the apoptosis;the results of this study provides a new molecular target for tumor radiotherapy.