吉林大学学报(医学版) ›› 2021, Vol. 47 ›› Issue (4): 904-910.doi: 10.13481/j.1671-587X.20210412

• 基础研究 • 上一篇    下一篇

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

秦丽晶1,韩冰2,李忠起3,索齐4,贾臻1,崔红丽1,徐维强1,方芳1(),王志成1()   

  1. 1.吉林大学公共卫生学院 国家卫健委放射生物学重点实验室,吉林 长春 130021
    2.吉林大学 第二医院放射线科,吉林 长春 130041
    3.吉林国际旅行卫生保健中心,吉林 长春 130062
    4.吉林省长春爱尔汇锋眼科医院,吉林 长春 130041
  • 收稿日期:2021-01-29 出版日期:2021-07-28 发布日期:2021-07-22
  • 通讯作者: 方芳,王志成 E-mail:fangfang7786@jlu.edu.cn;zhicheng@jlu.edu.cn
  • 作者简介:秦丽晶(1995-),女,内蒙古自治区兴安盟人,在读硕士研究生,主要从事辐射生物效应方面的研究。
  • 基金资助:
    吉林省卫健委卫生专项项目(2020SCZT066);吉林省教育厅科学研究项目(JJKH20211140KJ);吉林大学白求恩计划项目(2020B06)

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

Lijing QIN1,Bing HAN2,Zhongqi LI3,Qi SUO4,Zhen JIA1,Hongli CUI1,Weiqiang XU1,Fang FANG1(),Zhicheng WANG1()   

  1. 1.NHC Key Laboratory of Radiobiology,School of Public Health,Jilin University,Changchun 130021,China
    2.Department of Radiology,Second Hospital,Jilin University,Changchun 130041,China
    3.Jilin International Travel Healthcare Center,Changchun 130062,China
    4.Changchun AIER Huifeng Ophthalmology Hospital,Jilin Province,Changchun 130041,China
  • Received:2021-01-29 Online:2021-07-28 Published:2021-07-22
  • Contact: Fang FANG,Zhicheng WANG E-mail:fangfang7786@jlu.edu.cn;zhicheng@jlu.edu.cn

摘要: 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Annexin Ⅴ-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。

关键词: 辐射, α-地中海贫血/精神发育迟滞综合征X染色相关蛋白, 宫颈肿瘤, 细胞周期阻滞, 细胞凋亡, 细胞增殖

Abstract: 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 shCon-HeLa 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 AnnexinⅤ-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 after 0, 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 shCon-HeLa 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.

Key words: radiation, α-thalassemia/mental retardation syndrome X-linked protein, cervical neoplasms, cell cycle arrest, apoptosis, cell proliferation

中图分类号: 

  • R737.33