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

doi:10.13481/j.1671-587X.20210412

Abstract

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

CLC Number:

R737.33

Lijing QIN,Bing HAN,Zhongqi LI,Qi SUO,Zhen JIA,Hongli CUI,Weiqiang XU,Fang FANG,Zhicheng WANG. Effects of targeting-silenced ATRX on cell cycle arrest and apoptosis induced by radiation in cervical cancer HeLa cells[J].Journal of Jilin University(Medicine Edition), 2021, 47(4): 904-910.

Figures/Tables 5

Tab.1

Tab.2

Fig.1

Fig. 2

Fig. 3

References 21

1	CHEN S H， LUO T W， YU Q， et al. Isoorientin plays an important role in alleviating Cadmium-induced DNA damage and G0/G1 cell cycle arrest［J］. Ecotoxicol Environ Saf， 2020， 187： 109851.
2	SHIBATA A， JEGGO P A. Roles for 53BP1 in the repair of radiation-induced DNA double strand breaks［J］. DNA Repair （Amst）， 2020， 93： 102915.
3	CHEN C P， YIP H K， WANG L K， et al. Molecular genetic characterization of a prenatally detected 1.484-Mb Xq13.3-Q21.1 duplication encompassing ATRX and a literature review of syndromic intellectual disability and congenital abnormalities in males with a duplication at Xq13.3-Q21.1［J］.Taiwan J Obstet Gynecol，2017，56（3）：385-389.
4	WATSON L A， GOLDBERG H， BÉRUBÉ N G. Emerging roles of ATRX in cancer［J］. Epigenomics， 2015， 7（8）： 1365-1378.
5	NANDAKUMAR P， MANSOURI A， DAS S. The role of ATRX in glioma biology ［J］. Front Oncol， 2017， 7： 236.
6	唐庚，史耀挺，孔维轩，等. 电离辐射对沉默ATRX的HeLa细胞DNA损伤修复的影响［J］ . 吉林大学学报（医学版）， 2019， 45（5）：981-985.
7	LEUNG J W， GHOSAL G， WANG W Q， et al. Alpha thalassemia/mental retardation syndrome X-linked gene product ATRX is required for proper replication restart and cellular resistance to replication stress［J］. J Biol Chem， 2013， 288（9）： 6342-6350.
8	SMALL W JR， BACON M A， BAJAJ A， et al. Cervical cancer： a global health crisis［J］. Cancer， 2017， 123（13）： 2404-2412.
9	SHEN C J， SHARMA A， VUONG D V， et al. Ionizing radiation induces tumor cell lysyl oxidase secretion［J］. BMC Cancer， 2014， 14： 532.
10	KIM M Y， PARK S J， SHIM J W， et al. Naphthazarin enhances ionizing radiation-induced cell cycle arrest and apoptosis in human breast cancer cells ［J］. Int J Oncol， 2015， 46（4）： 1659-1666.
11	BURRAGE J， TERMANIS A， GEISSNER A， et al. The SNF2 family ATPase LSH promotes phosphorylation of H2AX and efficient repair of DNA double-strand breaks in mammalian cells ［J］. J Cell Sci， 2012， 125（Pt 22）： 5524-5534.
12	NAN X S， HOU J H， MACLEAN A， et al. Interaction between chromatin proteins MECP2 and ATRX is disrupted by mutations that cause inherited mental retardation［J］.Proc Natl Acad Sci U S A，2007，104（8）： 2709-2714.
13	BAUMANN C， OLSON M， WANG K， et al. Arginine methyltransferases mediate an epigenetic ovarian response to endometriosis［J］. Reproduction， 2015， 150（4）： 297-310.
14	SARMA K， CIFUENTES-ROJAS C， ERGUN A，et al.ATRX directs binding of PRC2 to Xist RNA and Polycomb targets ［J］. Cell， 2014， 159（4）： 869-883.
15	JUHÁSZ S， ELBAKRY A， MATHES A， et al. ATRX promotes DNA repair synthesis and sister chromatid exchange during homologous recombination［J］. Mol Cell， 2018， 71（1）： 11-24.e7.
16	KOSCHMANN C， CALINESCU A A， NUNEZ F J， et al. ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma［J］. Sci Transl Med， 2016， 8（328）： 328ra28.
17	HAIL N Jr， CARTER B Z， KONOPLEVA M， et al. Apoptosis effector mechanisms： a requiem performed in different keys［J］. Apoptosis， 2006， 11（6）： 889-904.
18	JI B C， HSU W H， YANG J S， et al. Gallic acid induces apoptosis via caspase-3 and mitochondrion-dependent pathways in vitro and suppresses lung xenograft tumor growth in vivo ［J］. J Agric Food Chem， 2009， 57（16）：7596-7604.
19	HUANG J， YU S N， JI C N， et al. Structural basis of cell apoptosis and necrosis in TNFR signaling［J］. Apoptosis， 2015， 20（2）： 210-215.
20	SONG X F， TIAN H， ZHANG P， et al. Expression of cyt-c-mediated mitochondrial apoptosis-related proteins in rat renal proximal tubules during development［J］. Nephron， 2017， 135（1）： 77-86.
21	WANG X Y， SIMPSON E R， BROWN K A. p53： protection against tumor growth beyond effects on cell cycle and apoptosis［J］. Cancer Res， 2015， 75（23）： 5001-5007.

Group	Proliferation activity
Group	(t/h) 0	6	10	24	48
shCon-HeLa
0 Gy	0.99±0.19	0.95±0.14	1.17±0.19	1.81±0.22	2.98±0.33
2 Gy	0.81±0.12	0.78±0.08	1.07±0.17	1.64±0.16	2.58±0.15
8 Gy	0.93±0.09	1.05±0.15	1.25±0.27	1.49±0.07	1.98±0.11
shATRX1-HeLa
0 Gy	0.73±0.12^*	0.92±0.07	0.87±0.14^*	1.38±0.06^**	2.16±0.16^*
2 Gy	0.67±0.09^*	0.62±0.08^*	0.80±0.14^*	1.37±0.12^*	1.97±0.10^**
8 Gy	0.74±0.06^*	0.71±0.16^*	0.80±0.04^**	1.13±0.13^**	1.70±0.18^*

Group	Percentage of cells			Apoptotic rate
Group	G₀/G₁	S	G₂/M	Apoptotic rate
shCon-HeLa
0 Gy	60.32±3.47	28.81±3.11	9.54±1.20	5.51±1.22
2 Gy	69.12±0.93	23.61±2.06	7.28±1.14	6.53±0.37
8 Gy	19.48±0.47	18.29±1.29	62.23±0.83	8.15±0.23
shATRX1-HeLa
0 Gy	57.60±0.42	29.51±0.16	12.89±0.26^*	10.17±1.34^*
2 Gy	62.01±0.72^*	26.91±1.89	11.09±1.59^*	13.43±1.63^*
8 Gy	36.25±2.29^*	17.83±0.39	45.82±1.90^*	15.84±1.23^**

[1]	Zhijuan WANG,Mingshu ZHANG,Liping YE. Effects of platelet-derived growth factor D on proliferation, migration and invasion of lung cancer H1299 cells through ERK signaling pathway and their mechanisms [J]. Journal of Jilin University(Medicine Edition), 2022, 48(4): 898-904.
[2]	Qingxu LANG,Xueshuang NIU,Kaiwen YANG,Ren ZHANG,Siteng WANG, ZUMIRETIGULI·Wumaier,Zhenqi WANG. Effects of sodium butyrate combined with ionizing radiation on apoptosis of lung cancer A549 cells and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(4): 915-921.
[3]	Yandi MA,Xiangyun LU,Shangfeng HE,Xueyan YU,Yunhua HU,Haixia GAO,Yunzhao CHEN,Jie YU,Wenjie WANG,Feng LI,Xiaobin CUI. Expression of m6A methylation binding protein YTHDF2 in esophageal carcinoma tissue and its effect on proliferation and migration of esophageal carcinoma cells [J]. Journal of Jilin University(Medicine Edition), 2022, 48(4): 962-970.
[4]	Guowu WANG,Yuan YAO,Yu ZHANG,Na XU,Fang LIU. Inhibitory effect of miR-152 on proliferation and invasion of endometrial carcinoma cells by reducing low-density lipoprotein receptor expression [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 591-599.
[5]	Guanhu LI,Qingxu LANG,Chunyan LIU,Qin LIU,Mengrou GENG,Xiaoqian LI,Zhenqi WANG. Inhibitory effect of valproic acid combined with X-ray irradiation on proliferation of breast cancer MDA-MB-231 cells and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 622-629.
[6]	Peiyu YAN,Aichen ZHANG,Hong ZHANG,Yang LI,Mengmeng ZHANG,Mengze LUO,Ying PAN. Therapeutic effect of adipose-derived mesenchymal stem cells on premature ovarian failure model rats and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 648-656.
[7]	Qiuting CAO,Jingchun HAN,Xiaofei ZHANG. Effect of silencing helicase BLM gene on chemotherapy sensitivity of irinotecan in colorectal cancer cells and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 657-667.
[8]	Cuilan LIU,Fengai HU,Jing LIU,Dan WANG,Changyun QIU,Dunjiang LIU,Di ZHAO. Effect of adiponectin receptor agonist AdiopRon on biological behaviors of glioma cells and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 702-710.
[9]	Ming xing YANG,Wen DONG,Ji LI. Inductive effect of peiminine on apoptosis of lung cancer A549 cells and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 711-717.
[10]	Zhuangzhi WU,Xiaoning HE,Siqi CHEN. Inhibitory effect of miR-124-3p on proliferation and invasion of oral squamous cell carcinoma cells and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 718-727.
[11]	Ming LI,Qiuting WANG,Shan CHEN,Huifang SHI. Improvement effect of p38 MAPK inhibitor on chronic obstructive pulmonary disease injury in mice through inhibiting cell pyrotosis mediated by NLRP3 pathway [J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 744-754.
[12]	Suxian CHEN,Zehui GU,Yangfei MA,Qi TAN,Qi LI,Yadi WANG. Promotion effect of rutin on apoptosis of human colon cancer SW480 cells and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(2): 356-363.
[13]	Zhihui ZHAO,Xianghua BAI,Jinling HE,Weiqin DUAN,Min LIU,Shengmao ZHANG. Inhibitory effect of sufentanil on apoptosis of myocardial cells in myocardial ischemia-reperfusion injury rats and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2022, 48(2): 364-373.
[14]	Xiaoyan LI,Wei ZHANG,Jie HE. Promotion effect of REG1A on proliferation and migration of lung adenocarcinoma cells by regulating Wnt/β-catenin signaling pathway [J]. Journal of Jilin University(Medicine Edition), 2022, 48(2): 444-453.
[15]	Guangsong XU,Haibing JIANG,Jing PAN,Guoqing LI. Inhibitory effects of betulinic acid on migration and invasion of gastric cancer MGC-803 cells and their mechanisms [J]. Journal of Jilin University(Medicine Edition), 2022, 48(1): 122-128.

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

RichHTML

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 5

References 21

Related Articles 15

Metrics

Comments

Recommended 1