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^**

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

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