Effect of ionizing radiation on DNA damage repair in hela cells silencing ATRX / 吉林大学学报(医学版)

ABSTRACT

Objective: To silence α-thalassemia/mental retardation syndrome XTinked gene (ATRX) in the cervical cancer HeLa cells, to detect the effect of ionizing radiation on the protein expressions of ATRX, γH2AX, Rad51 and γH2AX, Rad51 foci, and to explore the role of ATRX in DNA damage repair of the HeLa cells after irradiation. Methods: Three ATRX-shRNA and negative Control-shRNA lentiviral vectors were transfected into the 293T cells, and the Antiviruses were collected to infect the HeLa cells; puromycin was used to obtain the HeLa cells stably silencing ATRX named shAi-HeLa, shA2-HeLa, shA3-HeLa, and shCon-HeLa; the silencing efficiency was detected by Western blotting method. After ionizing radiation, the expressions of ATRX, γH2AX, and Rad51 proteins were measured by Western blotting method, and the numbers of γH2AX and Rad51 foci in shCon-HeLa and shAi-HeLa groups were observed and counted by immunofluorescence technique. Results: The ATRX protein expressed in shCon-HeLa cells, but did not express in shA1-HeLa, shA2-HeLa, and shA3-HeLa cells; it indicated that the silencing efficiency was higher. At 1, 6, and 24 h after 2 and 8 Gy irradiation, the ATRX protein expression levels in shCon-HeLa group were increased gradually; it was most at 24 h, and the ATRX was highly expressed at 1, 6, and 24 h after 8 Gy irradiation. Compared with shCon-HeLa group, at 0-6 h after 4 Gy irradiation, the number of γH2AX foci in shA1-HeLa group was significantly increased at 1 h (P<0. 05), then was gradually decreased, but the number of γH2AX foci in shA1-HeLa group was still higher at 6 h (P<0. 01). The number of Rad51 foci was consistent with the changes of γH2AX focus number. Compared with shCon-HeLa group, the number of Rad51 foci was significantly increased at 1 h (P<0. 05), and the number in shA1-HeLa group was still higher at 6 h (P<0.01). At 0-16 h after 4 Gy irradiation, compared with shCon-HeLa, the expression amounts of γH2AX and Rad51 proteins in shAi-HeLa group were increased. Conclusion: The HeLa cell models silencing ATRX are successfully obtained; ionizing radiation can cause the increase of ATRX expression level; the focus number and the protein expression amounts of γH2AX and Rad51 in HeLa cells silencing ATRX are higher than those in control group, which indicates that ATRX involves in the repair of radiation-induced DNA damage.