The repair role of RAD51 on lead-induced DNA double-strand break in TK6 cells
10.20001/j.issn.2095-2619.20250804
- VernacularTitle:RAD51对铅诱导TK6细胞DNA双链断裂损伤的修复作用
- Author:
Jialing ZHANG
1
;
Xiangquan LIU
Author Information
1. College of Public Health and Medical Technology, Xiamen Medical College, Xiamen, Fujian 361023, China
- Publication Type:Journal Article
- Keywords:
RAD51;
Lead;
TK6 cells;
DNA double-strand breaks;
DNA repair;
Gene silencing
- From:
China Occupational Medicine
2025;52(4):386-392
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the genotoxicity of lead on DNA damage and the repair role of homologous recombination repair protein RAD51 in lead-induced DNA double-strand breaks in TK6 cells. Methods i) TK6 cells in the logarithmic growth phase were divided into five groups: blank control group, low-dose group, medium-dose group, high-dose group, and positive control group. The first four groups were treated with lead acetate at concentrations of 0, 120, 240 and 480 μmol/L, respectively. The positive control group was treated with 100 μmol/L hydrogen peroxide solution incubated on ice for 24 hours. After the positive rate of phosphorylated histone H2AX (γ-H2AX) was detected by immunofluorescence. Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EDU) method. Cell cycle and apoptosis were detected by flow cytometry, and the relative expression of RAD51 protein was detected by Western blotting. ii) A short hairpin RNA (shRNA) silencing RAD51 model was constructed in TK6 cells, and the cells were divided into untreated group (normal TK6 cells), negative control group (sh-NC), and RAD51 silencing group (sh-RAD51). The mRNA expression of RAD51 was detected by real-time fluorescence quantitative polymerase chain reaction, and the protein expression of RAD51 was detected by Western blotting to verify the interference effect. iii) Normal TK6 cells, sh-NC, and sh-RAD51 were treated with 480 μmol/L lead acetate for 24 hours, and were divided into lead treatment group, lead+negative control group, and lead + RAD51 silencing group, respectively. Untreated normal TK6 cells was served as the control group. The positive rate of γ-H2AX and the expression of RAD51 protein in each group was detected by immunofluorescence and Western blotting, respectively. Results i) With the increase of lead acetate treatment concentration in TK6 cells, the positive rate of γ-H2AX increased (all P<0.05), the proportion of EDU positive cells decreased (all P<0.05), and the proportion of S-phase cells decreased (all P<0.05). The proportion of G1-phase cells in the high-dose group and the positive control group was higher than those in the low- and medium-dose groups (all P<0.05). The proportion of G2-phase cells in the medium-dose group, high-dose group, and positive control group increased compared with the blank control group (all P<0.05), although no significant differences were observed among these three dosing groups (all P>0.05). The apoptosis rate and the relative expression of RAD51 protein of TK6 cells in each group increased with the increase of lead acetate concentration (all P<0.05). ii) The relative expression levels of RAD51 mRNA and protein in the RAD51 silencing group were lower than those in the untreated group and the negative control group (all P<0.05) after RAD51 silencing. iii) The positive rate of γ-H2AX in the lead + RAD51 silencing group increased (all P<0.05), and the relative expression of RAD51 protein decreased (all P<0.05),compared with the control group, lead treatment group, and lead + negative control group. Conclusion Lead exposure induces DNA double-strand breaks in TK6 cells, inhibits cell proliferation, and causes cell cycle arrest and apoptosis in a dose-effect manner. Downregulation of the RAD51 gene can lead to inhibition of the homologous recombination repair pathway, thereby increasing the TK6 cell sensitivity to lead-induced DNA damage.
