Effect of Rad51 gene silencing on repair of lead-induced DNA double-strand breaks in TK6 cells

  Objective  To study the effect of Rad51 gene silencing on the repair of DNA double-strand breaks in human lymphoblastic cells (TK6 cells) induced by lead acetate.

  Methods  The TK6 cells were infected with Rad51 silent lentiviral vector to construct Rad51 gene silencing cell, which were confirmed by fluorescence quantitative PCR and Western blot. TK6 cells (control, shRNA-NC and shRNA-Rad51)were exposed to 480 μmol/L lead acetate for 24 hours. The expression of phosphorylated histone H2AX (γ-H2AX)and the expression of Rad51, BRCA1 and 53BP1 proteins in TK6 cells were detected by Western Blot.

  Results  The expression of Rad51 mRNA and Rad51 protein in the shRNA-Rad51 group were significantly lower than those in the Control group and the shRNA-NC group (P < 0.01). The positive rate of γ-H2AX in the shRNA-Rad51 group was (27.48 ± 1.66)%, which was higher than that in the Control group (14.77 ± 1.21)% and the shRNA-NC group (14.04 ± 1.31)% (P < 0.01). The expression level of BRCA1 protein in the shRNA-Rad51 group was (0.25 ± 0.03), which was lower than that in the Control group (0.55 ± 0.04) and the shRNA-NC group (0.51 ± 0.04) (P < 0.01). The expression level of 53BP1 was(3.24 ± 0.27)in the shRNA-Rad51 group, which was higher than that in the Control group(2.01 ± 0.19)and the shRNA-NC group(2.11 ± 0.17)(P < 0.01).

  Conclusions  After Rad51 gene silencing, HR repair pathway was inhibited and NHEJ repair pathway was activated in TK6 cells, and the sensitivity of TK6 cells to lead genotoxicity was enhanced.