TAB182 promotes DNA homologous recombination repair by stabilizing RPA2 mRNA
10.3760/cma.j.cn112271-20210930-00400
- VernacularTitle:TAB182维持RPA2 mRNA稳定性促进DNA同源重组修复的研究
- Author:
Gang LI
1
;
Shuting LAI
;
Yang HAN
;
Chenjun BAI
;
Hua GUAN
;
Shanshan GAO
;
Pingkun ZHOU
Author Information
1. 南华大学公共卫生学院，衡阳 421001
- Keywords:
TAB182;
RPA2;
DNA double-strand break;
HR repair;
Radiosensitivity
- From:
Chinese Journal of Radiological Medicine and Protection
2022;42(4):241-247
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the regulating molecules and acting mechanism of TAB182 in HR pathway.Methods:TAB182 in human breast cancer MCF-7 cells was knocked down by shRNA strategy, the TAB182 knockdown MCF-7 as the TAB182 knockdown group, and the MCF-7 cell using the shRNA negative control as the TAB182 negative control group. RNA sequencing and qRT-PCR were performed to screen and verify the differentially expressed genes of HR pathway related to TAB182 depression. Western blot was used to detect protein expression. Immunofluorescence staining of nuclear RAD51 and BrdU was used to check the 3′ ssDNA formation by the end resection. The cell cycle arrest and apoptosis were measured by flow cytometry. Cloning formation assay was used to evaluate the sensitivity TAB182-knockdown cells to radiation.Results:Both quantitative RNA sequencing and qRT-PCR assays showed that TAB182-knockdown significantly decreased the mRNA expression of RPA2( t=17.97, P<0.05). Compared with the TAB182 negative control group, the protein level of RPA2, the number of RAD51 foci, and the 3′ ssDNA-binding nuclear protein marker BrdU in TAB182-knockdown cells were significantly reduced. At 4, 8, and 12 h after actinomycin D treatment, the attenuation of RPA2 mRNA in the TAB182-knockdown cells was accelerated ( t=5.37, 3.79, 3.69, P<0.05). Compared with the TAB182 negative control group, the radiosensitivity and radiation-induced apoptosis in the TAB182-knockdown group were increased ( t=3.48, 11.05, P<0.05), and at 24 h after irradiation, the cell cycle block time was prolonged ( t=8.40, P<0.01). Conclusions:TAB182 plays a role in maintaining RPA2 mRNA stability, thereby promoting HR repair. TAB182 knockdown cells are highly sensitive to ionizing radiation.
