The Functional Role Of TAB182in Ionizing Radiati-on-induced DNA Damage And Repair In Hela Cell

AimTo study the biological functions and molecular mechanisms of TAB182involved in DNA damage induced by ironizing radiation in HeLa,to further understand the basis of human health and disease on the molecular level, and to provide experimental and theoretical evidences for maintaining human health and promoting disease therapy development, especially for the study of anticancer drugs.MethodThe TAB182gene silenced HeLa cell line (HeLa-TAB182shRNA) was established by shRNA. DNA damage of HeLa-TAB182shRNA was induced by ionizing radiation. Radiation sensitivity of HeLa-TAB182shRNA was determined by colony formation assay. Flow cytometry and western blotting were done to analyze cell cycle distribution and DNA damage response associated protein levels respectively in HeLa-TAB182shRNA after ionizing radiation. To study the role of TAB182in cell cycle progression, the cell cycle synchronization of HeLa-TAB182shRNA was achieved by TDR method. The PLPC-Myc-TAB182-FL vector was transiently transfected into HeLa cells, and western blotting and confocal laser scanning microscopy analysis were performed to examine the influence of TAB182on phosphorylation of DNA-PKcs. The interactions between TAB182and DNA-PKcs, TNKS and DNA-PKcs were detected by co-immunoprecipitation (Co-IP).Results1. The expression of TAB182was induced by ionizing radiation. After4Gy irradiation treatment, the expression of TAB182in HeLa cell reached a peak at the time point of2h. When treated with different doses of radiation and harvested2h later, HeLa cell showed the peak of TAB182expression in the4Gy irradiation group, indicating that the expression of TAB182is mainly affected by the low doses of radiation.2. Interactons between TAB182and DNA-Pkcs, TNKS and DNA-PKcs. The result of immunofluorescence in situ hybridization experiments revealed that TAB182co-localizated with DNA-PKcs phosphoryiated at2056sites. Co-immunoprecipitation also confirmed the interaction between TAB182and DNA-Pkcs, TNKS and DNA-Pkcs.3. TAB182knocking-down by shRNA increased the radiation sensitivity in HeLa cells. The result of colony formation assay showed that radiation sensitivity of HeLa-TAB182shRNA was increased when treated with low-dose gamma ray irradiation of4Gy, but not the high-dose of8Gy.4. TAB182involved in the regulation of cell cycle progression. After synchronized cell cycle by TDR method, the cells showed different TAB182expression levels in different cell cycle phases. Compared with control cells, HeLa-TAB182shRNA cells speed up S phase progression, indicating that TAB182involved in the regulation of cell cycle S phase progression.5. TAB182involved in the regulation of cell cycle arrest induced by ionizing radiation. Flow cytometric analysis of ionizing radiation-induced cell cycle arrest revealed that the G2/M phase arrest period of HeLa-TAB182shRNA cells was significantly longer than that of the control cells.6. The regulation of TAB182on the expression levels of DNA damage response proteins. Western blot analysis showed that the expression of DNA damage response proteins such as DNA-PKcs, ATM, Chk2, and p53in HeLa-TAB182shRNA cells was significantly lower than that of control cells.7. The impact of TAB182on the phosphorylation of DNA-PKcs. HeLa was transiently transfected with PLPC-Myc-TAB182-FL vector and then was processed to western bloting analysis and immunofluorescence microscopy experiments. The results showed that high expression of TAB182could increase the phosphorylation of DNA-PKcs at the site of S2056.Conclusions1. TAB182involved in DNA damage and repair pathway.2. TAB182interacts with DNA-Pkcs, and increases the phosphorylation of DNA-PKcs at S2056site. 3. TAB182involves in regulation of S phase progression and ionizing radiation-induced G2/M arrest.