Knockdown of the nucleoporin Nup50 protects cells against ionizing radiation through enhancing DNA-PKcs-mediated DNA damage repair

Abstract

Objective

To investigate the effect and mechanism of Nup50 on radiation-induced DNA damage repair to radiation and explore the potential role of Nup50 as radioprotective target.

Methods

The Nup50 gene was knocked down in HUVEC cells using lentiviruses. Colony formation, CCK-8, and flow cytometry were performed to determine the viability, proliferation and apoptosis of HUVEC cells treated with γ-rays，respectively. The extent of DNA damage was evaluated by using comet assay and immunofluorescence staining against γ-H2AX. In addition, we explored the role of Nup50 in DNA damage response (DDR) pathways through western blotting assay. Finally, nuclear and chromatin fractionation were performed to determine the potential molecular mechanism underlying the radiation protection function of Nup50 knockdown.

Results

Nup50 knockdown increased the cellular resistance to ionizing radiation. The CCK-8 data showed that cell viability was significantly increased in the Nup50 knockdown group after radiation (t ​= ​4.23, P ​< ​0.01). The Nup50 knockdown group also showed more survived colonies (t ​= ​10.06, P ​< ​0.001), less apoptosis rate (t ​= ​3.78, P ​< ​0.05) and less unrepaired DNA damage. Furthermore, Nup50 knockdown increased radiation-activated phosphorylation levels of DNA-PKcs in HUVEC cells. Finally, the nuclear and chromatin fractionation data showed that inhibiting Nup50 increased the recruitment of DNA-PKcs to chromatin after DNA damage.

Conclusions

Our findings revealed that Nup50 knockdown promoted radioresistance in normal HUVEC cells by regulating DNA-PKcs pathway, suggesting Nup50 as a potential target for radiation protection.