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 withγ-rays,respectively.The extent of DNA damage was evaluated by using comet assay and immu-nofluorescence staining againstγ-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.
Zhijie WanJingwen GuSongyun ZhaoHang JiaTingting LiuYuanyuan ChenYanyong Yang
Triple-negative breast cancer(TNBC)is highly malignant and refractory to immunotherapy through impeding the immune cell infiltration and inflammation in the tumor microenvironment(TME).1 DNA-dependent protein kinase catalytic subunit(DNA-PKcs)is a member of the phosphatidylinositol 3-kinase-related kinase family,which is required for the non-homologous end joining repair.2 The effect of DNA-PKcs on the generation of cytosolic DNA and inflammation response in tumor immuno-environment is not defined.We found a specific DNA-PKcs inhibitor,NU7441,induced cytosolic DNA,stimulator of interferon genes(STING),and retinoic acid-inducible gene I(RIG-I)signals in vitro.In Balb/c immune-competent mice bearing 4T1 TNBC cells,NU7441 impaired the tumor growth and metastasis,and increased the CD45+leukocytes,CD4+T cells,CD8+T cells,and CD1a+antigen-presenting cells,as well as MHC-I and interferon alpha receptor(IFNAR)in TME.However,in Balb/c athymic nude mice without IFNAR and CD8+T cells in TME,NU7741 did not influence tumor growth.These results show that inhibition of DNA-PKcs triggers cytosolic DNA sensing and induces an inflamed TME to promote anti-tumoral immunity,which provides a strategy to alter the inflammation and lymphocyte infiltration in TME to increase the efficacy of immunotherapy in TNBC and other cancers with an immune-suppressive TME.
