Effect Of ATF5 On EMT In Radiotherapy Resistance Cell Of Nasopharyngeal Carcinoma

Objective: To observe the effects of X-ray on the morphology,proliferation,invasion and colony formation of HONE-1 cells and the expression of ATF5,E-cadherin and N-cadherin protein.Further compare the expression levels of Activating transcription factor 5(ATF5)in recurrent and initial nasopharyngeal carcinoma(NPC)tissues from the same patients.Preliminarily explore the possible mechanism of ATF5 protein involved in radioresistance of NPC cells in order to provide a theoretical basis for improving the sensitivity of radiotherapy for NPC.Methods: The nasopharyngeal carcinoma cells,HONE-1 were irradiated by conventional fractionation to construct the radiotherapy resistant cells HONE-1R.HONE-1R cells were identified by colony formation assay,multi-target click model fitting survival curve and proliferation assay.We observed and recorded the morphological changes in HONE-1 cells under the microscope;Immunohistochemistry was used to detect the expression of ATF5 protein in primary tissues of initial and recurrent NPC and matched to normal tissues of nasopharynx in 6 patients with nasopharyngeal carcinoma.The impact of X-ray irradiation on the expression levels of ATF5,E-cadherin and N-cadherin in HONE-1 cells was detected by immunocytochemistry.Cellular immunofluorescence was used to detect the changes in cellular localization and expression of ATF5 protein in HONE-1 cells caused by X-ray irradiation;The shRNA-ATF5 lentiviral vector was re-constructed and packaged,and the interference efficiency of shRNA-ATF5 was verified by qRT-PCR.The effect of X-ray and shRNA-ATF5 on the ability of HONE-1 cells and HONE-1R cells to clone was detected by colony formation assay;Multi-target click model fit survival curve were used to analyze the effect of X-ray and shRNA-ATF5 on SF2 values of these two types of nasopharyngeal carcinoma cells,SF2 value refers to the survival fraction of cells after exposure to 2Gy X-ray(Survival fraction at 2Gy,SF2).CCK-8 proliferation assay and Transwell chamber assay were used to detect the effects of X-ray and shRNA-ATF5 on the survival rate,migration and invasiveness of these two types of nasopharyngeal carcinoma cells.Results:(1)X-ray could significantly promote the up-regulation of ATF5 protein level in HONE-1 cells,and the expression of ATF5 protein increased with the dose of X-ray irradiation;the ATF5 in subcellurs location also move from the cytoplasm to the nucleus.(2)The colony formation rate of HONE-1R cell was significantly higher than that of HONE-1 cell(P<0.05).The SF2 value of HONE-1R cells was 1.712 times that of HONE-1 cells,As for HONE-1R and HONE-1 cells through the 0-10 Gy conventional fractionation of X ray,the survival rate of the HONE-1R cells was significantly higher than that of the HONE-1 cell.(P<0.05)The migration and invasion ability of HONE-1R cells were significantly increased compared with HONE-1 cells(P<0.05).(3)The expression level of ATF5 protein in nasopharyngeal carcinoma tissues was significantly higher than that in normal nasopharyngeal tissues(P<0.05);the subcellular localization was mainly located in cytoplasm.The expression of ATF5 protein in recurrent nasopharyngeal carcinoma was significantly higher than that in initial nasopharyngeal carcinoma(P=0.032),and the subcellular localization was mainly located in the nucleus.(4)What’s more X-ray could also promote the morphology of epithelial-mesenchymal transition(EMT)in HONE-1 cells,inducing the expression down-regulation of E-cadherin,and the expression up-regulation of N-cadherin in a dose-dependent manner.(5)shRNA-ATF5 could significantly down-regulate the expression level of ATF5 mRNA and the colony formation rate in HONE-1R cells(P<0.05),and decreases the cell survival rate,migration and invasiveness of HONE-1R cells(P<0.05).Meanwhile,sensisizing the HONE-1R cells to X ray’s irradiation(P<0.05),significantly up-regulating the expression of E-cadherin in HONE-1R cells(P<0.05),and down-regulating the expression of N-cadherin in HONE-1R cells(P<0.05).Conclusion:(1)ATF5 was involved in the radioresistance of HONE-1 cells,and the EMT changes in HONE-1 radioresistance cells.(2)ATF5 silencing can reverse the EMT phenotype of radiotherapy resistant cells of NPC and increase the radiotherapy sensitivity of radiotherapy resistant cells of NPC,suggesting that ATF5 protein may be involved in the radiotherapy resistance of NPC through mediating EMT.