The Effect Of QSOX1 On Radiosensitivity And Radiation-induced Autophagy In Nasopharyngeal Carcinoma

Part?Expression of QSOX1 Protein in NPC Cells and Samples and Effectof QSOX1 Inhibition on Radiosensitivity of CNE-2 CellsObjectiveIn our previous study,we identified that QSOX1 is a differentially expressed protein in NPC cell lines with variable radiosensitivities.The present study aimed to investigate the biological behavior of QSOX1 in nasopharyngeal carcinoma(NPC)and its effect on radiosensitivity.MethodWestern blot was used to test the protein expression of QSOX1 in different NPC cell lines with variable radiosensitivities.Immunohistochemistry(IHC)and enzyme-linked immunosorbent assay(ELISA)assay were used to evaluate the expression levels of QSOX1 in NPC patient sera and tissue samples.The QSOX1 gene was designed and synthesized three interference targets,and then packed slow virus,application of RT-PCR and Western-blot showed the best target effect on silence to infection to CNE-2 cells,to obtain stable silencing cell line by purocymin screening.Furthermore,CCK-8 method,colony-forming assay,flow cytometry technology and Transwell assay were performed to detect changes of radiosensitivity of nasopharyngeal carcinoma CNE-2 cells after QSOX1 gene silencing.In the meantime,we observed QSOX1 gene silencing effects on the transplanted tumors growth of nude mouse induced by IR.ResultThe levels of QSOX1 detected by ELISA and IHC in radioresistant NPC patient sera and tissue samples were markedly lower than those in radiosensitive samples.Small hairpin RNAs(shRNAs)were employed to knock down endogenous QSOX1 expression in CNE-2 cells,and then,radiosensitivity,apoptosis,migration and invasion were assessed using colony formation,Cell Counting Kit-8(CCK-8),flow cytometry,and transwell assays,respectively.Tumor growth and radioresistance were also evaluated using a xenograft model in nude mice.The shRNA-mediated knockdown of QSOX1 significantly increased cell survival under irradiation(IR)and weakened radiosensitivity,which was likely due to a reduction in the cell apoptosis rate after IR.Moreover,QSOX1 silencing led to the suppression of cellular migration and invasion.Similar results were obtained with the xenograft mouse model.ConclusionQSOX1 protein is associated with radiosensitivity of NPC.Down regulation of QSOX1 gene can decrease the sensitivity of CNE-2 cells to radiation.Part?Effect of QSOX1 Protein on Radiation-induced Autophagy in CNE-2 CellsObjective The aim of this part was to investigate the effect of QSOX1 protein on radiation-inducedautophagy in CNE-2 cells,and to provide the theoretical basis for nasopharyngeal carcinoma radiosensitization.Method The level of microtubule-associated proteins light chain 3(LC3)was measured using a western blot after irradiation.Lentivirus-mediated overexpression of RFP-GFP-LC3 was constructed.The Confocal Laser Scanning Microscope(CLSM)was then used to evaluate the autophagy flow.In addition,cells were treated with chloroquine(CQ)to figure out the influence of QSOX1 on IR-induced cne-2 autophagy.Result The results of Western blot indicated that the LC3-II protein levels show a certain radiation dose and time dependenceafter IR in CNE-2.The LC3-II protein level in the QSOX1-interfering combined IR group was significantly higher than that in the IRalone groups(Control and NC)(F=6.88,P=0.032),and the p62 protein level was lower compared with the other two groups.After treated with chloroquine(CQ),the LC3-II protein levels were increased in both Control and NC groups,but there was no significant change in sh QSOX1 group(P>0.05).The results of dual-fluorescence RFP-GFP-LC3 detection showed that the number of both green and red spots in the sh QSOX1 group were increased significantly compared with the other two groups.After treated with CQ,the number of green spot in each group increased significantly(control: P=0.01;sh QSOX1: P<0.01).Compared with the Control+CQ group(P=0.045),the number of red spot in the sh QSOX1+CQ group did not decrease significantly(P>0.05).Comparing the ratios of red and green fluorescent spots in each group,the autophagic flux in the sh QSOX1 group was significantly lower than in the other two groups(F=13.859,P=0.002).Conclusion Down-regulation of QSOX1 can increase the number of autophagosomes in NPC cells after IR.Reduced QSOX1 can inhibit the autophagic flux throughthe inhibition of autophagosome/lysosome fusion and may decrease the degradation of autolysosomes.In addition,QSOX1 may affect radiosensitivity by regulating radiation-induced autophagy in nasopharyngeal carcinoma cells.