The Role Of EGLN3 In The Proliferation And Radioresistance Of Nasopharyngeal Carcinoma

Part ? Evaluation of the prognostic value of EGLN3 in NPCObjective:To explore the potential clinical significance of EGLN3 in NPC patientsMethods:Compare the difference of EGLN3 expression between nasopharyngeal carcinoma and normal nasopharyngeal cell lines.The expression of EGLN3 was evaluated by immunohistochemistry of tissue microarrays from a cohort of NPC patients.Then we explore the correlation between the expression of EGLN3 and patient survival through Kaplan-Meier assay.Results:EGLN3 was down-regulated in the cancer cells,it was mainly expressed in the cytoplasm.NPC patients with low EGLN3 expression had shorter overall survival and progression-free survival than those with high EGLN3 expression,and EGLN3 expression appeared to be an independent predictor of progression-free NPC survival.Conclusion:EGLN3 is downregulated in NPC cells and the downregulation is associated with poor prognosis in NPC patients.Part ? The effect of EGLN3 on the malignant behaviors of NPC cells.Objective:To explore the effects of EGLN3 on the malignant behaviors of NPC cells including proliferation and radiosensitivityMethods:We used Western Blot to determine the lentiviral transfection efficiency.We performed CCK8,clone formation assay and xenograft experiments to assess the effect of EGLN3 on tumor proliferation in NPC cell lines in vitro and in vivo.We used Clone formation,immunofluorescence,flow cytometry experiments to assess the effect of EGLN3 on tumor radio-sensitivity in NPC cell lines.Results:EGLN3 knockdown promoted NPC cell growth and colony formation,and EGLN3 upregulation inhibited proliferation;knockdown of EGLN3 contributed to proliferation in vivo in the xenograft mice;together,these data suggest that EGLN3 suppresses NPC proliferation.Exposed to different doses of irradiation the EGLN3knockdown cells showed less sensitivity to the irradiation than empty vector cells,and upregulation of EGLN3 increased the NPC cells'irradiation sensitivity;there were fewer yH2AX foci in the Sh-EGLN3 cells than in the empty-vector cells after irradiation at each time point;EGLN3 knockdown also decreased apoptosis after irradiation compared with the vector cells;these results suggest that knockdown of EGLN3 improves NPC cells'ability to repair DNA damage,desensitizing them to irradiation treatment.Conclusion:Our in vivo and in vitro experiments demonstrated that the knockdown of EGLN3 in NPC cells led to tumor growth and desensitize them to irradiation treatment.Part ? The mechanism of EGLN3 regulation in nasopharyngeal carcinoma cellsObjective:To explore the mechanism of EGLN3 regulation in nasopharyngeal carcinoma cellsMethods:To explore the functions of EGLN3,we extracted NPC's mRNA gene expression profiles from the GEO database(file GSE34573)and subjected them to gene set enrichment analysis.We performed Western Blot to validate the results obtained by the gene set enrichment analysis.We then used immunoprecipitation to explore the association between EGLN3 and RKIP.Futhermore,we treated NPC cell lines with prolyl hydroxylase inhibitor DMOG to inhibits hydroxylase activity and to test if the interaction between EGLN3 and RKIP are regulated by EGLN3 hydroxylase activity.Finally,we detect the phosphorylation of RKIP after treating with DMOG to explore the effect of EGLN3's hydroxylase activity on RKIP's activity.Results:EGLN3 levels were correlated with the gene signatures of the ERK pathway.EGLN3 knockdown increased the levels of P-ERK and upstream P-MEK as compared to the control group.Consistently,upregulation of EGLN3 in NPC cells inhibits the phosphorylation of ERK and MEK.However,the level of phosphorylation of C-Raf(ser338)protein,crucial for the activation of MEK and ERK,did not change.EGLN3 appeared to interact with RKIP and vice versa.Knockdown of EGLN3 decreased the association between RKIP and C-Raf.Moreover,the level of phosphorylation of RKIP(Ser153)increased after EGLN3 knockdown,indicating that the interaction between EGLN3 and RKIP activates the MEK/ERK pathway by influencing the phosphorylation of RKIP and the interaction between RKIP and C-Raf.We further found that DMOG treatment block the interaction between EGLN3 and RKIP.Upregulation of EGLN3 inhibited the phosphorylation of RKIP,and treating with DMOG restored the level of phosphorylation of RKIP(Ser153).These data indicate that EGLN3's hydroxylase activity is key to the binding of EGLN3 and RKIP as well as the phosphorylation of RKIP.Conclusion:EGLN3 could interact with RKIP and regulate RKIP inhibition of C-Raf by modulating the phosphorylation of RKIP in prolyl hydroxylation dependent manner to suppress the activity of MEK/ERK pathway.Part ? RKIP mediated the biological functions of EGLN3 in NPC cell linesObjective:To explore if RKIP mediates the biological functions of EGLN3 in NPC cell linesMethods:CNE2 and HNE1 cells were transfected with RKIP overexpression plasmid and shRNA targeting EGLN3.Western Blot was used to detect the the phosphorylation of MEK and ERK after transfection.CCK8 and clone formation assay were performed to explore the effect of RKIP overexpression on the proliferation of NPC cells.Results:EGLN3 knockdown resulted in increased P-ERK levels.However,upregulation of RKIP decreased the protein level of P-ERK in EGLN3-knockdown cells,indicating that the activation of the ERK pathway induced by EGLN3 knockdown is dependent on RKIP.Upregulation of RKIP did not affect the expression of EGLN3,and that NPC cell growth and proliferation induced by EGLN3 knockdown were restored by RKIP upregulation.Conclusion:RKIP mediates the biological effects of EGLN3,and the inhibitory effect of RKIP on the MAPK pathway is regulated by the interaction between RKIP and EGLN3,which in turn depends on the hydroxylase activity of EGLN3.