NOTCH2 Negatively Regulates Tumorigenicity And The Epithelial-Mesenchymal Transition In Nasopharyngeal Carcinoma Via TRAF6/AKT

Background:Nasopharyngeal carcinoma(NPC)is an epithelial malignancy of unknown pathogenesis,which is common in southern China,southeast Asia and some parts of north Africa.At present,EB virus infection,environmental and genetic factors are related to the occurrence and development of nasopharyngeal cancer.Radiotherapy alone and chemoradiotherapy are the main treatment means at present.However,the5-year recurrence rate of nasopharyngeal carcinoma patients with metastasis receiving standard treatment was 82%,and the 5-year survival rate was still very low.Therefore,it is extremely urgent to clarify the mechanism in the occurrence,development,invasion and metastasis of nasopharyngeal carcinoma and to find effective targeted drugs for the treatment of nasopharyngeal carcinoma.NOTCH signaling plays a role in promoting the angiogenesis,tumor invasion and metastasis of nasopharyngeal carcinoma.However,the exact role of these four NOTCH receptors and their mechanisms remain unclear.The purpose of this study was to investigate the effect of NOTCH2 on the growth,proliferation,invasion and metastasis of nasopharyngeal carcinoma cells;and to clarify the molecular mechanism of NOTCH2 in nasopharyngeal carcinoma cells.This study was divided into three parts:The first part:Effect of NOTCH2 on metastasis and survival time of nasopharyngeal carcinomaObjective:To investigate the effect of NOTCH2 expression on nasopharyngeal carcinoma metastasis ability,malignancy and patient survival time.Methods:1.To investigate the difference of NOTCH2 in nasopharyngeal carcinoma tissues between patients with cervical lymph node metastasis and those without cervical lymph node metastasis by immunofluorescence staining and immunohistochemical staining.2.To investigate the relationship between NOTCH2expression and survival time of patients by survival analysis.3.To investigate the relationship between the malignant degree and metastasis of nasopharyngeal carcinoma cells and the expression of NOTCH2 by western blot.Results:1.The expression of NOTCH2 in nasopharyngeal tissues of patients with cervical lymph node metastasis was lower than the expression of NOTCH2 in nasopharyngeal tissues of patients without cervical lymph node metastasis(t=6.485P<0.05).2.Through survival analysis,it was found that low expression of NOTCH2was significantly associated with poor prognosis of patients(P=0.001<0.05).3.The expression of NOTCH2 and N2ICD protein in CNE-1 cells was significantly lower than CNE-2 cells.The expression of NOTCH2 and N2ICD protein in 5-8F cells was significantly lower than 6-10B by western blot.Conclusion:The expression of NOTCH2 in the nasopharyngeal carcinoma tissues of patients with metastatic nasopharyngeal carcinoma,the nasopharyngeal carcinoma cells with high metastasis and low differentiation was low.The expression of NOTCH2 is negatively correlated with survival time of patients with nasopharyngeal carcinoma.The second part:NOTCH2 inhibits the growth and metastasis of nasopharyngeal carcinoma cells.Objective:To investigate the effect of inhibiting or overexpressing NOTCH2 on the growth and metastasis of nasopharyngeal carcinoma cells.Methods:1.shNOTCH2 nasopharyngeal carcinoma cells were constructed using short interference RNA technology,and NOTCH2-overexpressing nasopharyngeal carcinoma cells were constructed with LV-NOTCH2.2.The relationship between NOTCH2 and the growth of nasopharyngeal carcinoma was verified by clonal formation assay in vitro.3.Xenograft model of nude mice was used to verify the relationship between NOTCH2 and the growth of nasopharyngeal carcinoma cells in vivo.4.The relationship between NOTCH2 and the migration ability of nasopharyngeal carcinoma cells was verified by scratch test in vitro.5.Transwell assay was used to verify the relationship between NOTCH2 and invasion and metastasis of nasopharyngeal carcinoma cells in vitro.6.The metastatic tumor model was used to verify the relationship between NOTCH2 and metastasis capacity of nasopharyngeal carcinoma cells in vivo.7.Cytoskeleton was observed to verify the relationship between NOTCH2 and the morphology of nasopharyngeal carcinoma cells by phalloidin.8.Flow cytometry was used to verify the relationship between NOTCH2andstemcelltransformationofnasopharyngealcarcinomacells.9.Immunofluorescence staining and western blot were used to verify the relationship between NOTCH2 and the expression of EMT-labeled protein(E-cadherin and Vimentin)in nasopharyngeal carcinoma cells.Results:1.shNOTCH2 nasopharyngeal carcinoma cells(shNOTCH2 CNE-2 cell line,shNOTCH2 5-8f cell line)and NOTCH2-overexpressing nasopharyngeal carcinoma cells(oeNOTCH2 CNE-2 cell line,oeNOTCH2 5-8f cell line)were constructed.2.Cloning and formation experiments showed that the colony formation ability of shNOTCH2 nasopharyngeal carcinoma cell line was significantly faster than the colony formation ability of Ctrl nasopharyngeal carcinoma cell(P<0.05),and the colony formation ability of oeNOTCH2 nasopharyngeal carcinoma cell was significantly slower than the colony formation ability of oeVec nasopharyngeal carcinoma cell(P<0.05).3.Xenograft model in nude mice showed that the tumor volume in the shNOTCH2 group was significantly larger than the tumor volume in the control group(P<0.05).4.The migration distance of shNOTCH2 nasopharyngeal carcinoma cell was significantly longer than the migration distance of Ctrl nasopharyngeal carcinoma cell,and the migration distance of oeNOTCH2nasopharyngeal carcinoma cell was significantly shorter than the migration distance of oeVec nasopharyngeal carcinoma cell.5.The number of transmembrane cells in shNOTCH2 nasopharyngeal carcinoma cell was significantly higher than the number of transmembrane cells in control nasopharyngeal carcinoma cell(P<0.05),and the number of transmembrane cells in oeNOTCH2 nasopharyngeal carcinoma cell was significantly lower than the number of transmembrane cells in oeVec nasopharyngeal carcinoma cell(P<0.05).6.The number of nude mice with metastatic lesions on the lung surface in shNOTCH2 group was significantly higher than the control group(P<0.05),and the number of metastatic lesions on the liver surface of nude mice in shNOTCH2 group was significantly higher than the control group(P<0.05).7.The inhibition of NOTCH2 cause mesenchymal changes in nasopharyngeal carcinoma cells.8.The number of CD133~+cells in the shNOTCH2 group was higher than the Ctrl group(P<0.05).9.The low expression of E-cadherin and the high expression of vimentin in shNOTCH2 group compared to the Ctrl group.The high expression of E-cadherin and the low expression of vimentin in the oeNOTCH2 group compared to the oeVec group.Conclusion:NOTCH2 inhibits the growth,proliferation,invasion and metastasis of nasopharyngeal carcinoma cells in vitro and in vivo.NOTCH2 inhibits mesenchymal changes and stem cell transformation of nasopharyngeal carcinoma cells.The third part:The molecular mechanism of NOTCH2 regulateing the metastasis of nasopharyngeal carcinoma cellsObjective:To explore the molecular mechanism of NOTCH2 inhibiting the growth and metastasis of nasopharyngeal carcinoma cells.Methods:1.Bioinformatics analysis was used to screen the signaling pathway associated with NOTCH2 regulating the biological behavior of nasopharyngeal carcinoma.2.Western blotting was used to verify whether the signaling pathway was involved in the regulation of NOTCH2 in the biological behavior of nasopharyngeal carcinoma.3.Inhibiting the expression of the signaling pathway,cytoskeleton was observed to verify whether the inhibition of the signaling pathway could reverse the effect of NOTCH2 on the morphology of nasopharyngeal carcinoma cells by phalloidin.4.Transwell assay was used to observe whether inhibition of the signaling pathway could reverse the effect of NOTCH2 on the migration and metastasis of nasopharyngeal carcinoma cell.5.Western blotting was used to verify whether inhibition of the signaling pathway could reverse the effect of NOTCH2 on the expression of EMT-labeled proteins(E-cadherin and Vimentin)in nasopharyngeal carcinoma cells.6.Western blot assay was used to detect the upstream molecules(X)of the signaling pathway associated with NOTCH2 regulating the biological behavior of nasopharyngeal carcinoma.7.Western blot was performed to verify the success of the construction of CNE-2 cells that inhibit X expression and NOTCH2 expression.8.Cytoskeletal assay was used to detect whether inhibition of X could inhibit the morphological changes of nasopharyngeal carcinoma cells that inhibit NOTCH2expression.9.Transwell assay was used to verify whether inhibition of X could inhibit the effect of NOTCH2 on the migration and metastasis of nasopharyngeal carcinoma cells.10.Western blot was used to verify whether inhibition of X could activate the signaling pathway of nasopharyngeal carcinoma and reverse the EMT of nasopharyngeal carcinoma cells.11.The positions of NOTCH2 intracellular segment(N2ICD)and X were determined by immunofluorescence double staining.12.Immunoprecipitation was used to verify the relationship between N2ICD and X.Results:1.Bioinformatics analysis revealed that MAPK and PI3K/AKT signaling pathway associated with NOTCH2 regulating the biological behavior of nasopharyngeal carcinoma.2.Immunoblotting confirmed that the PI3K/AKT signaling pathway was involved in NOTCH2 regulating the biological behavior of nasopharyngeal carcinoma.3.Treating shNOTCH2 nasopharyngeal carcinoma cell with AKT inhibitor,AKT inhibitor blocked the morphological changes of nasopharyngeal carcinoma cells that inhibit NOTCH2 expression:the cells were still the epithelial morphological changes of the control CNE-2 cells.4.The migration(without matrix gel)and invasion ability(with matrix gel)of shNOTCH2+LY294002CNE-2 cells was the same as the control CNE-2 group(P>0.05),while the migration and invasion ability of shNOTCH2+LY294002 CNE-2 cells was weaker than shNOTCH2 CNE-2 cells(P<0.05).5.The expression of E-cadherin and Vimentin in shNOTCH2+LY294002 CNE-2 cells were same as the Ctrl CNE-2 cells(P>0.05).However,the expression of E-cadherin in shNOTCH2+LY294002 CNE-2 cells was significantly higher than the shNOTCH2 CNE-2 cells(P<0.05),while the expression of Vimentin in shNOTCH2+LY294002 CNE-2 cells was significantly down-regulated than the shNOTCH2 CNE-2 cells(P<0.05).6.The expression of TRAF6 was up-regulated when NOTCH2 was down-regulated.The expression of TRAF6 was down-regulated when NOTCH2 was up-regulated.7.Western blotting confirmed down-regulated expression of NOTCH2 and TRAF6 proteins in shNOTCH2+shTRAF6 CNE-2 cells.8.Inhibition of TRAF6 could reverse the effect of shNOTCH2on the morphology of nasopharyngeal carcinoma cells:The cells were still the epithelial morphology of the control CNE-2 cells.9.The migration(without matrix gel)and invasion ability(with matrix gel)of shNOTCH2+shTRAF6 CNE-2 cells was the same as the control CNE-2 group(P>0.05),while the migration and invasion ability of shNOTCH2+shTRAF6 CNE-2 cells was weaker than shNOTCH2 CNE-2 cells(P<0.05).10.The expression of E-cadherin and Vimentin inshNOTCH2+shTRAF6CNE-2 cells were same as the Ctrl CNE-2 cells(P>0.05).However,the expression of E-cadherin inshNOTCH2+shTRAF6 CNE-2 cells was significantly higher than the shNOTCH2 CNE-2 cells(P<0.05),while the expression of Vimentin in shNOTCH2+shTRAF6 CNE-2 cells was significantly down-regulated than the shNOTCH2 CNE-2 cells(P<0.05).11.Immunofluorescence double staining revealed that the intracellular segment of NOTCH2(N2ICD)was co-localized with TRAF6 in the cytoplasm of nasopharyngeal carcinoma cells.12.Immunoprecipitation revealed that the direct physical binding of N2ICD and TRAF6 was found by.Conclusion:In nasopharyngeal carcinoma cells,NOTCH2 regulates the PI3K/AKT signaling pathway.Inhibition of AKT or its upstream molecule TRAF6 can block the changes of cell morphology,invasion,metastasis ability and the occurrence of EMT after the inhibition of NOTCH2.Based on the above experimental results,this study confirmed that NOTCH2 was negatively associated with survival in patients with nasopharyngeal cancer;NOTCH2and TRAF6 directly bind to regulate the ETM of nasopharyngeal carcinoma cells through TRAF6/AKT signal axis,and ultimately inhibit the metastasis of nasopharyngeal carcinoma,which is a potential target for the treatment of nasopharyngeal carcinoma.