The Regulation Of NNMT In The Biological Behavior And Radiosensitivity Of Nasopharyngeal Carcinoma

Part Ⅰ: Correlation of NNMT expression with clinicopathologic characteristics and prognosis of nasopharyngeal carcinomaObjective: The differential expression of NNMT was analyzed and detected in normal nasopharyngeal tissues and nasopharyngeal carcinoma(NPC)tissues,and its correlation with clinicopathological characteristics and prognosis of patients with nasopharyngeal carcinoma was investigated.Methods: The expression difference of NNMT in normal nasopharyngeal tissues and nasopharyngeal carcinoma tissues was analyzed from Gene Expression Omnibus(GEO)database.Meanwhile,the expression difference of NNMT in nasopharyngeal carcinoma radiosensitive and radioresistant cell lines was analyzed.Limitations in GEO database included small nasopharyngeal carcinoma patient sample sizes.However,the Cancer Genome Atlas(TCGA)database lacked individual datasets of nasopharyngeal carcinoma patients.The assocication between NNMT expression and the prognosis of head and neck squamous cell carcinoma(HNSCC)patients was analyzed from TCGA database.The clinical tissue samples were tested to analyze the relationship of NNMT expression with clinicopathologic characteristics and prognosis of NPC patients by immunohistochemical staining.Results: Analysis of GSE12452 dataset revealed that the expression of NNMT in NPC tissues was significantly higher than that in normal nasopharyngeal tissues.Analysis of GSE48503 dataset found that the expression of NNMT in NPC radioresistant cell line was significantly higher than that in radiosensitive cell line.The HNSCC patients with high expression of NNMT had a significantly poor prognosis compared to those of low NNMT expression in TCGA database.The clinical tissue samples of 134 patients with NPC and 15 patients with benign nasopharyngeal lesions were further collected.According to the results of NNMT immunohistochemical staining,it was confirmed that the expression of NNMT was not associated with sex,N classification,M classification,histological type and EB viral infection.However,there was significant correlated with the age(P = 0.022),T classification(P = 0.000)and clinical staging(P = 0.005).Kaplan-Meier analysis suggested that NPC patients with higher NNMT expression level had a relatively poor overall survival rate(P = 0.043).The results suggested that high expression of NNMT was related to the poor prognosis of patients with NPC.Meanwhile,the study found that the high expression of NNMT was not significantly correlated with the progression free survival rate of patients with NPC.Conclusion: NNMT is highly expression in NPC,which is correlated with the poor prognosis of patients with NPC.Part Ⅱ: Correlation of the expression of NNMT and biological behavior of nasopharyngeal carcinomaObjective: To investigate the effects of NNMT expression on the biological behavior of NPC cells.Methods: The expression of NNMT was detected at different time points after irradiation of 6 Gy by Western blot and q RT-PCR in NPC cell lines.The radioresistant cells(CNE2R)were established by continuous irradiation of CNE2 cells,and the radiation resistance was verified.Stable NPC cell lines with overexpression and knockdown of NNMT were constructed by lentivirus transfection.The effect of NNMT expression on the proliferation of NPC cells was detected by CCK8 and colony formation assays.The effect of NNMT expression on the capabilities of NPC cells invasion and migration was detected by Transwell invasion assay and cell scratch test.The regulation of NNMT on EMT of NPC cells was detected by Western blot,q RT-PCR and immunofluorescence.Results: Western blot and q RT-PCR assays indicated that the expression of NNMT in CNE1,CNE2,HONE1 and HNE1 cells was greatly increased after irradiation of 6 Gy,with 24 hours after irradiation being most prominently increased.The colony formation assay confirmed that CNE2 R cells were more radioresistant than CNE2 cells.Western blot and q RT-PCR showed higher NNMT expression in CNE2 R cells compared to CNE2 cells,and the same results were obtained after irradiation of 6 Gy.In CNE2 R cells,the expression of E-cadherin was decreased,the expression of N-cadherin and Vimentin was increased,and the marker of DNA double-strand break γ-H2 AX expression was decreased compared to CNE2 cells.CCK8 and colony formation assays showed that NNMT overexpression promoted proliferation in CNE1 and CNE2 cell lines,and the difference was statistically significant.The knockdown of NNMT inhibited the proliferation ability of HONE1 and CNE2 R cells,and the difference was statistically significant.Transwell invasion assay and cell scratch test showed that the overexpression of NNMT significantly enhanced the invasion and migration ability of CNE1 and CNE2 cells,and the knockdown of NNMT significantly inhibited the invasion and migration ability of HONE1 and CNE2 R cells.Western blot,q RT-PCR and immunofluorescence showed that the overexpression of NNMT could promote the EMT of NPC cells,and NNMT knockdown could reverse the EMT of NPC cells.Conclusion: NNMT can regulate the proliferation,invasion,migration and epithelial mesenchymal transformation of nasopharyngeal carcinoma cells.Part Ⅲ: Correlation of the expression of NNMT and radiosensitivity of nasopharyngeal carcinoma in vitro and in vivoObjective: To explore the effects of NNMT expression on the radiosensitivity of NPC cells in vivo and in vitro.Methods: The effect of NNMT on radiosensitivity of nasopharyngeal carcinoma cell lines was detected by colony formation assays.The effect of NNMT expression on apoptosis rate and cell cycle distribution of nasopharyngeal carcinoma cell lines after irradiation was detected by flow cytometry.The effect of NNMT expression on DNA damage of nasopharyngeal carcinoma cell lines induced by radiation was analyzed by Western blot and immunofluorescence.Finally,the relationship between NNMT and radiosensitivity of nasopharyngeal carcinoma cell lines was evaluated in vivo by subcutaneous xenograft mouse mode.Results: The colony formation assays confirmed that overexpression of NNMT inhibited the radiosensitivity of nasopharyngeal carcinoma cells,and knockdown of NNMT enhanced the radiosensitivity of nasopharyngeal carcinoma cells.Apoptosis experiment revealed that the apoptotic rate of CNE1 and CNE2 cells was decreased significantly in the combined overexpression of NNMT and irradiation of 6 Gy,and the difference was statistically significant.The apoptotic rate of HONE1 and CNE2 R cells was increased significantly in the combined knockdown of NNMT and irradiation of 6 Gy,and the difference was statistically significant.Cell cycle analysis showed that 6 Gy irradiation induced cell cycle arrest at G2/M phase of nasopharyngeal carcinoma cells.The overexpression of NNMT could blunt the G2/M phase arrest,and the difference was statistically significant.In contrast,the knockdown of NNMT could promote the G2/M phase arrest of nasopharyngeal carcinoma cells.Western blot and immunofluorescence showed that the expression of γ-H2 AX decreased after irradiation of 6 Gy in the NNMT overexpression of NPC cells.In contrast,the expression of γ-H2 AX increased after irradiation of 6 Gy in the NNMT knockdown of NPC cells.Subcutaneous xenograft tumor model confirmed that the tumor volume was smaller and grew slowly in the irradiation group compared to the non-irradiation group,and the tumor volume of the knockdown NNMT group was significantly smaller and grew slowly than that of the control group.Conclusion: NNMT can regulate the radiosensitivity of nasopharyngeal carcinoma cells by regulating cell apoptosis,G2/M phase arrest and DNA double strand break in vivo and in vitro.Part Ⅳ: NNMT regulates the Wnt/β-catenin signaling pathway by interacting with β-cateninObjective: To explore the mechanism of NNMT regulating the biological behavior and radiosensitivity of nasopharyngeal carcinoma cells.Methods: Western blot assay was used to verify whether the interference with NNMT expression could active the downstream target gene of β-catenin.Co-transfection of CNE1 and CNE2 cells with NNMT overexpression and β-catenin knockdown lentivirus,the cell rescue experiments were carried out by CCK8 assays,Transwell invasion assay and cell scratch test and Western blot.Western blot and Co-immunoprecipitation were used to verify the relationship between NNMT and β-catenin.Results: Western blot showed that the expression of β-catenin was up-regulated in CNE1 and CNE2 cells unpon NNMT overexpression.The downstream target genes C-myc,Cyclin D1 and GSK-3β,the protein expression was also up-regulated.The expression of β-catenin was down-regulated in HONE1 and CNE2 R cells unpon NNMT knockdown.The downstream target genes C-myc,Cyclin D1 and GSK-3β,the protein expression was also down-regulated.Knockdown β-catenin could reverse the enhancement proliferation,invasion and migration of nasopharyngeal carcinoma cells caused by overexpression of NNMT,and could also reverse EMT caused by overexpression of NNMT.In CNE1 and CNE2 cells,NNMT could directly bind to β-catenin.After silencing NNMT in HONE1 and CNE2 R cells,the ubiquitination level of β-catenin was increased.Conclusion: NNMT interaction with β-catenin regulates ubiquitination β-catenin,which modulates the biological behavior and radiosensitivity of NPC cells.