Studies On The Effect And Mechanism Of 5-FU Sensitivity Regulated By Nnmt In Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC)is a common malignant tumor of digestive tract in China,with high degree of malignancy,easy metastasis and poor prognosis.The clinical treatment methods of ESCC mainly include surgery,radiotherapy and chemotherapy.Due to the lack of effective early diagnosis methods,many ESCC patients are diagnosed in advanced stage with lymph nodes or distant metastasis and lost the opportunity of surgical treatment.Chemotherapy is one of the common therapies of ESCC clinical treatment.However,the efficacy of chemotherapy is limited by the heterogeneity of tumor cells and the low sensitivity to drugs,leading to low survival rate.Therefore,it is necessary to reveal the molecular mechanism of ESCC metastasis and chemosensitivity,and further to explore potential biomarkers for ESCC.Metabonomics,as the complement of genomics and proteomics,can efficiently identify the metabolic spectrum of diseases.In recent years,metabonomics has become an important way to reveal the pathogenesis of diseases and to assist the prevention and treatment.Notably,it is wildly used in the exploration of tumor pathogenesis and the development of individualized treatment.The diagnosis and treatment evaluation based on the metabolic markers in blood and urine of tumor patients is also developed to clinical application.With the deepening of the research,metabonomics will play a greater role in the field of anti-tumor research.Nicotinamide N-methyltransferase(NNMT),a cytoplasmic enzyme belonging to N-methyltransferases family,catalyses N-methylation of nicotinamide and other pyridine compounds using S-adenosylmethionine as methyl donor.The enzyme is also involved in the balance of nicotinamide and the biotransformation of many drugs and xenobiotic compounds.In recent years,proteomics and genomics reaserch have found abnormal expression of NNMT in various cancers.Many studies reported that NNMT was overexpressed in malignant glioma,pancreatic cancer,renal cancer,colorectal cancer and so on.Furthermore,NNMT overexpression are closely related to cancer cell proliferation,apoptosis,cell cycle,migration,radiochemotherapy resistance and other biological functions.However,the study of NNMT in ESCC has not been reported.Accordingly,this study will identify the metabolic differences in ESCC cell lines with different 5-FU sensitivity by metabonomics analysis,and screen the different metabolic pathways and the key molecules involved.Then the effect of key molecules on the 5-FU sensitivity and other phenotypes of ESCC cells will be explored,and the molecular mechanism will be revealed.This study may provide theoretical basis for revealing the molecular mechanism of 5-FU sensitivity and the development of individualized medicine.There are three parts in this study:Part I:we detected metabolic molecules in ESCC cell lines with different 5-FU sensitivity,and screened the different metabolic pathways and the key enzyme.Subsequently,the expression of key enzyme in ESCC tissues(GEO database)and cell lines were analyzed.Part II:this part identified the expression of the key enzyme in ESCC clinical tissues and analyzed the correlation between the expression and clinicopathological parameters of patients.The effects of the key enzyme on the proliferation,apoptosis,cell cycle and migration of ESCC cells were studied by cell function experiment.Part III:In vitro and in vivo experiments were carried out to investigate the effect of the key enzyme on 5-FU sensitivity of ESCC cells.The role of Warburg effect in the regulation of 5-FU sensitivity was studied.MethodsPart I:Sensitivity analysis of ESCC cells to 5-FU and identification of the differential metabolism in ESCC cells1.CCK-8 assay was used to detect the sensitivity of EC1,Eca109 and TE1 cells to 5-FU.2.Metabonomics analysis of EC1,Eca109 and TE1 cells was carried out based on GC-MS detection,and the different metabolic pathways and key metabolic enzymes were screened.3.The expression of NNMT in ESCC clinical tissues was analyzed base on GSE111011 dataset in GEO database.4.The NNMT expression in EC1,Eca109 and TE1 cells was detected by q RT-PCR and Western blot.Part II:The expression of NNMT in ESCC and the effect of NNMT on cell phenotypes1.Immunohistochemical staining was performed to detect the expression of NNMT in 30 cases of ESCC clinical tissues and corresponding adjacent normal tissues.The correlation between the expression of NNMT and clinicopathological characteristics of the patients was statistically analyzed.2.q RT-PCR and Western blot were used to detect NNMT m RNA and protein expression in various ESCC cell lines.3.NNMT was downregulated in two ESCC cell lines with high NNMT level by transfection with si RNA.The optimal transfection concentration and time were determined.The downregulation efficiency of NNMT was detected by q RT-PCR and Western blot.4.The effects of NNMT knockdown on cell survival and proliferation in ESCC cells were respectively detected by CCK-8 and Ed U assays.5.Annexin V-FITC/PI double-staining was used to detect the effect of NNMT knockdown on apoptosis in ESCC cells.6.Flow cytometry was performed to examine the effect of NNMT knockdown on cell cycle in ESCC cells.7.Transwell and wound healing assays were employed to detect the effect of NNMT knockdown on cell migration abilities in ESCC cells.8.The expression of proteins involved to cell apoptosis,migration and epithelial-mesenchymal transformation was examined by Western blot.Part III:NNMT regulates 5-FU sensitivity of ESCC cells through Warburg effect1.NNMT was downregulated or upregulated in ESCC cells by transfection with si RNA or recombinant plasmid respectively.The effects of 5-FU on cell proliferation and apoptosis were respectively investigated by CCK-8,plate cloning assay and Annexin V-FITC/PI double-staining assay.2.The glucose consumption and lactate production after NNMT downregulated/upregulated and 5-FU treatment were detected by spectrophotometry.3.After treatment with glycolysis inhibitor 2-DG,the effects of NNMT and 5-FU on cell growth,glucose consumption and lactate production were detected.4.TE1 cells with stable NNMT downregulation were injected into the nude mice subcutaneously.The body weight of mice and tumor volume were measured every two days.When the tumor volume reached to approximately 100 mm~3,the treatment groups were received 5-FU treatment every two days.After treatment for three weeks,the mice were sacrificed and the xenografts and visceral tissues were collected.5.The expression of proteins involved in cell apoptosis and glycolysis in xenografts was detected by q RT-PCR and Western blot.ResultsPart I:Sensitivity analysis of ESCC cells to 5-FU and identification of the differential metabolism in ESCC cells1.CCK-8 results showed that the 5-FU sensitivity of TE1 cells was significantly lower than EC1 and Eca109 cells.2.The results of GC-MS detection revealed that the metabolic pattern of TE1cells was significantly different from EC1 and Eca109 cells.The differential metabolic pathways between TE1 and other two cells included nicotinate and nicotinamide metabolism and TCA cycle.3.Data analysis based on GSE111011 dataset demonstrated that NNMT,a key enzyme of nicotinate and nicotinamide metabolism pathway,was highly expressed in ESCC tissues compared with normal esophageal tissues.4.NNMT was significant highly expressed in TE1 cells compared with EC1 and Eca109 cells.Part II:The expression of NNMT in ESCC and the effect of NNMT on cell phenotypes1.Compared with adjacent normal tissues,NNMT was highly expressed in ESCC tissues and significantly correlated with lymph node metastasis.2.NNMT expression was significantly higher in EC9706 and TE1 cells than that in EC1,Eca109 and TE13 cells.3.The expression of NNMT was significantly downregulated both in EC9706and TE1 cells by transfected with si RNA for 48 h at a concentration of 50 n M.4.NNMT knockdown significantly suppressed the cell viability and proliferation,and increased cell apoptosis rate of EC9706 and TE1 cells.Furthermore,the expression of Bax and Cleaved-caspase3 were increased,while the expression of Bcl-2 was decreased.5.The cell cycle in EC9706 and TE1 cells was respectively arrested at S phase and G0/G1 phase after NNMT knockdown,and the expression of Cyclin D1 was decreased in both cells.6.NNMT knockdown significantly inhibited cell migration in EC9706 and TE1cells.Meanwhile,the expression of the intercellular adhesion molecule E-cadherin was upregulated,and the expression of mesenchymal markers N-cadherin and Vimentin was downregulated.Part III:NNMT regulates 5-FU sensitivity of ESCC cells through Warburg effect1.NNMT downregulation in TE1 cells significantly enhanced the inhibitory effect of 5-FU on cell viability and colony formation ability,and increased the cell apoptosis rate induced by 5-FU.The pro-apoptosis protein Bax and Cleaved-caspase3were further upregulated,the anti-apoptosis protein Bcl-2 was further downregulated.While NNMT overexpression in EC1 and Eca109 cells showed the opposite effects.2.Compared with EC1 and Eca109 cells,the glucose consumption and lactate production were significantly elevated in TE1 cells,and glycolysis-related enzymes(HK2,LDHA and PGAM1)were also higher expressed.3.NNMT downregulation in TE1 cells markedly suppressed the glucose consumption and lactate production,and the expression of glycolysis-related enzymes was also correspondingly decreased.In contrast,NNMT overexpression in EC1 and Eca109 cells resulted in the opposite effects.4.2-DG treatment markedly increased 5-FU sensitivity in EC1 and Eca109 cells with NNMT overexpression,and the enhanced glucose consumption and lactate production induced by NNMT overexpression was attenuated by 2-DG treatment.5.NNMT knockdown significantly increased the sensitivity of xenografts to5-FU.There was no significant change in the body weight of mice between different treatment groups.6.NNMT downregulation markedly decreased the expression of glycolysis-related enzymes in xenografts,and the expression was even lower after treated with 5-FU.Conclutions1.ESCC cells with different 5-FU sensitivity had different metabolic pathways,including nicotinate and nicotinamide metabolism and TCA cycle.NNMT,a key enzyme of nicotinate and nicotinamide metabolism pathway,was highly expressed in TE1 cells which showed lower sensitivity to 5-FU.2.Elevated NNMT expression was observed in ESCC tissues and associated with lymph node metastasis.NNMT knockdown can significantly suppressed cell viability,proliferation and migration,arrested cell cycle progression and promoted cell apoptosis.3.NNMT decreased 5-FU sensitivity of ESCC cells by promoting Warburg effect.4.NNMT knockdown significantly enhanced the anti-tumor effect of 5-FU in vivo.