The Effect And Mechanism Of Targeting Nicotinamide Phosphoribosyltransferase (NAMPT) In Inhibiting The Growth And Metastasis Of Esophageal Cancer

Esophageal cancer is one of the most common malignant tumors of digestive system in the world,of which squamous cell carcinoma accounts for more than 90%.The incidence of esophageal cancer has obvious regional,China is a country with high incidence of esophageal cancer,new cases and deaths are ranked first in the world.With the development of surgery,chemoradiotherapy and immunotherapy,the diagnosis and treatment of esophageal cancer have improved,but the 5-year survival rate of patients is still about 25%-30%.Therefore,it is of positive significance to find new therapeutic targets and drugs for comprehensive treatment of esophageal cancer.Nicotinamide phosphoribosyltransferase(NAMPT)is a pleiotropic protein that exists in two distinct forms: an intracellular form and an extracellular form.Intracellular NAMPT is the rate-limiting enzyme in the salvage synthesis pathway of mammalian nicotinamide adenine dinucleotide(NAD),while extracellular NAMPT as a growth factor and pro-inflammatory cytokine plays the important role on metabolism,inflammatory response,cell proliferation,apoptosis and so on.In recent years,studies have found that NAMPT is closely related to the occurrence,development and prognosis of tumors,but the role and mechanism of NAMPT in esophageal cancer are still unclear.Micro RNAs(mi Rs)are evolutionarily conserved non-coding RNAs with a length of about 22 nucleotides.They can regulate gene expression at the post-transcriptional level through sequence complementation,and have important biological effects.Studies have shown that mi R-206 has an important tumor suppressor effect in a variety of malignant tumors.In pancreatic cancer and breast cancer,mi R-206 plays an inhibitory role through targeted regulation of NAMPT expression.However,whether mi R-206 can affect the occurrence and development of esophageal cancer through targeted regulation of NAMPT has not been reported.Tumor-associated macrophages(TAMs)are important immune cells in tumor microenvironment(TME),which can promote tumor proliferation,invasion and metastasis,and are the important link between inflammation and tumors.Studies have confirmed that NAMPT as a pro-inflammatory cytokine,can participate in the regulation of tumor inflammatory microenvironment by promoting the inflammatory response of tumor cells,and the overexpression of NAMPT can significantly promote the proliferation and metastasis of a variety of tumor cells.However,the role and mechanism of NAMPT in the inflammatory microenvironment-mediated metastasis of esophageal cancer are still unknown.The purpose of this study is to preliminarily explore the regulatory mechanism of mi R-206 on intracellular NAMPT by studying the expression of NAMPT in esophageal cancer and its role in the growth and migration of esophageal cancer cells,and then using extracellular NAMPT as a target to study NAMPT antibodies The effect of therapy on esophageal cancer xenografts in nude mice and the role and molecular mechanism of NAMPT in tumor-associated macrophage-mediated esophageal cancer cell migration,in order to provide a scientific basis for the clinical treatment of esophageal cancer.1、The expression of NAMPT in esophageal cancer and its effect on the growth and migration of esophageal cancer cellsObjective To verify the high expression of NAMPT in esophageal cancer,and to study the role of NAMPT expression in the growth and migration of esophageal cancer cells.Methods(1)Online bioinformatics tools such as TIMER,GEPIA,and KaplanMeier were used to analyze the expression and survival of NAMPT in tumor tissues.(2)Real-time PCR and Western-blot were used to detect the m RNA and protein levels of NAMPT in fresh esophageal carcinoma and paracancerous tissues,respectively.(3)Immunohistochemistry was used to analyze the expression of NAMPT in protein tissue microarray of cancer tissues and paracancerous tissue of patients with esophageal cancer.(4)Flow cytometry was used to study the effect of NAMPT enzyme activity inhibitor FK866 on apoptosis of esophageal cancer TE-1 cells.(5)Esophageal cancer TE-1 cells were infected with lentivirus carrying NAMPT sh RNA,and TE1 cell line with low NAMPT expression was constructed.The inhibition effect of NAMPT expression on the proliferation ability of esophageal cancer TE-1 cells was detected by CCK-8 and plate cloning formation assay.(6)The effect of NAMPT knockdown on the migration of esophageal cancer cells was detected by wound healing test.Results(1)Bioinformatics analysis results showed that the expression of NAMPT in esophageal carcinoma,especially squamous cell carcinoma,was significantly increased,and the progress-free survival time and overall survival time of patients with high NAMPT expression group were significantly shorter than those with low NAMPT expression group.(2)The m RNA and protein levels of NAMPT in tissue samples from patients with esophageal cancer were significantly higher than those in adjacent tissues.(3)There were significant differences in the expression of NAMPT in tissue microarray of patients with esophageal cancer.The expression of NAMPT in cancer tissues was significantly higher than that in adjacent tissues,and showed an increasing trend with tumor progression.(4)The enzyme activity inhibitor FK866 of NAMPT can significantly promote the apoptosis of esophageal cancer TE-1 cells.(5)The expression of NAMPT in the cells obtained after aseptic sorting by flow cytometry was detected by Western-blot method,and the esophageal cancer TE1 cell line with NAMPT down-regulation was successfully constructed.(6)Down-regulation of NAMPT expression significantly inhibited the proliferation and migration of esophageal cancer TE1 cells.Conclusion In this part of the study,bioinformatics online network analysis tools were used to prove that NAMPT is abnormally high expression in esophageal cancer and is related to the prognosis of esophageal cancer patients.Further studies showed that NAMPT was highly expressed in tissue samples of patients with esophageal cancer,and the reduction of NAMPT expression could significantly promote the apoptosis of esophageal cancer cells and inhibit the proliferation and migration of esophageal cancer cells,confirming that NAMPT plays an important role in the occurrence and development of esophageal cancer and may become a new target for clinical treatment of esophageal cancer.2、Inhibitory effect and mechanism of mi R-206 targeting NAMPT gene on growth and migration of esophageal cancerObjective To investigate the regulatory mechanism of mi R-206 on intracellular NAMPT and its effect on the proliferation and migration of esophageal cancer cells.Methods(1)Bioinformatics was used to analyze the potential regulatory mechanism of NAMPT expression in esophageal cancer.(2)The mi R-206 mimic was transfected into esophageal cancer TE-1 cells,and the expression of mi R-206 was detected by real-time PCR.(3)The effect of mi R-206 on the proliferation of esophageal cancer TE-1 cells was detected by CCK-8 and plate clone formation assay,and the expressions of proliferation-related proteins Cyclin D1 and P21 were detected by Western-blot method.(4)Flow cytometry was used to analyze the effect of mi R-206 on the apoptosis of esophageal cancer TE-1 cells.(5)The effect of mi R-206 on the migration of esophageal cancer TE-1 cells was detected by cell wound healing test and Transwell migration assay,and the expressions of MMP-2 and MMP-9 were detected by Western-blot method.(6)The effect of mi R-206 on the expression and activity of NAMPT protein in esophageal cancer TE-1 cells was detected by Western-blot and NAD content assay respectively.Results(1)The expression of NAMPT in esophageal cancer is mainly regulated by gene amplification and mi RNA.(2)The expression of mi R-206 in esophageal cancer TE1 cells transfected with mi R-206 mimic was significantly higher than that in the control group.(3)mi R-206 can significantly inhibit the proliferation of esophageal cancer TE1 cells and promote cell apoptosis.(4)mi R-206 significantly inhibited the migration ability of esophageal cancer TE1 cells.(5)mi R-206 can regulate NAMPT and inhibit its expression and activity.Conclusion This part of the study preliminarily clarified the regulation and mechanism of NAMPT in the occurrence and development of esophageal cancer by confirming that mi R-206 can inhibit the proliferation and migration of esophageal cancer by targeting the regulation of NAMPT expression and enzymatic activity.3、The role and mechanism of targeting e NAMPT in regulating tumor-associated macrophage-mediated esophageal cancer metastasisObjective To explore the potential clinical application mechanism of targeted NAMPT in esophageal cancer metastasis,the role of NAMPT neutralizing antibody in esophageal cancer xenograft in nude mice and the role of NAMPT in tumor-associated macrophage-mediated migration of esophageal cancer cells were investigated.Methods(1)The effect of NAMPT neutralizing antibody on esophageal cancer xenografts in nude mice was analyzed by small animal in vivo optical imaging system.(2)ELISA was used to detect the expression levels of e NAMPT in peripheral blood of nude mice in different treatment groups.(3)Online bioinformatics analysis tools such as TIMER and TISIDB were used to analyze the correlation between NAMPT expression and immune cell infiltration in esophageal cancer.(4)Macrophage U937 was infected with lentivirus carrying NAMPT.After aseptic sorting by flow cytometry,the expression of NAMPT protein in U937 cells was detected by Western-blot method,and the expression level of e NAMPT in supernatants of different cell cultures was detected by ELISA method.(5)The expression of chemokines CCL3 and CCL22 secreted by U937 cells after infection with different lentiviruses as well as macrophage specific detection indicators i NOS and ARG-1 was detected by RT-PCR to identify the phenotype of TAMs.(6)U937 cells with stable expression of NAMPT were co-cultured with esophageal cancer TE-1 cells,and the effect of macrophages with stable over-expression of NAMPT on the migration ability of esophageal cancer cells was detected by cell wound healing assay and Transwell migration assay.(7)The expression of inflammatory factors such as IL-6,IL-8 and TGF-β in esophageal cancer TE-1 cells within co-culture system was detected by RT-PCR,and the expression of STAT3 signaling pathway protein and phosphorylated STAT3(P-STAT3)was detected by Western-blot method.Results(1)The use of NAMPT antibody can significantly inhibit the metastasis of esophageal cancer TE-1 cells in nude mice.(2)The level of e NAMPT in the peripheral blood of nude mice in the NAMPT antibody treatment group was significantly lower than that in the control group.(3)Bioinformatics analysis result showed that NAMPT may play a specific role in the immune invasion of esophageal carcinoma.(4)The lentiviral vector PWPXL-NAMPT was successfully constructed,and the U937 cell line with stable overexpression of NAMPT was established,and it was found that NAMPT overexpression could promote the secretion of e NAMPT.(5)Overexpression of NAMPT can promote the transformation of U937 cells into M2 macrophages.(6)Macrophages U937 with stable overexpression of NAMPT can significantly increase the migration ability of esophageal cancer cells.(7)Macrophages U937 with stable overexpression of NAMPT can promote the expression of inflammatory factors such as IL-6,IL-8 and TGF-β by activating the STAT3 signaling pathway in esophageal cancer cells.Conclusion NAMPT antibody can significantly inhibit the metastasis of esophageal cancer TE-1 cells in nude mice,and the mechanism may be related to e NAMPT as a pro-inflammatory cytokine that activates the STAT3 signaling pathway in esophageal cancer and promotes the transformation of macrophages into M2tumor-associated macrophages.These results suggest that NAMPT can be used as a potential drug target for esophageal cancer,and the targeted therapy of NAMPT can inhibit the metastasis of esophageal cancer,laying the foundation for a potential new treatment for clinical treatment of esophageal cancer.