Downregulation Of TNFAIP2 Suppresses Proliferation And Metastasis In Esophageal Squamous Cell Carcinoma Through Activation Of The Wnt/Β-catenin Signaling Pathway

Background: Esophageal cancer is one of the most common tumors of human being.The incidence of esophageal cancer in China has obvious regional differences.Esophageal squamous cell carcinoma is common in our country,its specific pathogenesis and mechanism are still unknown.The tumor microenvironment in tumor plays an indispensable role in the development,and tumor necrosis factor alpha(TNF-α)is an important inflammatory factor in tumor microenvironment,which can promote breast cancer,colon cancer,the occurrence and development of bladder cancer.Study of TNF-α in esophageal squamous cell carcinoma in the microenvironment of the specific role of esophageal squamous cell carcinoma has not been reported.Therefore,the TNF-a downstream gene TNF-α-induced protein 2(TNFAIP2)gene induced by TNF-a were detected in esophageal squamous cell carcinoma.It is expected to provide an alternative target for gene therapy of esophageal cancer,and to provide a theoretical basis for the clinical treatment of esophageal cancer.Methods: 1.qRT-PCR and immunohistochemistry were used to detect the expression level of target gene TNFAIP2 at the gene and protein level in cancer and adjacent tissues.2.Using qRT-PCR to screen high expression of target gene in esophageal squamous cell carcinoma cell lines,two strains with high expression of target gene cell lines by shRNA lentiviral vector Shanghai Jikai Gene Technology Co.Ltd.to build a low expression of target gene in two strains of selected cell lines.Experimental group lentivirus mediated RNA interference group(LV-RNAi),no load group(LV-CON).3.Using qRT-PCR and Western blot technology to verify the interference efficiency of target genes in gene and protein level.4.Recombinant human tumor necrosis factor alpha(rTNF-α)was used to detect the gene and protein expression level of target gene by qRT-PCR and Western blot technology in the selected cell line for 48 hours.5.The ability of cell proliferation was detected by clone formation and CCK8.6.Cell cycle and apoptosis were detected by flow cytometry.7.Transwell cell method was used to detect the migration and invasion ability of the cells.8.Western was used to detect the proliferation,apoptosis,cycle,migration and invasion related protein expression of blot.9.SPSS22.0 was for statistical analysis.Results: 1.The expression of TNFAIP2 in esophageal squamous cell carcinoma and adjacent tissues was different at the level of gene and protein.2.The interference efficiency of the selected cell lines at the level of gene and protein was over 75%.3.The expression of target gene and protein were increased after rTNF-α.4.Cloning tips and CCK8 results: interference group than in the control group can inhibit cell proliferation;cell cycle and apoptosis results suggest in the LV-RNAi group,the Eca109 and Kyse150 cells were mainly arrested in the G0/G1 phase and the percentages of cells in the G2 and S phases were decreased,but,there were no significant differences in apoptosis between the LV-RNAi and LV-CON groups;the migration and invasion of results show that the interference group than in the control group can inhibit cancer cell migration and invasive ability.5.Detection of Wnt/ β-catenin signaling pathway in the interference group was lower or higher than the control group.Conclusion: It is the first time that TNFAIP2 is highly expressed in esophageal squamous cell carcinoma,and it is confirmed that rTNF-α cytokines can promote the expression of target genes and products in esophageal squamous cell carcinoma cell lines at the level of gene and protein.Lentivirus mediated RNA interference technology confirmed that the low expression of TNFAIP2 had a significant effect on the biological function of esophageal squamous cell carcinoma cell lines.Further study on the effect of TNFAIP2 on the occurrence,development and biological function of esophageal squamous cell carcinoma(ESCC)indicates that TNFAIP2 may play an important role in the Wnt pathway,and may be a proto oncogene target in squamous cell carcinoma of the tube.