ENKUR Interacts With β-catenin To Suppress Cell Proliferation In Gastric Cancer

Background and ObjectivesGastric cancer is a malignant tumor originating from gastric mucosal epithelium and is one of the most common therioma in the world.According to the histopathological classification,gastric cancer can be divided into adenocarcinoma,adenosquamous carcinoma,squamous carcinoma,carcinoid,etc.Adenocarcinoma accounts for about 95%of gastric cancer.Although radiotherapy,chemotherapy,surgery and other new methods have been used to treat gastric cancer,the five-year survival rate of gastric cancer is still very low.Many researches have focused on the pathogenesis of gastric cancer.In lung cancer and colorectal cancer,ENKUR has been reported as a tumor suppressor gene,which can inhibit tumor cell proliferation,migration and invasion,but it has not been reported in gastric cancer.In our study,we explored the biological functions and related mechanisms of ENKUR in gastric cancer.Contents and methods(1)Preliminary verification of the biological function of ENKUR in lung cancer cells.(2)Transient transfection was used to overexpress ENKUR in lung cancer cells;(3)EDU experiment was used to detect the influence of ENKUR on the ratio of lung cancer cell DNA synthesis phase(S)and MTT assay was used to detect the effect of ENKUR on lung cancer cell proliferation ability.2.ENKUR expression and correlation analysis between ENKUR expression and clinical features in gastric cancer(1)The clinical data and expression data of gastric cancer were downloaded from the TCGA database for subsequent analysis.Chi-square test was used to detect the expression of ENKUR in gastric cancer tissues and para-cancer tissues.Survival analysis was used to explore the effect of ENKUR expression on the prognosis of gastric cancer;(2)RNA extraction experiment was applied to extract the RNA from GSE-1,AGS,SGC-7901 and BGC-823.qPCR experiment was applied to detect the expression level of ENKUR in gastric cancer cell lines and normal gastric mucosa cells.(3)Utilize immunohistochemical experiments to detect ENKUR expression in cancer tissues and normal tissues,and analyze ENKUR expression in adjacent tissues and cancer tissues.The correlation between ENKUR and clinical parameters(such as age,gender,TNM stage,pathological classification,AJCC stage,tumor size,lymph node metastasis)was explored by Chi-square test,too.(4)Detect the difference in survival rate between patients with high ENKUR expression and patients with low ENKUR expression through survival analysis,and explore the relationship between different ENKUR expression and survival rate in early tumor patients and advanced tumor patients through stratified survival analysis based on AJCC stage;(5)Determine whether ENKUR is an independent prognostic factor of gastric cancer through univariate Cox proportional hazard regression model analysis and multivariate Cox proportional hazard regression model analysis.3.ENKUR inhibits the proliferation of gastric cancer cells(1)ENKUR was overexpressed in gastric cancer cells AGS and SGC-7901 by transiently transfecting ENKUR plasmid;(2)The expression of ENKUR in ENKUR-overexpressed gastric cancer cells AGS and SGC-7901 was silenced by transiently transfecting ENKUR si-RNA.(3)Detect the influence of interference or overexpression of ENKUR on the percentage of AGS and SGC-7901 in the DNA synthesis phase(S)by EDU experiment.Detect the influence of interference or overexpression of ENKUR on the proliferation rate of AGS and SGC-7901 by MTT assay.(4)Viral vector containing ENKUR plasmid was infected into AGS by stable transfection technology.The difference in growth rate of AGS cells in vivo after overexpressing ENKUR was detected by subcutaneous tumor formation experiments.4.Related mechanisms of ENKUR in gastric cancer(1)Overexpress ENKUR in gastric cancer cells AGS and SGC-7901 by transiently transfecting ENKUR plasmid;(2)Interference ENKUR expression in gastric cancer cells AGS and SGC-7901 by transiently transfecting ENKUR si-RNA;(3)Western blotting was used to detect the changes in the protein levels of(3-catenin,c-Myc,CCND1 in ENKUR overexpressed AGS cells and SGC-7901 cells;(4)Use western blotting to detect whether the interference with ENKUR can reverse the changes of β-catenin,c-Myc,CCND1 protein levels in the cells with ENKUR overexpression;(5)Use immunoprecipitation experiment,immunofluorescence to detect whether ENKUR and β-catenin are interacting proteins;(6)Use immunoprecipitation to detect the binding domain of ENKUR toβ-catenin.(7)Use nuclear and cytoplasmic separation experiment to detect the effect of ENKUR on the expression of β-catenin in nucleus。Results1.The proliferation rate of lung cancer cells was significantly slowed down after overexpressing ENKUR;2.The expression of ENKUR is reduced in gastric cancer and the low expression of ENKUR is a poor prognostic factor for gastric cancer;3.Overexpression and interference of ENKUR can inhibit and promote the proliferation of gastric cancer,respectively;4.ENKUR shows anti-tumor effect which can inhibit the expression of c-Myc and CCND1 by inhibiting the interaction protein p-catenin via Enkurin domain;5.ENKUR and β-catenin bind to each other in the cytoplasm;6.Enkuin domain mediates the binding of ENKUR to β-catenin;7.ENKUR decreased the expression of β-catenin in the nucleus.Conclusions1.ENKUR inhibits the proliferation ability of lung cancer cells;2.ENKUR is downregulated in gastric cancer and low expressed ENKUR is a poor prognostic factor for gastric cancer patients;3.ENKUR inhibits gastric cancer proliferation in vivo and in vitro;4.ENKUR interacts with β-catenin via Enkurin domain to inhibit β-catenin entry into the nucleus,thus reducing the proliferation of gastric cancer cells.