上海交通大学学报（医学版）2025，Vol.45Issue(9)：1149-1160,12.DOI:10.3969/j.issn.1674-8115.2025.09.007
人去泛素化酶圆柱瘤蛋白截短体质粒的构建及其对胃癌细胞表型的调控研究
Construction of a truncated cylindromatosis tumor suppressor deubiquitinase plasmid and its regulation of the phenotypes of gastric cancer cells
摘要
Abstract
Objective·To construct truncations of CYLD,and to preliminarily analyze their effects on the proliferation of gastric cancer cells.Methods·TCGA,GTEx,and Kaplan-Meier Plotter databases were used to analyze the differences in the expression levels of CYLD between gastric cancer tissues and normal tissues,and their relationship with the prognosis of gastric cancer patients.Immunohistochemistry and Western blotting were used to detect the expression of CYLD in cancer tissues and adjacent noncancerous tissues.Western blotting and qRT-PCR were used to analyze the protein and mRNA expression levels of CYLD in gastric mucosal epithelial cells and gastric cancer cells.According to the sequence and structural characteristics of CYLD gene,primers were designed to construct its truncations.Their expression was detected and identified by agarose gel electrophoresis and Western blotting,and localization was observed by immunofluorescence.In the human gastric adenocarcinoma cells(AGS)with CYLD knockdown,blank NC was added to the control group,and the full-length CYLD,enzyme-inactivated mutant,and three truncated plasmids were added to the experimental group.The proliferation changes of cells in each group were detected by CCK-8 and plate cloning assays.Co-immunoprecipitation,deubiquitination,and Western blotting assays were performed to examine the binding ability of full-length CYLD,the enzyme-inactivated mutant,and the truncated variants to CAMK2A,the level of CAMK2A deubiquitination,and the expression of STAT3 and p-STAT3 proteins.Results·CYLD expression in normal gastric tissues and cells was significantly higher than in gastric cancer tissues and cells,and the prognosis of patients with high expression of CYLD was better.The truncations of human CYLD were successfully constructed,and full length CYLD,enzyme-inactivated mutant,and truncations were mainly localized in the cytoplasm.Knockdown of CYLD in gastric cancer cells significantly enhanced the proliferative ability of gastric cancer cells.Reconstitution of CYLD-knockdown cells with CYLD-WT,or truncated variants containing the CAP3 or USP domains significantly inhibited the proliferation of gastric cancer cells.In addition,CYLD bound to CAMK2A mediated K63 deubiquitination modification,and inhibited CAMK2A-induced phosphorylation of STAT3.Conclusion·The human CYLD truncation plasmids are successfully constructed,and the full length CYLD and its CAP3 and USP segments significantly inhibit the proliferation of gastric cancer cells.关键词
去泛素化酶/胃癌/圆柱瘤蛋白(CYLD)/截短体/增殖/CAMK2A/STAT3Key words
deubiquitinase/gastric cancer/cylindromatosis(CYLD)/truncation/poliferation/CAMK2A/STAT3分类
医药卫生引用本文复制引用
那迪娜·帕尔哈提,张鹏善,徐亦天,陈赟琪,黄陈..人去泛素化酶圆柱瘤蛋白截短体质粒的构建及其对胃癌细胞表型的调控研究[J].上海交通大学学报（医学版）,2025,45(9):1149-1160,12.基金项目
国家自然科学基金(82072662,82203751) （82072662,82203751）
上海交通大学"交大之星"计划医工交叉研究基金 （）
2023东方英才计划青年拔尖项目 （）
上海市第一人民医院特色国家领军人才培养计划 （）
上海交通大学医学院"双百人"项目(20191425). National Natural Science Foundation of China(82072662,82203751) （20191425）
Medical and Engineering Cross Research Fund from Shanghai Jiao Tong University"Jiao Tong University Star"Program （）
East Talents Program's Top-Notch Project(2023) （2023）
Featured National Leading Talents of Shanghai General Hospital （）
"Two-hundred Talents"Program of Shanghai Jiao Tong University School of Medicine(20191425). （20191425）
