摘要
目的·构建去泛素化酶圆柱瘤蛋白(cylindromatosis,CYLD)截短体质粒,初步分析CYLD对胃癌细胞增殖的影响。方法·收集癌症基因组图谱数据库(The Cancer Genome Atlas,TCGA)、基因型-组织表达数据库(Genotype-Tissue Expression,GTEx)及Kaplan-Meier Plotter数据库中CYLD在胃癌组织与正常组织中表达数据,以及与胃癌患者总生存期的相关性数据;免疫组织化学和Western blotting法检测胃癌组织与癌旁组织中CYLD的表达情况;Western blotting法和荧光实时定量PCR检测正常胃黏膜上皮细胞与胃癌细胞系中CYLD蛋白和mRNA表达情况。根据CYLD基因序列及结构特点,设计引物,构建其截短体真核表达质粒,琼脂糖凝胶电泳和Western blotting法检测及鉴定其表达情况,免疫荧光(immunofluorescence,IF)观察定位。在AGS细胞中敲低CYLD并且在敲低后分别回补CYLD野生型、酶失活突变型及截短体,细胞计数试剂盒-8(cell counting kit-8,CCK-8)和平板克隆实验检测CYLD野生型、酶失活突变型及截短体对细胞增殖的影响。免疫共沉淀(co-immunoprecipitation,Co-IP)、去泛素化实验、Western blotting法检测CYLD野生型、酶失活突变型及截短体与钙/钙调蛋白依赖性蛋白激酶Ⅱα亚基(calcium/calmodulin dependent protein kinaseⅡα,CAMK2A)的相互结合能力、CAMK2A去泛素化修饰水平及STAT3、p-STAT3蛋白表达情况。结果·胃正常组织中CYLD含量显著高于胃癌组织,正常胃黏膜上皮细胞中CYLD含量显著高于胃癌细胞系,高表达CYLD的胃癌患者预后较好。人CYLD截短体质粒构建成功,CYLD野生型、酶失活突变型以及3个截短体主要定位在细胞质的细胞中。敲低CYLD,AGS细胞增殖能力得到显著增强。而在敲低后分别回补CYLD野生型、酶失活突变型及截短体的细胞中,敲低CYLD后过表达CYLD野生型以及CAP3和USP区段能够显著抑制胃癌细胞的增殖。此外,CYLD能够与蛋白激酶CAMK2A相互结合并介导CAMK2A的K63去泛素化修饰,并且抑制CAMK2A对STAT3蛋白的磷酸化修饰。结论·成功构建人CYLD截短体真核表达质粒,CYLD野生型以及CAP3和USP区段显著抑制胃癌细胞增殖能力。
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.
作者
那迪娜·帕尔哈提
张鹏善
徐亦天
陈赟琪
黄陈
Nadina PAERHATI;ZHANG Pengshan;XU Yitian;CHEN Yunqi;HUANG Chen(Department of Gastrointestinal Surgery,General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200080,China)
出处
《上海交通大学学报(医学版)》
2025年第9期1149-1160,共12页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金(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)。
作者简介
通信作者:黄陈,主任医师,博士,电子信箱:richard-hc@hotmail.com。
