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

doi:10.3969/j.issn.1674-8115.2025.09.007

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (9): 1149-1160.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

那迪娜·帕尔哈提, 张鹏善, 徐亦天, 陈赟琪, 黄陈()

上海交通大学医学院附属第一人民医院胃肠外科，上海 200080

收稿日期:2024-12-13 接受日期:2025-04-08 出版日期:2025-09-28 发布日期:2025-09-30
通讯作者: 黄　陈，主任医师，博士；电子信箱：richard-hc@hotmail.com。
基金资助:
国家自然科学基金(82072662);国家自然科学基金(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)

Construction of a truncated cylindromatosis tumor suppressor deubiquitinase plasmid and its regulation of the phenotypes of gastric cancer cells

PAERHATI Nadina, ZHANG Pengshan, XU Yitian, CHEN Yunqi, HUANG Chen()

Department of Gastrointestinal Surgery, General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China

Received:2024-12-13 Accepted:2025-04-08 Online:2025-09-28 Published:2025-09-30
Contact: HUANG Chen, E-mail: richard-hc@hotmail.com.
Supported by:
National Natural Science Foundation of China(82072662);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(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);Featured National Leading Talents of Shanghai General Hospital

摘要/Abstract

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 kinase Ⅱα，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。

关键词: 去泛素化酶, 胃癌, 圆柱瘤蛋白（CYLD）, 截短体, 增殖, CAMK2A, STAT3

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.

Key words: deubiquitinase, gastric cancer, cylindromatosis (CYLD), truncation, poliferation, CAMK2A, STAT3

中图分类号:

R735.2

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.

PAERHATI Nadina, ZHANG Pengshan, XU Yitian, CHEN Yunqi, HUANG Chen. Construction of a truncated cylindromatosis tumor suppressor deubiquitinase plasmid and its regulation of the phenotypes of gastric cancer cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(9): 1149-1160.

图/表 9

表1 人 CYLD 基因截短体PCR引物序列

Tab 1 PCR primer sequences for truncated variants of the human CYLD gene

Primer name	Primer sequence(5'→3')
Flag-CYLD-Del2-320aa	F:CGCCATGTTTATGTCAAGAGGTGTTGGGGACAAAGG
	R:CTTGACATAAACATGGCGATCGCGGCGG
Flag-CYLD-Del321-592aa	F:CCAAACTTGCCGGCATCCAGGGTCATTACAATTCTTGTTACTTAGAC
	R:GGATGCCGGCAAGTTTGGGAGGCCTCCTTTCC
Flag-CYLD-Del593-956aa	F:GGAAGAAGAAAACGCGTACGCGGCCGCTC
	R:CGTACGCGTTTTCTTCTTCCCAATCATTATCTCCAAGCC

表2 实时荧光定量引物信息

Tab 2 Primer sequences for qPCR

Gene	Primer sequence (5'→3')
CYLD	F:GGTAATCCGTTGGATCGGTCAG
	R:AGTGCCTCTGAAGGTTCCATCC
β-actin	F:CACCATTGGCAATGAGCGGTTC
	R:AGGTCTTTGCGGATGTCCACGT

图1 去泛素化酶 CYLD 在胃癌组织及细胞系中的表达及其与胃癌患者预后的相关性分析Note： A. Relative expression of CYLD mRNA in STAD (stomach adenocarcinoma, gastric adenocarcinoma) and normal tissue samples from the TCGA and GTEx databases. ①P=0.002. B. CYLD protein expression levels in tumor and adjacent normal tissue samples from 5 gastric cancer patients were detected by Western blotting. T—tumor, N—normal. C. CYLDprotein expression in tumor and adjacent normal tissue samples from gastric cancer patients were detected by immunohistochemistry. D. Prognostic significance of CYLD in gastric cancer patients was analyzed using the Kaplan-Meier Plotter database. HR=0.68 (0.56-0.82), logrank P<0.001. E/F. CYLD expression levels in GES-1 and 7 gastric cancer cell lines were analyzed by qRT-PCR (E) and Western blotting (F). ①P=0.004, ②P<0.001, ③P=0.007, ④P=0.036, ⑤P=0.027, ⑥P=0.045, compared with GES-1.

Fig 1 Expression of CYLD in gastric cancer tissues and cell lines and its correlation with prognosis of gastric cancer patients

图2 去泛素化酶 CYLD 蛋白截短体的构建、表达验证及细胞定位分析Note： A. Schematic diagram showed the structure of Flag-CYLD full-length, enzymatically inactivated mutant, and truncated variants, with the three CAP domains (CAP1/CAP2/CAP3) and the USP catalytic domain marked. B. Agarose gel electrophoresis was performed to analyze the plasmids Flag-CYLD-WT, Flag-CYLD-C601S, Flag-CYLD-Del2-320aa, Flag-CYLD-Del321-592aa, and Flag-CYLD-Del593-956aa (M=DNA marker). C. Western blotting was conducted to analyze the protein expression of Flag-CYLD-WT, Flag-CYLD-C601S, Flag-CYLD-Del2-320aa, Flag-CYLD-Del321-592aa, and Flag-CYLD-Del593-956aa, with empty vector-transfected HEK-293T cells serving as controls. D. Immunofluorescence (IF) assay was carried out to detect the subcellular localization of Flag-CYLD-WT, Flag-CYLD-C601S, Flag-CYLD-Del2-320aa, Flag-CYLD-Del321-592aa, and Flag-CYLD-Del593-956aa.

Fig 2 Construction, expression verification, and subcellular localization of CYLD truncated variants

图3 CYLD 野生型、酶失活突变型及截短体对胃癌细胞系AGS增殖与克隆形成能力的影响Note： A. Western blotting was performed to analyze the basal CYLD expression in AGS cells transfected with shNC and shCYLD, as well as the protein expression after transfection of plasmids in each group. B. CCK8 assay was carried out to evaluate the cell proliferation of each group. ①P<0.001, ②P=0.007, ③P=0.004, ④P>0.05, ⑤P=0.033, ⑥P=0.041. C/D. Plate colony formation experiments were conducted to assess the colony-forming abilities of each group. ①P=0.036, ②P<0.001, ③P=0.002, ④P>0.05, ⑤P=0.002, ⑥P=0.041.

Fig 3 Effects of CYLD WT, enzyme-inactivated mutant, and truncated variants on proliferation and colony formation of AGS gastric cancer cells

表4 BioGRID 数据库鉴定的与 CYLD 相互作用的候选蛋白

Tab 4 Candidate proteins predicted to interact with CYLD， as identified in the BioGRID database

Protein ID	Candidate protein	Description
Q9UM82	SPATA2	Spermatogenesis-associated protein 2
Q9UBN7	HDAC6	Histone deacetylase 6
P04637	P53	Tumor protein p53
Q9UQM7	CAMK2A	Calcium/calmodulin-dependent protein kinase Ⅱα

图4 去泛素化酶CYLD与CAMK2A的相互结合验证Note： A. Co-immunoprecipitation (co-IP) assays were performed in HEK-293T cells transfected with HA-CAMK2A/Flag-CYLD (or negative control plasmids) to verify the interaction between CAMK2A and CYLD. B. The interaction between endogenous CYLD and CAMK2A was confirmed by co-IP assays in AGS cells.

Fig 4 Verification of the interaction between deubiquitinase CYLD and CAMK2A

图5 CYLD全长、酶失活突变体及截短体对CAMK2A的K63位泛素化修饰水平的调控Note： A. HEK-293T cells were co-transfected with NC, HA-CAMK2A, and/or Flag-tagged CYLD (wild-type, enzyme-inactivated mutant, or truncated mutant). Co-immunoprecipitation (co-IP) assays were performed to detect changes in K63-linked ubiquitination levels of HA-CAMK2A. B. AGS cells with CYLD knockdown were used, with shNC-transfected cells as controls. Co-IP assays were conducted to analyze changes in K63-linked ubiquitination levels of endogenous CAMK2A.

Fig 5 Regulation of K63-linked ubiquitination of CAMK2A by CYLD full-length, enzyme-inactivated mutant, and truncated variants

图6 CYLD全长、酶失活突变体及截短体对CAMK2A下游STAT3磷酸化修饰水平的调控Note： A. HEK-293T cells were co-transfected with NC, HA-CAMK2A, and/or Flag-tagged CYLD (wild-type, enzyme-inactivated mutant, or truncated mutant). Western blotting was performed to analyze changes in protein expression levels of p-STAT3 and STAT3. B. shNC-transfected AGS cells were used as controls. shNC, shCYLD-1, and shCYLD-2 AGS cells were transfected with HA-CAMK2A. Western blotting was conducted to detect changes in protein expression levels of p-STAT3 and STAT3.

Fig 6 Regulation of phosphorylation of STAT3 (downstream of CAMK2A) by CYLD full-length, enzyme-inactivated mutant, and truncated variants

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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

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