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

doi:10.3969/j.issn.1674-8115.2025.09.007

Abstract

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

CLC Number:

R735.2

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.

Figures/Tables 9

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

Tab 2 Primer sequences for qPCR

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

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

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

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

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

Fig 4 Verification of the interaction between deubiquitinase CYLD and CAMK2A

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

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

TrendMD

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