USP5 silencing inhibits the proliferation of bladder cancer cells and induces cell apoptosis and ferroptosis by destabilizing COL14A1 expression

Background: Bladder cancer (BC) remains a prevalent malignant tumor of the urinary system with high morbidity and mortality rates. Despite advances in therapeutic strategies, the underlying molecular mechanisms driving BC progression are not fully understood, necessitating the identification of novel biomarkers and therapeutic targets. Ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, has emerged as a critical player in tumor suppression. However, the specific roles of ferroptosis-related genes and their regulatory mechanisms in BC progression require further elucidation. The study aimed to analyze ferroptosis-related genes and their regulatory mechanisms in BC progression.

Methods: This study integrated bioinformatics analysis with experimental validation. Differentially expressed genes (DEGs) between BC and normal tissues were identified using The Cancer Genome Atlas (TCGA) and GSE13507 datasets. These DEGs were intersected with ferroptosis-related genes to screen potential candidates. Weighted gene co-expression network analysis (WGCNA) was employed to identify hub modules associated with BC phenotypes, followed by least absolute shrinkage and selection operator (LASSO) regression and support vector machine recursive feature elimination (SVM-RFE) algorithms to pinpoint key genes. Ubiquitination-related databases were utilized to predict upstream regulators. In vitro and in vivo experiments, including co-immunoprecipitation (Co-IP), ubiquitination assays, functional assays (proliferation, apoptosis, and ferroptosis markers), and xenograft mouse model assay, were conducted to validate the molecular mechanisms and biological functions.

Results: Through integrated analysis, 84 ferroptosis-related DEGs were identified. WGCNA and machine learning algorithms further screened and identified collagen type XIV alpha 1 chain (COL14A1) as a critical hub gene. Functional enrichment analysis indicated that these genes were primarily involved in extracellular matrix organization and cell division. Mechanistically, COL14A1 was predicted and validated as a substrate of the deubiquitinase ubiquitin specific peptidase 5 (USP5). USP5 was found to be upregulated in BC cells and interacted with COL14A1 to inhibit its ubiquitination, thereby stabilizing its protein expression. Functional experiments demonstrated that USP5 knockdown significantly promoted COL14A1 degradation, leading to suppressed cell proliferation, induced apoptosis, and increased accumulation of reactive oxygen species (ROS), malondialdehyde (MDA), and Fe²⁺, while decreasing glutathione (GSH) levels. Crucially, the effects induced by USP5 silencing were effectively reversed by COL14A1 overexpression. In vivo studies further confirmed that USP5 knockdown inhibited tumor growth and reduced COL14A1 expression.

Conclusions: This study unveiled a novel USP5/COL14A1 regulatory axis that promotes BC progression by inhibiting ferroptosis and apoptosis. These findings highlight USP5 and COL14A1 as promising therapeutic targets.