Abstract: Objective To elucidate the molecular mechanisms through which ubiquitin⁃specific peptidase 41 (USP41) modulates the malignant biological behavior of head and neck squamous cell carcinoma (HNSCC). Methods The Gene Expression Profiling Interactive Analysis (GEPIA) database was used to examine expression profiling of USP41 across various tumors, including HNSCC. HNSCC cells lines (UM2, UM1, and HN31), along with normal oral keratinocyte (HOK) cells were cultured in vitro. Plasmid transfections were conducted to achieve knockdown of USP41 utilizing shRNA constructs (shRNA#1 and shRNA#2) and to facilitate the overexpression of signal transducer and activator of transcription 1 (STAT1). and USP41. Corresponding empty vector controls were included concurrently. Reverse transcription quantitative polymerase chain reaction (RT⁃qPCR) was used to quantify the mRNA expression levels of USP41, epithelial⁃mesenchymal transition (EMT) markers (E⁃cadherin, vimentin, and N⁃cadherin), and STAT1. Western blot analysis was used to determine the protein expression and ubiquitination levels of USP41and STAT1. Cell Counting Kit⁃8 (CCK⁃8), colony formation, and Transwell assays were conducted to assess the cells proliferation, migration, and invasion capabilities. Co⁃immunoprecipitation combined with Western blot analysis was used to validate the interaction between USP41 and STAT1 in UM1 cells. Results USP41 expression was upregulated in various tumor types, including HNSCC (P<0.05). Both mRNA and protein levels of USP41 were elevated in UM1 and HN31 cell lines (all P<0.05). Knockdown of USP41 resulted in the inhibition of cell proliferation, migration and invasion, accompanied by an increase in E⁃cadherin expression and a decrease Vimentin and N⁃cadherin expression (all P<0.01). These effects were reversed upon overexpression of STAT1. USP41 overexpression led to a reduction in  ubiquitinated protein levels while enhancing the expression of STAT1 and USP41 protein in UM1 cell lines. Co⁃immunoprecipitation and Western blot confirmed the interaction between USP41 and STAT1 in UM1 cell lines. Conclusions USP41 is markedly overexpressed in HNSCC cell lines and facilitates cell proliferation, migration, invasion, and EMT progression through STAT1 deubiquitination, suggesting its potential as a therapeutic target for HNSCC.

Key words:  Head and neck squamous cell carcinoma, Deubiquitination, Ubiquitin?specific peptidase 41, Sigal transducer and activtor of transcription, Cell proliferation, Epithelial?mesenchymal transition