Shuping Wu ¹ Yu Liu ² Yu Zeng ² Xianhui Ruan ² Mei Tao ² Wenrong Lin ^3* Chang Liu ^1* Hongbin Chen ¹ Hui Liu ^1* Yu Wu ^1*

• ¹ Department of Head and Neck Surgery, Clinical Oncology School of Fujian Medical University & Fujian Cancer Hospital, Fuzhou, China
• ² Department of Thyroid and Neck Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China
• ³ Department of Ultrasound, Clinical Oncology School of Fujian Medical University, Fuzhou, China

Background:The EMT is a very important process involved in cancer invasion and metastasis. Additionally, CTSK gene is closely related to the degradation of the extracellular matrix, which is a critical component of the EMT. The purpose of this study was to determine the relationships between EMT-related genes and immune cell infiltration and their prognostic value in Thyroid carcinoma.Methods:Within the framework of the present study, the THCA cohort was analyzed in detail based on data obtained from The TCGA database in the context of the EMT. The TCGA-THCA cohort was then divided into two groups, high-and low-risk groups, based on the calculated EMT scores.Finally, based on the findings from the WGCNA algorithm, LASSO regression analysis, and Kaplan-Meier plotter, we selected five genes (CTSK, C3ORF80, FBLN2, PRELP and SRPX2) associated with patient prognosis. Furthermore, this study examined the presence of various immune cells within the THCA samples using three distinct algorithms, ssGSEA, xCell, and MCPcounter. Additional studies have been conducted to establish the roles of CTSK in THCA cell proliferation and migration using various assays, such as CCK8, colony formation, EdU proliferation, Transwell migration and wound healing assays.The involvement of CTSK in the regulation of various EMT-related markers was confirmed using Western blot.Results:Based on EMT scores, TCGA-THCA patients were further divided into two groups, and the study revealed that patients in the high-risk group had a worse prognosis than those in the low-risk group. Among the five genes linked to the prognostic value of EMT (CTSK, C3ORF80, FBLN2, PRELP, and SRPX2), CTSK exhibited notably elevated expression in the high-risk cohort. This group also exhibited pronounced immune cell infiltration, with a marked correlation observed between CTSK expression and the levels of macrophages, MDSCs, and various T-cell subtypes.Furthermore, in vitro studies demonstrated that reducing CTSK expression led to significant reductions in THCA cell viability; clonogenic, proliferative, motility and migratory capacities; and the expression of key EMT-related proteins, including N-cadherin, vimentin, slug, and snail. Conclusion:Our results suggest that the expression of CTSK, a gene associated with the EMT, may be associated with THCA onset and progression and thus may serve as a promising prognostic biomarker.