Functional Characterization of Plasminogen Activator Urokinase as a Key Gene in Papillary Thyroid Carcinoma Lymph Node Metastasis

Abstract

Objective: Herein, we addressed the clinical challenge of high lymph node metastasis rates despite the lack of reliable diagnostic biomarkers in papillary thyroid carcinoma (PTC) by employing bioinformatics approaches to identify key biomarkers, aiming to provide new strategies for clinical diagnosis and treatment. Methods: Through bioinformatics analysis, plasminogen activator urokinase (PLAU) was identified as a key biomarker for lymph node metastasis in PTC. Immunohistochemistry (IHC) was performed to validate PLAU expression in tumor and adjacent normal tissues and its correlation with clinicopathological features. PLAU cellular expression was further confirmed by immunocytochemistry (ICC), Western blotting, and quantitative real-time PCR (qRT-PCR). Cell Counting Kit-8 and Transwell assays were used to assess its role in PTC tumor cell proliferation, migration, and invasion. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed on PLAU-related genes; immune cell infiltration in PTC was evaluated using the TIMER database and CIBERSORT algorithm. Results: Bioinformatics analysis showed that PLAU expression was significantly elevated in the PTC lymph node metastasis group [area under the receiver operating characteristic curve, 75.3%]. IHC results demonstrated significantly elevated PLAU expression in tumor tissues. Clinicopathological correlation analysis indicated that PLAU was associated with lymph node metastasis, particularly lateral cervical lymph node involvement. ICC, qRT-PCR, and Western blotting confirmed that PLAU was highly expressed in PTC tumor cells. After transient knockdown of PLAU, proliferation, migration, and invasion of PTC tumor cells were significantly reduced. GO and KEGG enrichment analyses showed that PLAU-related genes were primarily involved in signal transduction, inflammatory response, and P53, PI3K-Akt, and Mitogen-activated protein kinase (MAPK) signaling pathways. Immune cell infiltration was significantly higher in PTC tissues than in adjacent normal tissues; PLAU expression positively correlated with B and CD8⁺ T cell infiltration and Programmed cell death protein 1 (PD-1) and Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression. Conclusions: PLAU enhances PTC cell proliferation, migration, and invasion while promoting immune escape through the Th1/Th2 imbalance and PD-1/CTLA-4 upregulation, serving as a potential biomarker for lymph node metastasis in PTC.

Keywords: PLAU; bioinformatics; immune response; lymph node metastasis; papillary thyroid carcinoma.