Correlation of carbonic anhydrase 9 (CA9) with pathological T-stage and prognosis in patients with oral tongue squamous cell carcinoma

BACKGROUND: We explored the mechanisms underlying tumorigenesis in oral tongue squamous cell carcinoma (OTSCC) with the goal of uncovering prognostic molecular biomarkers. METHODS: An mRNA sequencing dataset was obtained from The Cancer Genome Atlas (TCGA) database, and differentially expressed genes (DEGs) were selected using R language software packages. Functional enrichment analysis was conducted with DAVID software and protein-protein interaction (PPI) networks were constructed using the STRING database. The relationship between hub genes and overall survival (OS) was evaluated by Kaplan-Meier analysis and Cox proportional hazard regression models. Expression of the candidate gene, carbonic anhydrase 9 (CA9), was verified by real-time RT-PCR, western blotting, and immunohistochemistry. RESULTS: DEGs (n=581) were obtained from 11 OTSCC samples and corresponding adjacent non-tumor tissues. Gene ontology (GO) analysis revealed that most DEGs were implicated in anterior/posterior pattern specification, embryonic skeletal system morphogenesis, and multicellular organism development, and pathway analysis suggested that DEGs were associated with neuroactive ligand-receptor interaction, calcium signaling pathway and transcriptional misregulation in the cancer. A PPI network consisting of 301 nodes and 2011 edges was constructed and 71 hub genes, with high degrees of connectivity in the network, were identified. Kaplan-Meier analysis of the hub genes indicated that high expression of CA9, LHX1, and KISS1R and low expression of CCKAR were associated with poor OS in OTSCC; however, only CA9 was a significant prognostic factor influencing survival in OTSCC on multivariate analysis. High expression of CA9 was associated with poor pathological T-stage. CA9 tumor specificity was confirmed using the Gene Expression Omnibus (GEO) database and further molecular tests. CONCLUSIONS: We identified key DEGs that may assist in the molecular understanding of OTSCC. CA9 warrants further exploration as potential prognostic biomarker and therapeutic target in OTSCC.