Integrated analysis identified MARK4 as a prognostic and immunomodulatory biomarker in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity and is characterized by high invasiveness and a poor prognosis. Although a number of molecular biomarkers have been investigated, their clinical applications remain limited. Despite microtubule affinity-regulating kinase 4 (MARK4) having been implicated in a number of cancers, its role in OSCC remains unclear. RNA-sequencing data and corresponding clinical information for patients with OSCC were obtained from The Cancer Genome Atlas database. MARK4 expression levels were analyzed in OSCC and corresponding noncancerous tissues. Associations between MARK4 expression and clinicopathological characteristics and survival outcomes, including overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI), were evaluated using univariate Cox regression and Kaplan-Meier analyses. Immune infiltration was assessed using single-sample Gene Set Enrichment Analysis, Cell-type Identification by Estimating Relative Subsets of RNA Transcripts and Tumor Immune Estimation Resource 2.0 analyses. To evaluate its functional role, two independent small interfering RNA was used to silence MARK4 expression in human squamous carcinoma-3 (HSC-3) cells. Results indicated that MARK4 was notably upregulated in OSCC and was associated with advanced T and clinical stages. High MARK4 expression was associated with poorer OS, DSS and PFI in the unadjusted survival analyses and showed discriminatory ability between tumor and normal tissues (area under the curve=0.841). Functional enrichment analysis indicated that MARK4 and its co-expressed genes were mainly involved in cytoskeletal organization, immune response and metabolic pathways. Immune infiltration analysis showed that MARK4 expression was negatively correlated with CD8⁺ T, B, dendritic and regulatory T cells. Functional experiments further demonstrated that MARK4 knockdown markedly suppressed HSC-3 cell proliferation and invasion, with minimal effects on cell migration. In conclusion, MARK4 is upregulated in OSCC and is associated with clinicopathological progression, survival outcomes, immune infiltration and tumor-cell aggressiveness. These findings support MARK4 as a potential survival-associated biomarker and candidate therapeutic target; however, its independent prognostic value remains to be established through multivariable analyses and external clinical validation.