Integrative Multi-Cohort Analysis of hsa-miR-1277, hsa-miR-301b, and hsa-miR-519a-1 Reveals Diagnostic Potential and Regulatory Associations in Oral Squamous Cell Carcinoma

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous malignancy characterized by extensive molecular dysregulation, including aberrant microRNA (miRNA) expression. Identification of clinically relevant miRNAs and their associated regulatory networks may improve understanding of oral squamous cell carcinoma (OSCC) biology and facilitate biomarker discovery. OBJECTIVE: The present study aimed to identify novel dysregulated miRNAs associated with OSCC and to investigate their potential diagnostic relevance, predicted target interactions, and functional pathway associations through integrative transcriptomic and clinical validation analyses. METHODS: Mature miRNA expression profiles from The Cancer Genome Atlas (TCGA)-HNSCC cohort were retrieved through the UCSC Xena Browser and analyzed using DESeq2 following anatomical site-based filtering to obtain HPV-negative OSCC samples. Differentially expressed miRNAs were identified using adjusted p-value < 0.05 and |log2FoldChange|≥ 0.5 thresholds. Based on differential expression patterns and limited prior characterization in OSCC, hsa-miR-1277, hsa-miR-301b, and hsa-miR-519a-1 were selected for downstream analyses. Kaplan-Meier survival, receiver operating characteristic (ROC), integrative target prediction, Pearson correlation, STRING protein-protein interaction network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were subsequently performed. Independent validation was carried out using four Gene Expression Omnibus (GEO) cohorts and qRT-PCR analysis in Indian OSCC clinical specimens. RESULTS: Differential expression analysis identified 283 significantly expressed miRNAs in OSCC, including 167 upregulated and 116 downregulated transcripts. ROC analysis demonstrated strong discriminatory performance for hsa-miR-301b (Area Under the Curve = 0.868) and moderate performance for hsa-miR-1277 (AUC = 0.735), whereas hsa-miR-519a-1 exhibited comparatively limited diagnostic utility. Integrative target prediction identified five high-confidence targets each for hsa-miR-1277 and hsa-miR-301b, while hsa-miR-519a-1 demonstrated a broader overlap profile comprising 24 candidate genes. Functional enrichment analyses demonstrated enrichment of neuronal signaling, synaptic regulation, ion channel activity, neuroactive ligand-receptor interaction, and cadherin-associated pathways among candidate target genes. Validation across independent GEO cohorts and clinical specimens demonstrated heterogeneous expression patterns with partial concordance to TCGA-derived observations. CONCLUSION: This integrative multi-cohort analysis identifies hsa-miR-1277, hsa-miR-301b, and hsa-miR-519a-1 as novel miRNA candidates associated with OSCC-related molecular dysregulation. The findings further highlight the substantial biological heterogeneity underlying HNSCC and emphasize the challenges associated with translating large-scale transcriptomic signatures into reproducible clinical biomarkers.