Differential Expression of Selected miRNAs in CD44 ‐Enriched Cell Populations of Oral Squamous Cell Carcinoma

Background Oral squamous cell carcinoma (OSCC) remains a clinically challenging malignancy characterized by marked biological heterogeneity, high recurrence rates, and limited progress in survival outcomes. MicroRNAs (miRNAs) have emerged as key post‐transcriptional regulators involved in carcinogenesis and cancer progression, yet their expression patterns within phenotypically distinct tumor cell subpopulations, particularly CD44 + cancer stem cells‐like (CSC‐like), remain insufficiently characterized. Objective This study investigated the expression of five candidate miRNAs (hsa‐miR‐26a‐5p, hsa‐miR‐31‐5p, hsa‐miR‐133a‐5p, hsa‐miR‐491‐3p, and hsa‐miR‐532‐3p) in OSCC on multiple levels: public dataset analysis, whole‐tissue expression profiling, and evaluation within sorted CD44 + and CD44 − primary cell populations. Results TCGA‐HNSC dataset analysis revealed upregulation of hsa‐miR‐31‐5p and hsa‐miR‐532‐3p ( p < 0.0001), alongside downregulation of hsa‐miR‐26a‐5p and hsa‐miR‐133a‐5p in tumor tissue ( p < 0.0001, p = 0.0015, respectively), whereas hsa‐miR‐491‐3p showed no significant difference ( p = 0.925). In our OSCC cohort ( n = 20 OSCC, n = 10 controls), hsa‐miR‐31‐5p was significantly upregulated ( p = 0.0086), while hsa‐miR‐26a‐5p, hsa‐miR‐133a‐5p, and hsa‐miR‐491‐3p were markedly downregulated in tumor samples compared with normal mucosa ( p = 0.0363, p = 0.0029, p < 0.0001, respectively). Although hsa‐miR‐532‐3p exhibited an upward trend, this change did not reach statistical significance. CD44 expression was significantly higher in OSCC tissues, particularly in advanced TNM stages ( p = 0.0319), confirming its association with tumor aggressiveness. To explore miRNA dysregulation within CD44‐defined subpopulations, CD44 + and CD44 − cells were isolated from primary OSCC cultures ( n = 5 primary cultures). hsa‐miR‐133a‐5p, hsa‐miR‐491‐3p, and hsa‐miR‐532‐3p were significantly less expressed in CD44 + cells than in CD44 − cells ( p = 0.0142, p = 0.0015 and p = 0.0002), indicating that their downregulation characterizes the CD44 + ‐enriched fraction. These findings suggest that the tumor‐suppressive miRNAs hsa‐miR‐133a‐5p, hsa‐miR‐491‐3p, and hsa‐miR‐532‐3p are selectively dysregulated within CSC‐like cells, potentially contributing to stemness‐associated signaling and therapy resistance. Conclusion Overall, this study integrates bioinformatic, tissue‐level, and cell‐specific analyses to identify miRNAs implicated in CSC‐like biology in OSCC, highlighting their potential as biomarkers and targets for therapeutic strategies aimed at eradicating chemotherapy‐resistant CSC‐like populations.