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MicroRNAs and their role in cancer have been extensively studied for the past decade. Here, we analyzed the biological role and diagnostic potential of <i>miR-154-5p</i> and <i>miR-154-3p</i> in head and neck squamous cell carcinoma (HNSCC). miRNA expression analyses were performed using The Cancer Genome Atlas (TCGA) data accessed from cBioPortal, UALCAN, Santa Cruz University, and Gene Expression Omnibus (GEO). The expression data were correlated with clinicopathological parameters. The functional enrichment was assessed with Gene Set Enrichment Analysis (GSEA). The immunological profiles were assessed using the ESTIMATE tool and RNAseq data from TCGA. All statistical analyses were performed with GraphPad Prism and Statistica. The study showed that both <i>miR-154-5p</i> and <i>miR-154-3p</i> were downregulated in the HNSCC samples and their expression levels correlated with tumor localization, overall survival, cancer stage, tumor grade, and HPV p16 status. GSEA indicated that individuals with the increased levels of <i>miR-154</i> had upregulated AKT-MTOR, CYCLIN D1, KRAS, EIF4E, RB, ATM, and EMT gene sets. Finally, the elevated <i>miR-154</i> expression correlated with better immune response. This study showed that <i>miR-154</i> is highly involved in HNSCC pathogenesis, invasion, and immune response. The implementation of <i>miR-154</i> as a biomarker may improve the effectiveness of HNSCC treatment.