Advanced laryngeal squamous cell carcinoma (LSCC) is the second most prevalent type of head and neck squamous cell carcinoma (HNSCC). Identifying microRNAs (miRNAs) related to key regulatory molecules or mechanisms could offer an alternative approach to developing new treatment strategies. The aim of our study is to evaluate significant correlations among deregulated miRNAs in advanced laryngeal carcinoma and to analyze, in silico, their strength of association, targets, and the most deregulated pathways. Several miRNAs demonstrated promising co-expression results, specifically miR-93-5p, miR-145-5p, and miR-210-3p. Their expressions were explored and further validated in a large set of in vivo advanced LSCC samples, which were subsequently used for bioinformatics and enrichment analyses. Our results highlight the significant roles of miR-93-5p, miR-145-5p, and miR-210-3p in regulating major pathways linked to the cell cycle via epithelial-to-mesenchymal transition (EMT), PI3K/Akt signaling, hypoxia, metabolism, apoptosis, angiogenesis, and metastasis. The associations between the expressions of these miRNAs and patients' clinical features could be central to the progression of advanced LSCC. Overall, our study provides important insights into the co-expression and regulatory networks of miR-93-5p, miR-145-5p, and miR-210-3p in advanced laryngeal carcinoma, underscoring their potential as therapeutic targets or biomarkers for this aggressive cancer. Further research is needed to elucidate the specific mechanisms through which these miRNAs contribute to the pathogenesis and progression of laryngeal carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a significant global health burden, for which there has been limited evidence of improved survival rates. Although the roles of hypoxia‑inducible factor (HIF)1α and HIF2α have been well documented in hypoxia, the involvement of HIF3α, particularly in LSCC, has been inadequately explored. The present study aimed to investigate the correlation between HIFα subunits and the hypoxia‑related long noncoding RNAs (lncRNAs) MALAT1 and HOTAIR in 63 patients diagnosed with LSCC. Total RNA was extracted from fresh‑frozen laryngeal tumor and adjacent normal tissues, and was subjected to reverse transcription‑quantitative PCR for target detection. Statistical analyses were conducted using SPSS software, with significance set at P<0.05. The present study is the first, to the best of our knowledge, to report a positive moderate monotonic correlation (rs=0.347) and moderately strong positive linear correlation (r=0.630) between HIF3α mRNA and lncRNA MALAT1 in LSCC. Regression analysis revealed a direct association between 39.6% of both variables. Additionally, a positive correlation was observed between lncRNAs MALAT1 and HOTAIR (rs=0.353); HIF2α mRNA and lncRNA MALAT1 (rs=0.431); HIF3α mRNA and lncRNA HOTAIR (rs=0.279); and HIF3α mRNA and HIF2α mRNA (rs=0.285). Notably, a significant negative correlation (rs=‑0.341) was detected between HIF3α mRNA and HIF1α mRNA, potentially indicative of the HIF switch or negative regulation. In addition, the present study investigated the association between HIFα subunits and the clinicopathological characteristics of patients. The results revealed a notable association between HIF1α transcript levels and the location of LSCC; specifically, subglottic tumors exhibited elevated HIF1α levels compared with glottic and supraglottic LSCC. Furthermore, a significant association was identified between HIF3α transcript levels and patient age (P=0.032) and positive family history (P=0.047). In conclusion, the present findings suggested a pivotal role for HIF3α in LSCC development, potentially involving direct regulation of lncRNA MALAT1. However, further research is warranted to elucidate its precise mechanisms.

Background and Objectives: Laryngeal cancer is a common head and neck tumor burden, with no significant improvements in long term patient survival. Despite the progress of molecular genetics and oncology strategies, there is still a lack of biomarker use in routine clinical practice for early laryngeal cancer screening or diagnosis. miRNAs are explored as promising molecules, that could serve as liquid biopsy. Our goal is to explore the screening potential of miR-31-3p and miR-196a-5p in early- and advanced-stage laryngeal HPV-negative plasma samples. Methods: In this study, 50 plasma samples obtained from early and advanced HPV-negative laryngeal cancer patients were included. The expression levels of mir-31-3p and miR-196a-5p were analyzed via TaqMan RT-qPCR. SPSS v27.0 was used for statistical analysis. Results: For the first time, miR-31-3p and miR-196a-5p were analyzed in plasma samples from early HPV-negative primary LSCC patients. Both circulating miRNAs showed significantly elevated expression levels in early and advanced laryngeal cancer samples. miR-31-3p was significantly associated with T stages (p < 0.001) and N stages (p = 0.009). The ROC analysis revealed that miR-31-3p could significantly discriminate early-stage from advanced-stage LSCC with an AUC of 0.850 (95% CI: 0.743–0.956, p < 0.001) at an RQ cutoff of 2.03, achieving a sensitivity of 95.5% and a specificity of 64%. Nevertheless, miR-196a-5p was found to be significantly overexpressed in early-stage LSCC, which could contribute to the development of its screening potential. For the first time, both miRNAs revealed a significant positive correlation, which indicates that miR-31-3p and miR-196a-5p could coregulate cancerogenesis. Conclusions: In conclusion, the data revealed that miR-31-3p has greater potential as an LSCC screening marker in comparison to miR-196a-5p. Still, miR-196a-5p also showed promising results in early-stage laryngeal cancer monitoring. The utilization of circulating miR-31-3p or miR-196a-5p analysis could enable liquid biopsy approaches, with results potentially informing treatment monitoring strategies, personalized oncological protocols, and early diagnosis. These advancements could ultimately benefit patient outcomes by improving laryngeal organ preservation and survival rates.

Supplementary Table 2. H-scores for VEGF Expression in Individual Tumor Samples.

Subgroup of patients with VEGF-A upregulation at depth (RQ > 2) and HIF1α downregulation (RQ ≤ 2) showed markedly higher recurrence rates (log-rank P = 0.0039; χ2 = 8.346).

A, Distribution of recurrent cases by molecular subgroup. The VEGF-A/HIF1α subgroup (blue) and the miRNA co-expression subgroup (red) were independent and together accounted for 76% of recurrences. B, Kaplan–Meier analysis confirmed significantly poorer outcomes for both subgroups (log-rank P = 0.0015; χ2 = 12.95).