A Group of Highly Secretory miRNAs Correlates with Lymph Node Metastasis and Poor Prognosis in Oral Squamous Cell Carcinoma

MicroRNAs (miRNAs) in oral squamous cell carcinoma (OSCC)-derived small extracellular vesicles (sEVs) play a pivotal role in modulating intercellular communications between tumor cells and other cells in microenvironment, thereby influencing tumor progression and the efficacy of therapeutic interventions. However, a comprehensive inventory of these secretory miRNAs in sEVs and their biological and clinical implications remains elusive. This study aims to profile the miRNA content of OSCC cell lines sEVs and computationally elucidate their biological and clinical relevance. We conducted miRNA sequencing to compare the miRNA profiles of OSCC cells and their corresponding sEVs. Our motif enrichment analysis identified specific sorting motifs that are implicated in either cellular retention or preferential sEVs secretion. Target cell analysis suggested that the sEVs miRNAs potentially interact with various immune cell types, including natural killer cells and dendritic cells. Additionally, we explored the clinical relevance of these miRNAs by correlating their expression levels with TNM stages and patient survival outcomes. Intriguingly, our findings revealed that a distinct sEVs miRNA signature is associated with lymph node metastasis and poorer survival in patients in TCGA-HNSC dataset. Collectively, this research furthers our understanding of the miRNA sorting mechanisms in OSCC and underscores their clinical implications. Extracellular vehicles (EVs) were harvested from cell culture supernatants using a ultracentrifugation protocol. First, we centrifuged the supernatants twice at 3000 g for 20 minutes at 4 °C to remove cell debris. Then, the cleared supernatant underwent ultracentrifugation at 120,000 g for 70 minutes at 4 °C using a Beckman Coulter Optima XE-100, facilitating EV isolation. The EV pellet was resuspended in phosphate-buffered saline (PBS). The quantitative and qualitative analysis of EVs was conducted through nanoparticle tracking analysis (NTA, Particle Metrix, Germany). For transmission electron microscopy (TEM) examination, EV samples were applied onto carbon-coated grids for 2 minutes, followed by dual PBS washes. Post-blotting and air-drying, the specimens were stained with 2% uranyl acetate for enhanced contrast and visualized using a Hitachi transmission electron microscope. miRNAs were extracted from cell and sEVs. miRNA-seq were performed to profiling miRNA transcriptome.