The aim of this study is to investigate whether tongue squamous cell carcinoma (TSCC)-derived exosomal microRNAs (miRNAs) promote the conversion to tumor-associated fibroblasts (CAFs) and to elucidate the underlying molecular mechanisms. First, we isolated and characterised exosomes from the CAL27 tongue squamous cell carcinoma cell line. These exosomes were then co-cultured with human oral mucosal fibroblasts (hOMFs). We evaluated the expression of inflammatory cancer-associated fibroblasts (iCAFs)-related markers using Western blotting (WB), quantitative real-time PCR (qRT-PCR) and ELISA. We then conducted miRNA sequencing of the exosomes to identify the top five highly expressed miRNAs. WB was then employed to assess which of the corresponding miRNA mimics most effectively promotes the transformation of hOMFs into iCAFs. Finally, we predicted the potential target genes of the selected miRNA through bioinformatics analysis and validated them using a dual-luciferase reporter assay. Under transmission electron microscopy, the isolated exosomes exhibited a characteristic cup-shaped morphology with a double-membrane structure. Nanoparticle tracking analysis (NTA) revealed a size distribution ranging from 30 to 150 nm. Western blotting confirmed positive expression of the exosomal markers CD9, CD81, HSP90 and TSG101, while microRNA (miRNA) sequencing identified the most highly expressed miRNAs as miR-21-5p, miR-122-5p, miR-148a-3p, miR-143-3p and let-7i-5p. Of these, miR-122-5p induced the most significant changes in the expression levels of iCAF-related markers. Bioinformatic prediction combined with dual-luciferase reporter assays confirmed that GNPDA1 is a direct target of miR-122-5p. CAL27-exo may facilitate the conversion of hOMFs into iCAFs by targeting glucosamine-6-phosphate deaminase 1 (GNPDA1) via exosomal miR-122-5p.
Xia et al. (Tue,) studied this question.