Effects of Exosomal microRNAs on Oral Mucosal Epithelial Cells Cocultured with Limbal Niche Cells

Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important method for the treatment of limbal stem cell deficiency (LSCD), but the appearance of peripheral corneal neovascularization after COMET has prevented its widespread use in clinical practice. Using limbal niche cells (LNCs) as feeders in the process of coculturing could inhibit the postoperative corneal neovascularization. However, the specific mechanism is still unclear. In this study, LNCs were used as feeder cells to alter the phenotype of cultured oral mucosal epithelial cells (COMECs) by mimicking the primitive limbal microenvironment. The high-throughput sequencing of COMECs cocultured with LNCs or 3T3 cells (named LNCs group and 3T3 groups) was performed, and differential miRNA expression was analyzed. A total of 99 known and 1 newly predicted miRNAs were significantly upregulated in the LNCs group, while 101 known and 8 newly predicted miRNAs were significantly downregulated. A total of 3000 target genes with the 60 most significantly differentially expressed miRNAs were predicted, and 7 upregulated and 7 downregulated miRNAs were ultimately screened. The supernatants obtained from both cocultures were found to be rich in exosomes, indicating that the intercellular communication between COMECs and LNCs or 3T3 cells was highly active. Furthermore, the expression levels of rno-miR-200-5p, rno-miR-204-5p, rno-miR-126a-3p, rno-miR-192-5p, rno-miR-211-5p, rno-miR-143-3p, and rno-miR-184 were significantly higher in the LNCs group compared to the 3T3 group, and the expression levels had a similar trend in exosomes. Meanwhile, sequencing of the cell lines revealed 7 miRNAs that were significantly downregulated in the LNCs group. Interestingly, in that case, rno-miR-23a-3p, rno-miR-379-5p, and rno-miR-127-5p were also significantly downregulated in the exosomes. In summary, this study suggested that signal transduction between cells mediated by exosomal miRNAs may be an important factor for the inhibition of angiogenesis by LNCs nourished COMECs.