Eye Center, Renmin Hospital of Wuhan University, Wuhan 430000, China.
Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China.
Contrast Media Mol Imaging. 2022 Aug 16;2022:9794950. doi: 10.1155/2022/9794950. eCollection 2022.
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.
自体培养口腔黏膜上皮移植（COMET）是治疗角膜缘干细胞缺乏症（LSCD）的重要方法，但 COMET 术后周边角膜新生血管的出现阻止了其在临床实践中的广泛应用。在共培养过程中使用角膜缘龛细胞（LNCs）作为饲养细胞可以抑制术后角膜新生血管化。然而，其具体机制尚不清楚。在这项研究中，LNCs 被用作饲养细胞，通过模拟原始角膜缘微环境来改变培养的口腔黏膜上皮细胞（COMECs）的表型。对与 LNCs 或 3T3 细胞共培养的 COMECs 进行高通量测序（分别命名为 LNCs 组和 3T3 组），并分析差异表达的 miRNA。LNCs 组中共有 99 个已知和 1 个新预测的 miRNA 显著上调，而 101 个已知和 8 个新预测的 miRNA 显著下调。共预测到 3000 个具有 60 个最显著差异表达 miRNA 的靶基因，并最终筛选出 7 个上调和 7 个下调的 miRNA。发现两种共培养物的上清液中富含外泌体，表明 COMECs 与 LNCs 或 3T3 细胞之间的细胞间通讯非常活跃。此外，rno-miR-200-5p、rno-miR-204-5p、rno-miR-126a-3p、rno-miR-192-5p、rno-miR-211-5p、rno-miR-143-3p 和 rno-miR-184 在 LNCs 组中的表达水平明显高于 3T3 组，在外泌体中的表达水平也有相似的趋势。同时，细胞系测序显示，LNCs 组中有 7 个 miRNA 表达明显下调。有趣的是，在这种情况下，rno-miR-23a-3p、rno-miR-379-5p 和 rno-miR-127-5p 在外泌体中也明显下调。综上所述，本研究表明，LNCs 滋养的 COMECs 细胞间通过外泌体 miRNA 介导的信号转导可能是抑制血管生成的一个重要因素。
