Winkler Michael A, Dib Christian, Ljubimov Alexander V, Saghizadeh Mehrnoosh
Eye Program, Board of Governors Regenerative Medicine Institute, Departments of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.
Eye Program, Board of Governors Regenerative Medicine Institute, Departments of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California, United States of America; David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America.
PLoS One. 2014 Dec 9;9(12):e114692. doi: 10.1371/journal.pone.0114692. eCollection 2014.
Limbal epithelial stem cells (LESC) residing at the corneal periphery are largely responsible for maintaining corneal optical transparency by continuously supplying new corneal epithelial cells, which mature during their radial migration to the central cornea. Diabetes mellitus (DM) affects all the structures of the eye including the cornea. Frequent epithelial erosions, delayed wound healing, and microbial infections are common alterations of the diabetic eye that can result in vision loss. MicroRNAs (miRNAs) are short non-coding oligonucleotides that regulate gene expression by repressing translation. Our purpose was to understand the role of miR-146a in the human limbal versus central corneal epithelial compartment in normal and pathological conditions such as diabetes mellitus. Using quantitative real-time PCR (QPCR) we found miR-146a enrichment in the limbal corneal compartment. This miRNA was also expressed at higher levels in the diabetic vs. normal limbus. Cell migration and wound closure were significantly delayed in normal and diabetic primary limbal epithelial cells (LEC) transfected with miR-146a. Cells treated with miR-146a had decreased levels of phosphorylated (activated) p38 and EGFR, mediators of epithelial wound healing. Conversely, inhibition of miR-146a significantly enhanced cell migration in both normal and diabetic primary LEC and in diabetic organ-cultured corneas by nearly 40% vs. scrambled miRNA control, accompanied by increased phosphorylated signaling intermediates. Transfection of miR-146a in cultured LEC resulted in an increased immunoreactivity for putative LEC markers Frizzled-7 and K15, whereas inhibition of miR-146a decreased their expressions. These data suggest that miR-146a plays a role in LEC maintenance at the corneal periphery, and its expression is downregulated during their migration towards the central cornea and accompanying terminal differentiation. Furthermore, abnormal miR-146a upregulation may be an important mechanism of delayed wound healing in the diabetic cornea.
位于角膜边缘的角膜缘上皮干细胞（LESC）通过持续供应新的角膜上皮细胞，在很大程度上负责维持角膜的光学透明度，这些新细胞在向角膜中央的径向迁移过程中成熟。糖尿病（DM）会影响包括角膜在内的眼睛所有结构。频繁的上皮糜烂、伤口愈合延迟和微生物感染是糖尿病眼部常见的病变，可导致视力丧失。微小RNA（miRNA）是短的非编码寡核苷酸，通过抑制翻译来调节基因表达。我们的目的是了解miR-146a在正常和糖尿病等病理条件下，在人角膜缘与中央角膜上皮区室中的作用。使用定量实时PCR（QPCR），我们发现miR-146a在角膜缘区室中富集。在糖尿病患者的角膜缘与正常角膜缘相比，这种miRNA的表达水平也更高。用miR-146a转染的正常和糖尿病原代角膜缘上皮细胞（LEC）的细胞迁移和伤口闭合明显延迟。用miR-146a处理的细胞中，上皮伤口愈合的介质磷酸化（活化）p38和表皮生长因子受体（EGFR）的水平降低。相反，与乱序miRNA对照相比，抑制miR-146a可使正常和糖尿病原代LEC以及糖尿病器官培养角膜中的细胞迁移显著增强近40%，同时磷酸化信号中间体增加。在培养的LEC中转染miR-146a会导致假定的LEC标志物卷曲蛋白-7（Frizzled-7）和角蛋白15（K15）的免疫反应性增加，而抑制miR-146a则会降低它们的表达。这些数据表明，miR-146a在角膜边缘的LEC维持中起作用，并且在它们向角膜中央迁移及伴随的终末分化过程中其表达下调。此外，miR-146a的异常上调可能是糖尿病角膜伤口愈合延迟的重要机制。
