Abstract
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.
Highlights
Diabetes mellitus (DM) is a metabolic disease currently affecting nearly 29 million people in the United States alone [1]
In the present study, using total RNA from six normal and five diabetic human limbi and central corneas, we demonstrated that in addition, miR-146a was upregulated in the limbal compartments of the diabetic vs. normal cornea (Fig. 1A)
Using a telomerase-immortalized human corneal epithelial cell (HCEC) line we have previously shown that miR-146a plays a major role in the corneal epithelial wound healing [25]
Summary
Diabetes mellitus (DM) is a metabolic disease currently affecting nearly 29 million people in the United States alone [1]. This number is projected to increase with the expanding population. Corneal epithelium is constantly renewed by limbal epithelial stem cells (LESC) located at the corneal periphery [6,7,8]. In normal corneal homeostasis LESC give rise to progeny, transient amplifying (TA) cells, which differentiate into mature corneal epithelium during their radial migration towards the central cornea [9]. The consistent generation of new corneal epithelium provides a means for maintaining a transparent cornea, which is required for optimal visual clarity. We have previously shown that diabetic corneas have significantly decreased expression of several LESC markers, which could contribute to their dysfunction and lead to diabetic keratopathy [10, 11]
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