Limbal MicroRNAs Differentially Expressed in Normal and Diabetic Human Corneas

Abstract

Purpose: To understand the roles of differentially expressed microRNAs in human limbal epithelial stem cells (LESC) versus mature central corneal epithelial cells in normal and pathological conditions such as diabetes mellitus. To examine how miRNAs function in regulatory pathways involved in corneal epithelial self-renewal, differentiation and wound healing processes in normal and disease states.

Methods: Deep sequencing analysis was performed on 10 normal and 12 diabetic central and limbal corneas. Differentially expressed miRNAs were validated by quantitative RT-PCR and in situ hybridization in independent samples. Primary LESC were transfected with 10-30 nM of human pre-miRTM miRNA precursors or miRNA inhibitors (antagomirs). Expression of signaling proteins and limbal markers was detected by immunostaining. Confluent transfected cell cultures were scratch-wounded and wound closure was monitored by photography and analyzed with ImageJ software. Organ-cultured human diabetic corneas were transfected with 30 nM antagomir of diabetes-upregulated miR-146a for 48 hr. The other cornea from each donor received scrambled Cy3-labeled control miRNA. 5-mm epithelial wounds were created using n-heptanol, and healing was monitored microscopically.

Results: Deep sequencing revealed several upregulated miRNAs (> 2 cutoff; p < 0.05) in limbal versus central corneas including miR-145-5p, -126-3p, and -136-3p. Some miRNAs (e.g., 146a-5p) were even more pronounced in the LESC-harboring basal cell layer suggesting their roles in stem cell maintenance. MiR-146a was also upregulated in diabetic versus normal limbus. Cell migration and wound closure were significantly delayed in normal primary LESC transfected with miR-146a, reminiscent of the situation in diabetic cornea with slow epithelial wound healing. Cells treated with miR-146a had decreased p-p38 and p-EGFR staining. MiR-146a antagomir significantly enhanced cell migration vs. scrambled control both in primary LESC and in diabetic organ-cultured corneas, with nearly 40% decrease in wound healing time.

Conclusions: These data suggest that miR-146a is associated with undifferentiated LESC at the corneal periphery, and its expression is downregulated following their radial 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.

Keywords: 482 cornea: epithelium • 721 stem cells • 765 wound healing