Wound Healing Related microRNAs Differentially Expressed in Diabetic Cornea

Abstract

Purpose: : To identify by miRNA microarray analysis miRNAs abnormally expressed in diabetic corneas and determine their effects on wound healing in human corneal epithelial cell (HCEC) line.

Methods: : Total RNA was extracted from 6 normal and 6 diabetic age-matched human central corneal buttons.Labeled RNA was hybridized to Affymetrix GeneChip® miRNA Arrays1.0 containing 1813 human probe sets including 521snoRNAs and scaRNAs, and 847miRNAs. Supervised analysis was used to identify expression differences, with 2-fold cutoff. Select differentially expressed miRNAs were validated by quantitative RT-PCR (Q-PCR). Cultures of 60% confluent telomerase-immortalized HCEC were transfected with 30-100 nM of human pre-miR miRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000 (Invitrogen). Total RNA extracted 72 hrs post transfection was analyzed by Q-PCR. High-density transfected HCEC were scratch-wounded with P200 tip.Wound closure was monitored at regular intervals over 24 hrs by digital photography and analyzed with ImageJ. Wound areas from 3 independent triplicate assays relative to 0 hr were compared to negative control (cells transfected with Cy3-labeled h-miR). Phosphorylated p38 MAP kinase (p-p38) was detected by immunostaining.

Results: : We identified several differentially expressed miRNAs in diabetic vs. normal corneas and confirmed these changes by Q-PCR. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure. Respective antagomirs significantly enhanced wound healing vs. controls. Q-PCR showed increased miRNA expression in transfected HCEC but decreased expression when using antagomirs. Treatment with h-miR-424 decreased p-p38 staining, but it increased upon antagomir treatment over control in cells close to wound edge. Wound healing in high glucose (30 mM) was similar to control, with or without miRNA.

Conclusions: : Several miRNAs with increased expression in human diabetic central corneas were found using Q-PCR validated miRNA microarrays. Two miRNAs inhibited wound healing in cultured corneal epithelial cells, and specific antagomirs enhanced this process. Abnormal miRNA expression may be an important mechanism of diabetic changes in corneal wound healing. Manipulating miRNA levels in diabetic corneas could help alleviate symptoms of diabetic keratopathy.

Keywords: cornea: epithelium • diabetes • wound healing