Significant Role of Differentially Expressed miR-10b in Normal and Diabetic Limbus in Corneal Epithelial Homeostasis

Abstract

Purpose : Limbal epithelial stem cells (LESC) are indispensable for normal function of corneal epithelium. MicroRNAs play a major role in modulating a plethora of biological processes through post-transcriptional gene regulation; nevertheless, their role in maintenance and differentiation of LESC remains unclear. A comprehensive analysis of miRNA expression profile and a thorough investigation of specific differentially regulated miRNAs can reveal their role in corneal limbus in normal and diseased state.

Methods : Age-matched human autopsy normal and diabetic corneas were received from the National Disease Research Interchange (NDRI) in Optisol medium (Chiron Vision) within 24 hours of donor death. Ten normal and 12 diabetic corneas were used for miRNA expression profiling using deep sequencing. The in situ hybridization for localization of specific miRNA, and immunohistochemistry experiments for specific proteins were performed. The in vitro experiments were performed with either primary limbal epithelial cells (LEC) or telomerase-immortalized human corneal epithelial cells (tHCEC). Transfection was performed using lipofectamine RNAiMAX (Thermo Fisher) following the manufacturer’s instructions. Experiments were analyzed using Prism6 software (GraphPad Software) by Student’s t-test for two groups, or ANOVA for three or more groups with p < 0.05 considered as significant.

Results : Of many differentially regulated miRNAs, miR-10b was one of the most abundant miRNA in limbus (91.844 fold increase compared to central cornea, adjusted p-value <1.18E-07) and was also up-regulated in diabetic limbus. In situ hybridization confirmed this finding and further showed that miR-10b expression was more pronounced in the basal and suprabasal epithelial cell layers. Overexpression of miR-10b in human organ-cultured corneas and primary limbal cells increased the number of Ki-67 positive cells and colony formation, respectively. miR-10b transfected human organ-cultured corneas showed downregulation of Pax6, Klf-4 and Dkk1, and upregulation of limbal keratins K15 and K17.

Conclusions : Distinct miRNA expression profile in limbus vs. central cornea suggests a major role of miRNAs in the limbus. It can also be concluded that miR-10b is involved in the maintenance and/or early differentiation of LESC. The role of miR-10b in diabetes and therapeutic potential therein needs further investigation.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.