Abstract
Small non-coding RNAs, in particular microRNAs (miRNAs), regulate fine-tuning of gene expression and can impact a wide range of biological processes. However, their roles in normal and diseased limbal epithelial stem cells (LESC) remain unknown. Using deep sequencing analysis, we investigated miRNA expression profiles in central and limbal regions of normal and diabetic human corneas. We identified differentially expressed miRNAs in limbus vs. central cornea in normal and diabetic (DM) corneas including both type 1 (T1DM/IDDM) and type 2 (T2DM/NIDDM) diabetes. Some miRNAs such as miR-10b that was upregulated in limbus vs. central cornea and in diabetic vs. normal limbus also showed significant increase in T1DM vs. T2DM limbus. Overexpression of miR-10b increased Ki-67 staining in human organ-cultured corneas and proliferation rate in cultured corneal epithelial cells. MiR-10b transfected human organ-cultured corneas showed downregulation of PAX6 and DKK1 and upregulation of keratin 17 protein expression levels. In summary, we report for the first time differential miRNA signatures of T1DM and T2DM corneal limbus harboring LESC and show that miR-10b could be involved in the LESC maintenance and/or their early differentiation. Furthermore, miR-10b upregulation may be an important mechanism of corneal diabetic alterations especially in the T1DM patients.
Highlights
Many forms of corneal conditions can be sight threatening, such as diabetic keratopathy and limbal stem cell deficiency (LSCD) that is hard to treat even with corneal transplantation
In the limbus, which is more metabolically active than central cornea, most miRNAs had much higher expressions suggesting their possible roles in limbal epithelial stem cells (LESC) maintenance and differentiation both in normal and diseased tissue
Using deep sequencing with quantitative reverse transcribed (RT)-PCR (QRT-PCR) validation, we have identified a number of differentially expressed miRNAs in limbus vs. central cornea, some of which have not been documented previously (Table S1)
Summary
Many forms of corneal conditions can be sight threatening, such as diabetic keratopathy and limbal stem cell deficiency (LSCD) that is hard to treat even with corneal transplantation Such diseases are related to abnormal self-renewal and wound healing due to epithelial dysfunction. We examined how certain limbal-enriched and diabetes-increased miRNAs function in regulatory pathways involved in corneal epithelial self-renewal and differentiation in normal and disease states. To our knowledge, this is the first study using genome-wide microRNA expression profiling in normal vs T1DM and T2DM limbal and central human corneas by high throughput sequencing. We provide novel data on the role of miR-10b, one of the most abundant limbal miRNAs, in the corneal epithelium with possible function in LEC homeostasis
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