Abstract
Purpose :
There is a strict boundary between the vascularized limbus and avascular cornea. MiRNA (miR)-184 is prominently expressed in corneal epithelial cells, and acts as a negative regulator of corneal angiogenesis. However, how limbal vascularity is maintained is unclear. Ephrin-A3, a ligand for EphA receptor tyrosine kinases, is a surface protein that is expressed specifically on limbal basal and superbasal cells. We show that Ephrin-A3 overexpression reduces miR-184 levels in limbal epithelial cells. It has been shown that epigenetic regulations such as methylation and histone acetylation play key roles in miRNA expression. Therefore, we hypothesize that Ephrin-A3 negatively regulates miR-184 levels in the limbal epithelium by epigenetic silencing of its promoter activity to maintain vascularity.
Methods :
Inhibitors and activators of methylation and histone acetylation were used in primary human limbal and corneal epithelial cell (HLEC and HCEC) cultures and a cell line (hTCEpi). miR-184 levels were determined by Taqman PCR. RNAseq analysis was performed in HLEC overexpressing Ephrin-A3 and data were validated by qPCR.
Results :
miR-184 levels were increased in HLECs and hTCEpi by: (i) pharmacological inhibition of methylation by 5-Aza; (ii) siRNA reduction of DNA methyl transferase 1; or (iii) siEphrin-A3. Ephrin-A3-induced suppression of miR-184 was also reversed by inhibition of DNA methylation. In addition, inhibition of NFkB1, an activator of histone acetylation, suppressed miR-184 levels in HCECs. NFkB1 has a binding site on miR-184 promoter. Conversely, PMA, an activator of this modification increased miR-184 and reversed Ephrin-A3 suppression of miR-184 in HLECs. RNAseq data showed changes in the levels of SIK1, an inhibitor of histone deacetylases (HDAC), and ATF3, a transcription factor that has a binding site on miR-184 promoter and shown to recruit HDAC.
Conclusions :
Collectively, these observations strongly support our hypothesis that miR-184 levels are regulated by DNA methylation in the limbal epithelium. We also demonstrate a positive role for histone acetylation on the miR-184 promoter in HCECs, and an Ephrin-A3-dependent inhibition of histone acetylation in HLECs to reduce miR-184 levels. The functional significance of such regulation is the suppression of the angiostatic miR-184 in the limbus, which insures proper limbal vascularity and maintenance of the limbal stem cell niche.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.