Abstract
Purpose :
There is a strict boundary within the anterior segmental epithelium where more differentiated corneal epithelial cells are clearly segregated from their limbal epithelial counterparts. Expression patterns of Eph/Ephrins as well as miRNAs show compartmentalization at the limbal/corneal junction. We have shown that reciprocal expression patterns of EphA2 and Ephrin-A1 are likely to contribute to normal limbal-corneal epithelial compartmentalization. Moreover, miRNA (miR)-184 is prominently expressed in corneal epithelial cells, and acts as a negative regulator of corneal angiogenesis. Therefore, we investigated whether Eph/Ephrin signaling is responsible for the miR-184 expression pattern in cornea.
Methods :
RNA-seq analysis was performed on human limbal and corneal epithelial cells (HLEC and HCEC) treated with antagomirs or Ephrin-A3 overexpression, and validated by real time qPCR and western blotting. Retroviral transduction was used to manipulate Ephrin-A levels. Antagomirs and siRNA knock-down were used to study changes in miR-184 levels by Taqman PCR.
Results :
We show that similar to miR-184, miR-210 is prominently expressed in corneal epithelial cells while expression of its well-known target, Ephrin-A3, is restricted to limbal basal cells. Interestingly, antago-210 markedly decreased miR-184 levels, suggesting that miR-210 positively regulates miR-184. Ephrin-A3 overexpression reduced miR-184 levels in HLECs, while Ephrin-A3 knocked down in antago-210 treated cells reversed the reduction seen in miR-184 levels. A transcription factor with a putative binding side on the miR-184 regulatory region was increased both with Ephrin-A3 and antago-210 treatment. Knock-down to reduce ATF3 levels showed increased miR-184 levels, suggesting a role for ATF3. Although antago-210 treatment resulted in increased EphA2 and reduced Ephrin-A1 levels, manipulation of EphA2/Ephrin-A1 expression had no effect on miR-184 levels. However, EphA1, a close relative of EphA2, is enriched in limbal epithelium and siRNA depletion of EphA1 in limbal epithelial cells enhances miR-184 levels and prevents inhibition of miR-184 expression by Ephrin-A3.
Conclusions :
Our data clearly demonstrate a link between miR-210 and EphA1/Ephrin-A3 signaling in restricting miR-184 expression within corneal epithelial cells. The functional significance of such regulation insures proper limbal vascularity while keeping the cornea avascular.
This is a 2021 ARVO Annual Meeting abstract.