Abstract
Purpose :
Fuchs endothelial corneal dystrophy (FECD) is a vision threatening disorder characterized by endothelial cell dysfunction. Excessive production of extracellular matrix (ECM) proteins is one mechanism predicted to contribute to ER stress and cellular dysfunction. Our previous work found a number of micro-RNAs (miRs) to be differentially methylated in FECD patients compared with controls. Specifically, miR-199-3p was significantly hypermethylated in FECD endothelial cells. Here we investigate the expression of miR-199-3p in FECD endothelial cells and explore mechanistically how miR-199-3p may be implicated in ECM production.
Methods :
Corneal endothelium was collected from FECD patients undergoing endothelial keratoplasty. Non-FECD corneal endothelial samples (controls) were obtained from an eye bank. RNA was isolated from tissue samples using PureLink RNA mini kit (Ambion). Reverse transcription, pre-amplification and qPCR were performed using TaqMan Micro-RNA kits with predesigned primer/probes (Applied Biosystems). To explore the functional potential of miR-199-3p in FECD pathogenesis, in silico analysis was performed to predict putative target genes and corresponding binding sites using computational prediction algorithms (Targetscan and miRmap).
Results :
RNA was isolated from endothelial cells from 9 patients with FECD and 10 control patients. qPCR expression data was available for 4 FECD samples and 7 control samples. Expression of miR-199-3p was decreased in FECD patients compared with controls. FECD samples expressed 0.28 the amount of miR-199-3p (CI 0.23-0.50) compared with control samples. In silico analyses predicted highly conserved binding sites for miR-199-3p in SP1, DNMT3 and FN1 mRNAs across species (prediction scores 92-94). Sequence alignment analyses revealed a highly conserved binding motif in the 3′-UTR of SP1 (position 886-893), DNMT3 (position 837–843) and FN1 (position 646–652).
Conclusions :
This study contributes to the evidence supporting epigenetic regulation of ECM production in patients with FECD. The miR-199 family is hypermethylated in FECD endothelial cells, resulting in reduced expression of miR-199-3p. Downregulation of miR-199-3p increases expression of its target genes including predicted targets SP1/DNMT3A (enhancing further DNA methylation) and FN1 (contributing to increased ECM production).
This is a 2020 ARVO Annual Meeting abstract.