Abstract
Purpose :
Glaucoma is associated with elevated levels of the profibrotic cytokine transforming growth factor-β2 (TGF-β2). TGF-β2 has been implicated in glaucoma damage to the optic nerve head (ONH), which undergoes changes such as increased ECM synthesis and deposition. The disruption of the normal balance between ECM deposition and degradation have an important role in the integrity of the ONH. ONH studies have focused on the regulators of the ECM including matrix metalloproteinases, growth factors, and cross-linking enzymes. However, miRNAs have added additional complexity as post-transcriptional regulators of gene expression that modulate cell functions. The purpose of this study was to determine: (a) whether there are differences in expression of miRNAs between normal and glaucoma ONH cells, (b) whether expression of ONH cell miRNAs are regulated by TGF-β2 and (c) differentially expressed miRNA target predictions.
Methods :
Primary human ONH cell strains from normal (NONH=3) and glaucomatous (GONH=3) donor eyes were grown to 100% confluency and treated with or without TGF-β2 (5ng/ml) for 24 hours. RNA was isolated and cDNA synthesis performed and subjected to miRNA PCR arrays. miRNA target predictions were based on the miRanda algorithm.
Results :
The miRNA PCR arrays were analyzed and comparisons were made between NONH and GONH cells and most of the affected miRNAs were down regulated (hsa-miR-9-5p, hsa-miR-32-5p, hsa-Let-7c, hsa-Let-7f-5p, and hsa-miR-124-3p). NONH cells exposed to TGF-β2 showed that the most affected miRNA was up regulated (hsa-miR-424-5p). GONH cells exposed to TGF-β2 showed that the most affected miRNAs were down regulated (hsa-miR-9-5p, hsa-miR-26b-5p, hsa-miR-200c-3p). The most prominent targets of miRNAs included LOX and gremlin, which previously were shown to have altered expression in GONH cells or with TGF-β2 treatment.
Conclusions :
These data indicate that miRNAs are expressed by ONH cells and may be regulated by TGF-β2. Therefore, miRNAs may target BMP antagonist proteins and cross-linking enzymes to modify the ECM in the ONH. These results may provide a new understanding on the role of miRNA epigenetics in molecular pathogenesis of the ONH.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.