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Rudolf Fuchshofer, Gunter Meister, Markus Kretz, W Daniel Stamer, Mark Johnson; Role of microRNAs in glaucomatous outflow resistance dysregulation. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6012.
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© ARVO (1962-2015); The Authors (2016-present)
MicroRNAs (miRNAs) have been implicated in many different diseases including neurodegenerative disorders, but their role in regulating conventional outflow resistance has not been explored. In the present study, we analyzed the miRNA expression profiles of normal and glaucomatous Schlemm's canal (SC) cells to identify differentially regulated miRNAs and the affected pathways.
SC cell strains were isolated from 4 normal and 3 glaucomatous human donors. Small RNA sequencing was performed to assess differences in miRNAs between normal and glaucomatous SC cells. Briefly, isolated RNA was ligated to an adenylated 3’ adapter and a 5’ RNA adapter, using a truncated T4 RNA ligase 2 as well as T4 RNA ligase 1. The ligated product was reverse-transcribed and size-separated using urea gel electrophoresis. Libraries for each sample were generated, sequenced and mapped against the human miRNA database. The average expression levels of glaucomatous SC cell lines were compared with the average of normal SC cell lines using a Student’s t-test.
Differential expression analysis carried out revealed 1317 miRNAs with altered expression patterns, among which were 138 differentially regulated miRNAs with an expression ratio differences in glaucomatous strains (relative to normals) of either greater than 0.8 (log2) or less than -0.8 (log2). Using these data sets the pathway analysis of the miRNAs and their possible target differentially expressed genes revealed novel networks. The results show that TGF-β, WNT, MAPK, PI3-AKT and most significantly the nitric oxide (NO) signaling system are affected. We identified 4 miRNAs with a validated role and 8 miRNAs with a predicted role in NO regulation. These miRNAs include miR-138 (+1.9 log2-fold; p=0.006), miR-221/222 (+1.1 log2-fold, p=0.037) and miR-27b (-0.8 log2-fold, p=0.037), which were shown to play a role in regulation of NO production. miR-221/222 were shown to be highly expressed in endothelial cells and are negatively associated with eNOS expression, whereas miR-138 has a negative impact on the NO production by downregulation of the Ca2+-binding protein S100A1, an essential factor required for eNOS activity.
We report strong preliminary evidence showing that miRNAs that regulate NO synthase expression are altered in glaucomatous SC cells. We suggest that such changes are a key step in loss of IOP regulation characteristic of primary open-angle glaucoma.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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