Transforming Growth Factor β2 Regulates the Expression of microRNAs (miRNAs) in Human Optic Nerve Head Cells

Purpose: microRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. miRNAs are known to modulate cellular functions such as extracellular matrix (ECM) turnover. There is evidence that dysregulation of miRNA expression has a role in the pathogenesis of fibrotic diseases including glaucoma. Glaucoma is the leading cause of irreversible blindness and is associated with fibrotic changes to the optic nerve head (ONH), the initial site of glaucomatous damage to the retina and optic nerve. Our previous study showed that expression of the pro-fibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes compared to age-matched normal eye. However, there currently is little knowledge regarding the roles of miRNAs in the ONH. The purpose of this study was to determine if there are differences in expression of pro-fibrotic and anti-fibrotic miRNAs in normal ONH cells treated with or without TGFβ2. Methods: Primary human ONH cell strains derived from normal donor eyes were grown to 100% confluency. ONH cells were treated with 5ng/ml TGFβ2 or with control medium for 24hrs. RNA was isolated and cDNA synthesis performed for miRNA qPCR arrays to compare expression levels of pro-fibrotic and anti-fibrotic miRNAs in normal human ONH cells treated with or without TGFβ2. Results: Normal ONH cells exposed to TGFβ2 showed that several anti-fibrotic miRNAs were downregulated (hsa-miR-107, hsa-miR-132-3p, hsa-miR-141-3p hsa-miR-18a-5p, hsa-miR-194-5p, hsa-miR-204-5p) compared to control cells. In contrast, only one pro-fibrotic miRNA was upregulated (hsa-miR-34a-5p) in ONH cells treated with TGFβ2 compared to control. The most prominent targets of these miRNAs include connective tissue growth factor (CTGF), gremlin 2 and lysyl oxidase-like 3 (LOX-L3). Conclusions: Our results suggest that miRNAs expressed by ONH cells may be regulated by TGFβ2. These miRNAs may target CTGF, crosslinking enzymes and BMP antagonists to modify the ECM in the ONH.