Abstract
Purpose :
Glaucoma is a neurodegenerative disease of the optic nerve that affects more than 60 million people worldwide. Elevated intraocular pressure is a major risk factor for glaucoma and leads to pathological fibrotic changes at the optic nerve head (ONH). Pro-fibrotic cytokine TGFβ2 is elevated in the ONH of glaucoma eyes and has been shown to induce the synthesis of extracellular matrix (ECM) including collagen types I and IV, fibronectin and post translational modifiers of ECM proteins such as tissue transglutaminase 2 (TGM2). MicroRNAs (miRNAs) are a class of small, endogenous non-coding RNAs that epigenetically regulate post-transcriptional gene expression. The purpose of this study was to determine whether TGFβ2 modulated the expression of miRNAs in ONH cells and whether transfection with candidate miRNA mimics targeted ECM and ECM related proteins in ONH cells.
Methods :
Primary human glial fibrillary acidic protein (GFAP) positive ONH astrocytes (ONA) and GFAP negative lamina cribrosa (LC) cells were grown to 100% confluency. LC cells and ONA were treated with 5ng/ml TGFβ2 or with control to determine differentially expressed miRNAs. Human ONA and LC cells were transfected with candidate miRNA mimics (1, 5, and 10nM) or non-targeting siRNA to determine ECM or ECM related protein expression levels in human ONH cells.
Results :
miRNA PCR arrays showed that TGFβ2 treatment downregulated the expression of miR-26a-5p, miR-26b-5p, miR-27b-3p and miR-29c-3p in LC cells, while miR-200b-3p, miR-211-3p, miR-217 were downregulated in ONA. Transfection with miR-26a-5p mimics downregulated the expression of TGM2 and FN in cultured LC cells and ONA respectively. Transfection with miR-29c-3p mimics downregulated the expression of FN and Gremlin1 in cultured ONA. Transfection with miR-217 mimics downregulated FN, TGM2 expression in cultured ONA.
Conclusions :
TGFβ2 is capable of modulating the expression of several miRNAs in cultured human ONH cells. Transfection of candidate miRNAs downregulated ECM and ECM associated proteins including FN, TGM2 and Gremlin 1 in human ONH cells. Thus, TGFβ2 modulation of miRNAs could lead to an increase in ECM deposition and covalent crosslinking in the glaucomatous ONH.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.