Influence of Extracellular Matrix Stiffness on Micro-RNA Expression in Human Trabecular Meshwork Cells

Abstract

Glaucoma is a chronic neurodegenerative eye disease characterized by a loss of retinal ganglion cells associated with structural changes in the ocular outflow tract, disturbed intraocular pressure regulation and changes in tissue biomechanics. Earlier studies revealed a strong influence of ECM stiffness on protein expression patterns and growth factor signaling in cells of the ocular outflow tract (trabecular meshwork). To gain further insight into possible regulatory mechanisms, we asked whether ECM stiffness would alter expression of short non-coding microRNAs.Human trabecular meshwork cells were plated on tissue culture plastic or polyacrylamide gel substrata (E-moduli approx. 40 or 120kPa) of different stiffness and allowed to adjust for 7 days. Subsequently, miRNA expression levels were studied by Affymetrix arrays and qPCR. Genes targeted by at least five mechanoresponsive miRNAs (stiffness-related change >2 fold) were determined using R software. Protein expression of selected targets was assessed by western blot. At least 26 miRNAs were differentially expressed and 24 genes were predicted targets of 5 or more mechanoresponsive miRNAs. ECM stiffness-dependent protein expression changes were confirmed for the predicted targets NF-kB, AKT, E2F1, p53, p21 and cyclin-D1. These data suggest a mechanoresponsive regulation of miRNA levels with a possible role in cell differentiation and disease.