miRNA Profiling of Optic Nerve Head Astrocytes Exposed to Cyclic Stretch

Introduction: Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, a leading cause of irreversible blindness consequent to retinal ganglion cell (RGC) degeneration. Elevated IOP induces biomechanical aberrations within ocular tissues - including the transmission of biomechanical stretch through the lamina cribrosa (LC) region of the optic nerve head (ONH), the site where RGC axon damage first occurs. LC cells and ONH astrocytes (ONHA), the primary cells of the LC, respond to stretch in a manner that promotes pathological extracellular matrix (ECM) remodeling (fibrosis) and mechanical damage of RGCs within the ONH. A complex set of molecular mechanisms regulate ECM remodeling. Part of this regulation may involve microRNAs (miRNAs), small molecules that can inhibit protein expression by binding to and silencing mRNA. In this study, we examined miRNA expression profiles of ONHA exposed to cyclic stretch. We hypothesized that cyclic stretch would induce upregulation of miRNAs that silence anti-fibrotic protein translation and downregulation of miRNAs that silence pro-fibrotic protein translation, promoting a net-fibrotic molecular signaling environment. Methods: Primary human normal ONHA cell strains (n=3) were exposed to 0-12% cyclic stretch for 24 hours; controls were exposed to 0% stretch. RNA samples were collected from stretched and control cells, and miRNA PCR arrays were used to determine expression changes for miRNAs associated with fibrosis. Expression fold changes were normalized to SNORD68. The bioinformatics tool TargetScan was used to predict mRNA targets for any dysregulated miRNAs. Induction of fibrotic cellular changes by cyclic stretch was confirmed by western blotting of conditioned media for secreted proteins. Results: miR-146b-5p was found to be significantly upregulated by +5.97-fold (P = 0.029) in stretched ONHA. Predicted mRNA targets for miR-146b-5p are known to be involved in fibrosis and cell survival, among other functions. Preliminary data indicates upregulation of secreted proteins associated with fibrosis (TGFβ2, Fibronectin, Transglutaminase 2) by stretched ONHA. Conclusions: Stretch modulates miRNA expression in cultured human ONHA, miR-146b may mediate ECM alterations and other pathological changes at the LC. Future experimental directions will include assessing co-expression of other miR-146 family miRNAs, validating putative mRNA targets and elucidating the mechanisms by which specific miRNA and their targets modulate ECM remodeling.