The TGF-beta microRNAome in glaucoma

Abstract

MicroRNAs (miRNAs) are essential regulators of many biological processes, therefore abnormal miRNA expression can play a role in both the onset and progression of diseases, such as glaucoma, via key disease-related pathways, such as the TGFβ signalling pathway. TGFβ-induced fibrosis is driven by NOX4-mediated redox imbalance, with evidence suggesting that NOX4-dependent redox signalling may regulate TGFβ/Smad signalling in a feed-forward manner. Our hypothesis is that unexplored interactions between miRNAs and their targets in glaucoma can lead to fibrosis and serve as disease indicators. Furthermore, miRNA modulation of NOX4 may be a therapeutic target for glaucoma fibrosis. This project aimed to investigate alterations in miRNA expression in primary non-glaucomatous human TM cells and in non-glaucomatous and glaucomatous Tenon’s ocular fibroblasts (NTF and GTF, respectively) and function regarding their involvement in TGFβ signalling and NOX4expression in glaucoma.

Small RNA-Seq was performed in TM cells treated with TGFβ1 or -β2, and in TGFβ1-NTF and GTF cells to determine changes in miRNA expression. A panel of enriched miRNAs were validated through RT-qPCR. Functional enrichment analyses were performed to determine enriched pathways. NOX4 mRNA expression in both cell types was studied in response to TGFβ stimulation. Six miRNAs; hsa-miR-9-5p, hsa-miR-21-3p, hsa-miR-25-3p,hsa-miR-92a-3p, hsa-miR-146a-5p and hsa-miR-146b-5p were selected for investigation based on in-silico analysis and literature evidence of available NOX4 data. The expression of these miRNAs was investigated in response to TGFβ1 or -β2 and NOX4 activity was studied, using an Amplex Red Assay, following cell transfection with miRNA mimics (miR-21-3p, miR25-3p, miR-92a-3p and miR-146a-5p), a NOX4 siRNA and GKT137831.

Overall results identified multiple differentially expressed TGFβ-induced miRNAs in the TM and in NTF and GTF tissue. The differentially expressed miRNAs were predominantly enriched in the TGFβ signalling pathway, focal adhesion, Hippo, MAPK and Wnt pathways, and regulation of the actin cytoskeleton. NOX4 mRNA expression was significantly upregulated in TGFβ1 and -β2 treated TM cells and in TGFβ1-stimulated NTF and GTF cells.miR-21-3p and miR-92a-3p inhibited NOX4 at early timepoints while miR-146a-5p is more effective after 48 hours of treatment.

In summary, this thesis presents original data evidencing that NOX4 is involved in TGFβ-induced fibrosis in both the TM and TFs. Furthermore, targeting NOX4 with miRNAs could be a promising therapeutic approach for suppressing the fibrotic effects of TGFβ in these cells.

Thesis is embargoed until 31st May 2026

Date of Award	May 2024
Original language	English
Supervisor	Andrew Nesbit (Supervisor), Colin Willoughby (Supervisor) & Declan McKenna (Supervisor)