Abstract
Purpose :
Primary open angle glaucoma (POAG) results from defective extracellular matrix (ECM) deposition and turnover by trabecular meshwork (TM) cells, driven by an increase in TGFβ signalling. Connective tissue growth factor (CTGF) is a key downstream mediator of the ECM effects of TGFβ, and has been shown to also have increased expression in the aqueous humor of POAG patients. Identifying regulators of this response could lead to new therapeutic targets. MiRNA-18a, -19a, -19b, -199a, -199b, -26a, -26b, -133b and -205 have been shown to regulate the TGFβ/CTGF response in other tissues. Here we investigated the change in expression of these miRNAs in human TM (HTM) after TGFβ treatment, and what effect they have on CTGF expression in response to TGFβ.
Methods :
TM cells from 5 human donors were treated with 5 ng/ml TGFβ for 24 hours and the expression of miRNAs, CTGF mRNA and protein were measured. HTM cells from three donors were treated with 5 ng/ml TGFβ and cotransfected with 100 nM of miRNA mimic, miRNA inhibitor or a scrambled control for 24 hours. The mRNA and protein expression of CTGF was measured, and compared to HTM treated with TGFβ without cotransfection.
Results :
Although the expression of CTGF increased after TGFβ treatment in HTM cells from all donors, miRNA responses were variable between donors with only miR-18a showing a consistent upregulation, with an average 1.44±0.22-fold increase (mean±SD, n=5, p<0.001). With this in mind, we further investigated the effect of miR-18a on the TGFβ-induced expression of CTGF. Cells transfected with a miR-18a mimic showed a 0.57±0.30-fold reduction in the transcript abundance of CTGF (mean±SD, n=3, p<0.01), and a 0.411±0.020-fold reduction in the protein abundance (mean±SD, n=3, p<0.0001). Inhibiting miR-18a had no significant effect on CTGF expression.
Conclusions :
These data suggest that increasing miR-18a expression can decrease the TFGβ-induced expression of CTGF in HTM cells. MiR-18a mimics or upregulation represent a viable therapeutic strategy for treating the TGFβ-induced changes to the TM in glaucoma.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.