Abstract
Purpose :
Pathologic retinal neovascularization commonly causes blindness. Our preliminary study found that miR-18a-5p was highly expressed in developing mouse retina. In order to explore the role of miR-18a-5p in retinal neovascularization, we conducted functional analysis using human retinal microvascular endothelial cells (HRMECs) and rescue study using oxygen-induced retinopathy (OIR), a mouse proliferative retinopathy model.
Methods :
Agomir-18a-5p was transfected into HRMECs using Lipofectamine MAX and followed proliferation, wound healing, transwell, and tube formation assays. C57BL/6J mice were used to generate OIR model. 1.5 μg of agomir-18a-5p were intravitreously injected to the right eyes of P12 pups. The contralateral eyes with vehicle injection were used as controls. Retinas were stained with Isolectin B4 and quantified retinal neovascularization. The expression of miR-18a-5p, hypoxia-inducible factor 1 (HIF1α) and fibroblast growth factor 1 (FGF1) were measured by real-time qPCR and western blot. Dual-luciferase report assay system was used to verify the target genes of miR-18a-5p. Image J or Adobe Photoshop were used for semi-quantification. Two-tailed Student'st-test was used for statistical analysis andp<0.05 was considered significant. All animal experiments were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Results :
Agomir-18a-5p significantly reduced HRMECs proliferation (p=0.027), migration (p=0.0009) and invasion (p=0.0033) compared to scrambled control. Moreover, agomir-18a-5p significantly reduced tube formation ability of HRMECs in tubule length (p=0.0051) and mesh numbers (p=0.0058), and inhibited the expression of HIF1α (p=0.0138) and FGF1 (p=0.032). While miR-18a-5p levels increased significantly (p=0.021), the expression of HIF1α (p=0.0143) and FGF1 (p=0.0322) were suppressed in the retina of OIR mice (n=7) at P17. Luciferase report assay conformed that both of HIF1α and FGF1 were the target genes of miR-18a-5p. Retinal flat mount analysis (n=6) showed that agomir-18a-5p significantly inhibited pathologic retinal neovascularization compared to control (p=0.0013).
Conclusions :
Our data suggests that miR-18a-5p inhibits retinal neovascularization by targeting HIF1α and FGF1. miR-18a-5p can be a promising new therapeutic target for neovascular eye diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.