Abstract
Purpose :
Ocular fibrosis can cause vision loss without treatment available. Subretinal fibrosis occurs in response to choroidal neovascularization (CNV) in wet Age-related macular degeneration (AMD), which is not treatable by anti-VEGF therapy. We have reported that miR-24 inhibits CNV in mice by regulating actin cytoskeleton remodeling. The purpose of the project is to define the function of miR-24 in subretinal fibrosis. We hypothesize that miR-24 overexpression can repress RPE-derived epithelial-mesenchymal transition (EMT) and subretinal fibrosis in AMD disease.
Methods :
ARPE-19 cells or primary human RPE (hRPE) cells were treated with pre-miR-24 or adenovirus expressing miR-24 (Ad-miR-24). miR-24 overexpression was quantified by qRT-qPCR. EMT-associated gene expression in TGF-β2 treated RPE cells was measured by Western blot and immunostaining. The expression of potential miR-24 target genes, SMAD3, LIMK2 and PAK4, were measured by Western Blot after miR-24 overexpression with or without TGF-β2 treatment. Novel miR-24 target gene, SMAD3, was confirmed by luciferase assays. Lentivirus-mediated overexpression of target genes were used to rescue miR-24 overexpression phenotype. Stress fiber formation was visualized by Phalloidin staining and quantified by measuring the ratio of G-actin/F-actin. Student’s t-test was used for statistical analysis.
Results :
MiR-24 was significantly increased in APRE-19 or hRPE cells using Ad-miR24 or pre-miR-24. Markers of myofibroblast and fibrosis, including α-SMA, Fibronectin, Collagen III and Collagen I, were induced an in vitro EMT and fibrosis model. Overexpression of miR-24 repressed expression of these proteins as shown by Western blot analyses and immunostaining. SMAD3, a major protein critical for fibrosis by mediating TGF- β signaling, was identified as a miR-24 target gene, and was downregulated by miR-24 overexpression. Overexpression of miR-24 target genes SMAD3 or LIMK2 partially rescued miR-24 overexpression phenotype. miR-24 also repressed stress fiber formation and increased ratio of G-actin/F-actin, consistent with downregulation of its verified target protein LIMK2 and PAK4.
Conclusions :
MiR24 overexpression prevents EMT and fibrosis and actin cytoskeleton dynamics through targeting SMAD3, LIMK2 and PAK4 in RPE cells. MiR-24 can be investigated as a therapeutic target for subretinal fibrosis in vivo.
This is a 2021 ARVO Annual Meeting abstract.