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Yinga Wu, Katherine Brynn, Jing Ma, Shusheng Wang; MiRNA-24 represses TGF-β2 induced epithelial-mesenchymal transition (EMT) and fibrosis in RPE cells. Invest. Ophthalmol. Vis. Sci. 2021;62(8):309.
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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.
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.
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.
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.
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