June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
MiRNA-24 represses TGF-β2 induced epithelial-mesenchymal transition (EMT) and fibrosis in RPE cells
Author Affiliations & Notes
  • Yinga Wu
    Tulane University, New Orleans, Louisiana, United States
  • Katherine Brynn
    Tulane University, New Orleans, Louisiana, United States
  • Jing Ma
    Tulane University, New Orleans, Louisiana, United States
  • Shusheng Wang
    Tulane University, New Orleans, Louisiana, United States
  • Footnotes
    Commercial Relationships   Yinga Wu, None; Katherine Brynn, None; Jing Ma, None; Shusheng Wang, None
  • Footnotes
    Support  2R01EY021862-07A1
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 309. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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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.


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