June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
microRNA-192 Expression Induced by TGF-β1 Contributes to Epithelial-Mesenchymal Transition of RPE by activating AKT Kinases
Author Affiliations & Notes
  • Daniel E. Maidana
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Cambridge, MA
  • Haijiang Lin
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Cambridge, MA
  • Bernard Dib
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Cambridge, MA
  • John B Miller
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Cambridge, MA
  • Peggy Bouzika
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Cambridge, MA
  • Bo Tian
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Cambridge, MA
  • Joan W Miller
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Cambridge, MA
  • Demetrios Vavvas
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Cambridge, MA
  • Footnotes
    Commercial Relationships Daniel Maidana, None; Haijiang Lin, None; Bernard Dib, None; John Miller, None; Peggy Bouzika, None; Bo Tian, None; Joan Miller, None; Demetrios Vavvas, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4231. doi:https://doi.org/
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      Daniel E. Maidana, Haijiang Lin, Bernard Dib, John B Miller, Peggy Bouzika, Bo Tian, Joan W Miller, Demetrios Vavvas; microRNA-192 Expression Induced by TGF-β1 Contributes to Epithelial-Mesenchymal Transition of RPE by activating AKT Kinases. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4231. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Fibrosis can complicate several retinal diseases, including epiretinal membranes, proliferative vitreo-retinopathy, and age-related macular degeneration. MicroRNA-192 is a known mediator of transforming growth factor-β1 (TGF-β1) pro-fibrotic effects in kidney diseases. However, the role of miR-192 in Epithelial-Mesenchymal Transition (EMT), a key process in tissue repair and fibrosis, has not been yet investigated. The purpose of this study was to assess the role of miR-192 in TGF-β1-induced EMT of RPE cells.

Methods: ARPE-19 cells were treated with TGF-β1 (2.5, 5, 10 ng/mL), and RNA was extracted at 72 hours. miR-192 expression was quantitated by qRT-PCR. Following, ARPE-19 cells were treated with TGF-β1, and pretreated an AKT-specific inhibitor MK-2206. Expression of epithelial (E-Cadherin and ZO-1) and mesenchymal (Smooth Muscle Actin and Vimentin) EMT markers, and phosphorylated AKT, were assessed by Immunostaining and Western Blot. In addition, cells were transfected with miR-192 mimic, and EMT markers and ratio of activated AKT were assessed by Western Blot. Acetylation of ETS-1 transcription factor, which binds and represses miR-192 gene, was assessed by Immunoprecipitation. All experiments were performed 3 times, in duplicates. Statistical analyses were performed by Student’s t-tests or ANOVA followed by Tukey’s post hoc test. A p value < 0.05 was considered statistically significant.

Results: miR-192 expression was significantly upregulated 2-fold after treatment with 5 and 10 ng/mL of TGF-β1 (p=0.011 and p=0.015, respectively). Treatment with TGF-β1 down-regulated epithelial markers, up-regulated Mesenchymal markers, and increased phosphorylation of AKT. Pre-treatment with MK-2206 significantly abrogated TGF-β1 induced-EMT and arrested phosphorylation of AKT kinases. Transfection with miR-192 mimic significantly increased the expression of Vimentin and Smooth Muscle Actin, and phosphorylation of AKT kinases. In addition, the ratio of acetylated ETS-1 was significantly increased after treatment with TGF-β1, and was correlated to increased mir-192 expression.

Conclusions: miR-192 can contribute to EMT in ARPE-19 cells by activation of AKT kinases and inducing the expression of Mesenchymal EMT markers. This suggests that miR-192 may have a role in the development of intraocular fibrosis, and may be a potential therapeutic target for retinal and macular diseases.

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