June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
MicroRNA-124-3p regulates TGFβ1-induced epithelial-mesenchymal transition in retinal pigment epithelium by down-regulating RhoG expression
Author Affiliations & Notes
  • JongHwa Jun
    Department of Ophthalmology, School of Medicine, Keimyung University, Daegu, Korea (the Republic of)
  • Yu Cheol Kim
    Department of Ophthalmology, School of Medicine, Keimyung University, Daegu, Korea (the Republic of)
  • Kwang-Soo Kim
    Department of Ophthalmology, School of Medicine, Keimyung University, Daegu, Korea (the Republic of)
  • Youngkyun Lee
    Department of Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu, Korea (the Republic of)
  • Jae-Young Kim
    Department of Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu, Korea (the Republic of)
  • Choun-Ki Joo
    Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Department of Ophthalmology and Visual Science, Seoul, Korea (the Republic of)
  • Footnotes
    Commercial Relationships JongHwa Jun, None; Yu Cheol Kim, None; Kwang-Soo Kim, None; Youngkyun Lee, None; Jae-Young Kim, None; Choun-Ki Joo, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4235. doi:
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      JongHwa Jun, Yu Cheol Kim, Kwang-Soo Kim, Youngkyun Lee, Jae-Young Kim, Choun-Ki Joo; MicroRNA-124-3p regulates TGFβ1-induced epithelial-mesenchymal transition in retinal pigment epithelium by down-regulating RhoG expression. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4235.

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

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Abstract

Purpose: Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) is a crucial experimental pathophysiology of proliferative vitreoretinopathy (PVR), and actin reorganization plays a key role as a major alteration in fibroblastic transformations of RPE in EMT. RhoG has been reported a key regulator in the reorganization of actin.

Methods: After transfection of MicroRNA (miR)-124-3p on ARPE-19 cells, TGF-β1 (10ng/mL) was treated for 36 hours. Western blot analysis and immunocytochemistry were performed to detect the alteration in expression/localization of EMT/epithelial markers. For identifying direct targeting of miR-124-3p on RhoG 3’ UTR, luciferase assay was adopted. In addition, RhoG siRNA was transfected and evaluated the change of EMT-related factors by western blotting/immunostaining.

Results: MiR-124-3p, a putative regulator of RhoG, was downregulated with progression of EMT that is induced by transforming growth factor (TGF)-β1. Exogenous overexpression by miR-124-3p impeded the typical fibroblastic alterations of RPE. Furthermore, it preserved epithelial markers, both ZO-1 and RPE65, and down regulated fibroblatoid phenotypic factors, fibronectin, α-SMA, vimentin, and N-cadherin in RPE. TGF-β1 increased RhoG expressions/localization but miR-124-3p overexpression blocked these and its downstream effector, Rac1 was also decreased and its distribution in cytoplasm was altered by miR-124-3p overexpression. In addition, RhoG overexpression by inhibiting endogenous miR-124-3p using anti-miR-124-3p showed up regulation of mesenchymal markers and decreased factors that represent epithelial phenotype. miR-124-3p overexpressions blocked TGF-β1 induced collagen gel contraction and it caused by alterations of cell spreading/cell-to-cell adhesion of RPE by RhoG downregulations. With an in silico analysis, web-base program, Targetscan predicted two well-conserved and two vertebrates-only conserved sequences in RhoG that would possible to form four seed matchs by hsa-miR-124-3p, respectively. Hsa-miR-124-3p is targeting to 3’UTR region of RhoG mRNA was evident by luciferase assay. Direct silencing induced by RhoG siRNA showed identical results in EMT regulations.

Conclusions: In present study, we demonstrated the regulations of EMT by miR-124-3p/RhoG/Rac1 axis and it is accompanied by crosstalk with TGF-β/SMAD signaling cascades.

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