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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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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.
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.
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.
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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