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.