Differential Expression of MeCP2 and its Regulation by miR132 in Human RPE Cells

Abstract

Purpose: : Peroxisome proliferator-activated receptor-γ (PPAR-γ) plays an important role in the regulation of epithelial cell differentiation and fibrosis. It has been known that expression of PPAR-γ is regulated by MeCP2, which is regulated by microRNA 132 (miR132). We investigated the expression and regulation of PPAR-γ, MeCP2, and miR132 in human RPE cells showing different levels of polarity.

Methods: : Cultured, early-passage human fetal RPE cells and polarized RPE monolayers were used. The expression of PPAR-γ, MeCP2, and miR132 expression was studied in non-polarized and polarized RPE monolayers by immunoblot and real-time PCR analysis. To elucidate the interaction between miR132 and MeCP2, RPE cells were transfected with miR132 mimic (2 µM) and MeCP2 expression was examined. The effects of TGF-β on PPAR-γ expression were analyzed by immunoblot analysis in cultured RPE cells after exposure to TGF-β2 (5-15 ng/ml) for 3 days.

Results: : miR132 was differentially expressed in polarized vs non-polarized human fetal RPE and its expression was significantly (P<0.001 vs non-polarized) upregulated in polarized RPE monolayer. On the other hand, MeCP2 expression was reduced in polarized RPE monolayers when compared with non-polarized RPE cells. We found that MeCP2 protein expression was regulated by miR132. Overexpression of miR132 decreased MeCP2 expression level in RPE cells. In addition, PPAR-γ expression depends on cell polarity; the expression showed progressive reduction when cells attain polarity. Furthermore, TGF-β treatment reduced PPAR-γ expression in RPE cells without altering MeCP2 expression.

Conclusions: : Our data provide evidence that 1) MeCP2 expression is regulated by miR132; increased miR132 significantly reduced MeCP2 expression; 2) PPAR-γ expression depends on cell polarity; and 3) TGF-β treatment reduced PPAR-γ expression in RPE cells. Our results suggest that regulation of miR132 may be a therapeutic strategy in preventing RPE transdifferentiation and fibrosis.

Keywords: retinal pigment epithelium • growth factors/growth factor receptors • proliferative vitreoretinopathy