Microphthalmia-associated Transcription Factor (MITF) Promotes Differentiation of Human Retinal Pigment Epithelium (RPE) by Regulating microRNAs-204/211 Expression

Jeffrey Adijanto,John J Castorino,Zi-Xuan Wang,Arvydas Maminishkis,Gerald B Grunwald,Nancy J Philp

doi:10.1074/jbc.m112.354761

Jeffrey Adijanto, John J Castorino + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m112.354761

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Abstract

The retinal pigment epithelium (RPE) plays a fundamental role in maintaining visual function and dedifferentiation of RPE contributes to the pathophysiology of several ocular diseases. To identify microRNAs (miRNAs) that may be involved in RPE differentiation, we compared the miRNA expression profiles of differentiated primary human fetal RPE (hfRPE) cells to dedifferentiated hfRPE cells. We found that miR-204/211, the two most highly expressed miRNAs in the RPE, were significantly down-regulated in dedifferentiated hfRPE cells. Importantly, transfection of pre-miR-204/211 into hfRPE cells promoted differentiation whereas adding miR-204/211 inhibitors led to their dedifferentiation. Microphthalmia-associated transcription factor (MITF) is a key regulator of RPE differentiation that was also down-regulated in dedifferentiated hfRPE cells. MITF knockdown decreased miR-204/211 expression and caused hfRPE dedifferentiation. Significantly, co-transfection of MITF siRNA with pre-miR-204/211 rescued RPE phenotype. Collectively, our data show that miR-204/211 promote RPE differentiation, suggesting that miR-204/211-based therapeutics may be effective treatments for diseases that involve RPE dedifferentiation such as proliferative vitreoretinopathy.

Highlights

MicroRNAs 204/211 regulate retinal pigment epithelial cell phenotype
We examined the purity of our human fetal RPE (hfRPE) cultures and showed that our dedifferentiated hfRPE cells did not express collagen type I, which was highly expressed in choroidal fibroblast cells
To determine whether miR-204/211 down-regulation was the primary cause for the loss of retinal pigment epithelium (RPE) phenotype in Microphthalmia-associated transcription factor (MITF) KD cells, we examined whether addition of pre-miR-204/211 could prevent RPE dedifferentiation caused by MITF KD. hfRPE cells (30% density) were transfected with MITF siRNA (30 nM) ϩ control miRNA (30 nM), MITF siRNA (30 nM) ϩ pre-miR-204/ 211 (15 nM each), or control miRNA and siRNA (30 nM each) and cultured for 7 days

Summary

Introduction

MicroRNAs 204/211 regulate retinal pigment epithelial cell phenotype. Results: In RPE, MITF regulates miR-204/211 expression and down-regulation of MITF results in loss of RPE phenotype, which can be prevented by overexpressing miR-204/211. RNA was extracted, and TaqMan qRT-PCR was performed to verify increased expression of mature miR-204/211 in samples transfected with corresponding miRNAs. In samples treated with pre-miR-204, -211, or both, the mRNA levels of several putative miR-204/211 target genes (CREB5, RAB22A, ELOVL6, and TCF12) were down-regulated (Fig. 2B).

Results

Conclusion