MicroRNA-204/211 Regulates Human Retinal Pigment Epithelial Physiology

Abstract

Purpose: : To determine a set of microRNAs that are enriched in human RPE compared to retina and choroid. Using a human fetal retinal pigment epithelial (hfRPE) culture model, we studied the regulation of miR-204/211 on RPE physiology.

Methods: : Primary hfRPE cultures were obtained as described previously. miRNA in human RPE, retina and choroid were profiled using the TaqMan® MicroRNA Assays Human Panel Early Access Kit. Q-RT-PCR was carried out with TaqMan® microRNA assay. miRNA Northern blots were done with the LNA^TM probes and in situ hybridization was performed with digoxigenin (Dig)-labeled probes. Direct miRNA targets were determined by a dual luciferase reporter assay using pEZX-MT01- target constructs. RPE physiology on intact monolayers of hfRPE was studied as previously described.

Results: : We determined eight miRNAs that are enriched (>10 fold) in RPE (miRs-184,187, 200a/200b, 204/211, 221/222) compared to neuroretina or choroid (p<0.05). Five of these miRNAs are enriched in RPE compared to 20 tissues throughout the body and are >10,000 fold more highly expressed (p<0.005) and miR-204/211 are the most highly expressed. We determined that TGFβR2 and Snail2 are direct targets of miR-204. Addition of anti-miR-204 significantly decreased: (1) transepithelial resistance (TER); (2) cell membrane potentials; (3) claudin expression (RNA and protein); (4) Kir 7.1 (K channels) (protein).

Conclusions: : miR-204 regulation of RPE membrane physiology is mediated through a TGFβ /SNAI2 signaling pathway that controls the expression of claudins in the tight junctions and Kir 7.1 in the plasma membranes. The present experiments provide the basis for understanding how miR-204/211 regulates RPE tight junction integrity and maintain the blood/retina barrier in a quiescent state, which is a hallmark of epithelia.

Keywords: gene/expression • retinal pigment epithelium • electrophysiology: non-clinical