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C. Zhang, F. Wang, A. Maminishkis, L. Dong, C. Zhi, J. Zhao, V. Majerciak, S. Chen, S. S. Miller; MicroRNA-204/211 Regulates Human Retinal Pigment Epithelial Physiology. Invest. Ophthalmol. Vis. Sci. 2010;51(13):478.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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 LNATM 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.
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).
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.
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