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Nishantha Gunawardena, Nady Golestaneh, Maria Kokkinaki, Mia Gunawan; MicroRNA 184 regulates Ezrin expression and potentially impacts Ezrin-dependent functions in human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1600.
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The purpose of this study is to investigate the role of microRNA 184 (miR-184) in regulating RPE functions. It is known that miR-184 plays a key role in neurological development and is highly expressed in mice corneal epithelium and in human fetal RPE, however, its role in RPE is largely unknown.
Proteomic analysis of RPE transfected with hairpins for miR-184 was performed and the data were analyzed to identify the genes that were highly affected by the down-regulation of miR-184. Since RPE express miR-184, Hela cells were used to analyze the binding of miR-184 to the 3’UTR of the targeted genes. Luciferase assays were performed after transfection of Hela cells with both plasmids for EZR 3’UTR in pEZX-MT01 vector and miR-184 coding DNA sequence in pEZX-MR04. Real Time PCR was used to relatively quantify the mRNA expression levels of the targeted gene. Western blot was used to measure the protein levels of ezrin in miR-184 transfected cells. RNAi technology was used to verify the effect of EZR down-regulation in RPE function. Phagocytosis assay and ultrastructural analysis using electron microscopy were performed.
Our proteomic data showed that EZR is a potential target for miR-184. Ezrin is an actin-binding cytoplasmic peripheral membrane protein that functions as a protein-tyrosine kinase substrate in the apical microvilli and as an intermediate between the plasma membrane and the actin cytoskeleton. Luciferase assay revealed that miR-184 binds to the 3’UTR of EZR and directly down-regulates EZR gene expression. In addition, real time PCR revealed that EZR gene expression is inhibited by miR-184. Western blot confirmed the down-regulation of ezrin protein by miR-184. Using EZR siRNA, we showed that RPE morphology and phagocytosis function were affected by ezrin down-regulation.
Our results suggest that miR-184 affects actin depolymerization and in turn impacts membrane trafficking and RPE functions through down-regulation of EZR. In addition, since ezrin is expressed in the apical region of polarized RPE, its regulation by miR-184 might be crucial for RPE cell polarity and related biological functions. Further research is required to delineate the post-translational regulation of EZR mRNA by miR-184 and its possible role in RPE related diseases such as Age-related Macular Degeneration.
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