June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
MicroRNA 184 regulates Ezrin expression and potentially impacts Ezrin-dependent functions in human Retinal Pigment Epithelium
Author Affiliations & Notes
  • Nishantha Gunawardena
    Georgetown University, Washington, DC
  • Nady Golestaneh
    Georgetown University, Washington, DC
  • Maria Kokkinaki
    Georgetown University, Washington, DC
  • Mia Gunawan
    Georgetown University, Washington, DC
  • Footnotes
    Commercial Relationships Nishantha Gunawardena, None; Nady Golestaneh, None; Maria Kokkinaki, None; Mia Gunawan, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1600. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 701 retinal pigment epithelium • 606 NaK ATPase • 493 cytoskeleton  
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