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