The RPE is a highly specialized epithelium that is crucial for maintaining photoreceptor function and visual cycle 
. Previous studies have shown a critical role for miRNAs in eye development, function, and survival in vivo
. Dicer-deficient mice show multiple defects, indicating an important role for miRNAs in many tissues including the eye 
. Previous miRNA profiles in RPE have primarily focused on mouse RPE or the human cell line ARPE19 
, however, miRNA profiles in human RPE are still limited. MiRNAs are proposed to act as fine tuners of gene expression either through inhibiting translation in the cytoplasm, or promoting mRNA degradation in the nucleus 
. More recently, miRNAs have been shown to primarily repress genes through transcriptional control 
. Therefore, we used different software to predict the mRNA target of differentially expressed miRNA. Many of the down-regulated miRNAs potentially target a generous portion of RPE signature genes. However, a recently published study comparing various miRNA prediction softwares show very low concordance between three major prediction algorithms (miRanda, TargetScan, PicTar), indicating the standard for predicting miRNA target genes are still not robust 
. To the best of our abilities and available tools, we employed these prediction algorithms to provide putative targets of key miRNAs.
Through profiling miRNA expression at four defined time points during RPE differentiation, our study demonstrated that each stage of RPE differentiation has a unique subset of miRNAs that are significantly differentially expressed compared to all other stages. We identified miRNAs that were exclusively enriched in ESCs, intermediate stages of spontaneously differentiated ESCs, and fully derived RPE. In addition, we were able to show that a portion of the miRNA become gradually increased or decreased, or transiently increased during the differentiation process, suggesting the expression level of particular miRNAs may be used as an indicator for RPE maturation. Thus, our study indicated that the degree of RPE differentiation can be gauged by profiling the specific mRNA expression patterns during RPE differentiation as shown in . Furthermore, by incorporating miRNA profiles from various somatic tissues, we were able to find a portion of miRNA that are specifically expressed in human RPE cells. Functional annotation of the predicted targets using DAVID revealed RPE-specific miRNAs are primarily associated in regulating the epithelial barrier and TGF-β pathway. TGF-β signaling pathway is essential for epithelium-mesenchymal transition in RPE cells, indicating suppression of the TGF-β pathway contributes to maintaining the epithelium in RPE 
. Future study shall validate the function of these miRNA in RPE differentiation through gain or loss of function experiments. We also find a portion of human RPE-specific miRNAs also share enrichment in mouse RPE, indicating a potentially conserved functional role for these miRNAs. We suggest that the human RPE-specific miRNA signature may serve as molecular markers for characterizing functional RPE.
Age-related macular degeneration (AMD) is characterized by malfunction and degeneration of RPE. Recently, Kaneko et al. 
discovered a miRNA-independent cell survival function for DICER1 in the context of AMD pathology, demonstrating upregulation of Alu elements in the absence of DICER1 promoted RPE cell death. However, this does not exclude the possibility for miRNAs in maintaining other features of RPE. Indeed, other studies have shown that miRNA may play important roles in AMD pathogenesis. Lin et al. discovered that miR-23a was down regulated in AMD eyes while upregulation of miR-23a can protect RPE cell from oxidative damage in ARPE19 cells 
. In our dataset, miR-23a was enriched in RPE cells, and its expression was significant increased during development, supporting the hypothesis that miR-23a expression is necessary for maintaining healthy RPE. Overall, our data uncovered a unique set of miRNAs that are expressed in RPE, suggesting a small number of key miRNAs may contribute to promoting RPE survival and function.