The long term goal of this project is to understand how microRNAs (miRNAs) function in regulating the corneal and limbal epithelia. MiRNAs are a family of endogenous, small RNAs that acting as post-translational repressors, have been shown to function in cell development, differentiation, stem cell regulation and the progression of cancer. Despite the importance of these regulatory RNAs, little attention has been directed towards characterizing miRNAs in the mammalian ocular anterior segment. We have shown that microRNA- 184 (miR-184) is special in that it is the most abundant miRNA in the corneal epithelium and is not expressed in the limbal or conjunctival epithelia. MiR-205, the second most abundant miRNA in the corneal epithelium exhibits a broad range of expression throughout the anterior segmental epithelia as well as in many other stratified squamous epithelia. Most importantly, our preliminary data indicates that miR-205 undergoes a unique form of regulation through an interaction with the corneal-preferred miR-184. This is the first demonstration that one miRNA plays a major role as an antagonist of another and highlights the distinctiveness of the corneal epithelium. We successfully identified the lipid phosphatase SHIP2 as one of the targets of miR-205 and observed that miR-205 functions to enhance cell survival and alter cytoskeletal organization. Given the importance of these two processes to corneal epithelial homeostasis, we propose to investigate the functional significance of the unique regulation of miR-205 in corneal epithelium. We will also test our hypothesis that the stem cell-enriched limbal basal cells have a distinct miRNA signature. Towards these ends, we focus on how modulating miR-184 and miR-205 levels in corneal epithelial cells will: (i) affect their survival and growth capabilities and determine whether these events are acting through SHIP2;and (ii) affect their adhesive and migratory abilities. We will also define the miRNA expression patterns of the limbal basal cells and identify targets of those miRNAs that are unique to the limbal epithelium. To accomplish these goals we will capitalize on our ability to downregulate specific miRNAs with antagomirs and assess the functional consequences of such miRNA modulation with a combination of biochemical, molecular biological and cell biological approaches. Using laser capture microdissection, we will isolate relatively pure populations of limbal and corneal epithelial basal cells and define their miRNA expression patterns by microarray profiling, qRT-PCR and in situ hybridization. Information from these studies will provide insight into the how the corneal- enriched miRNAs affect cell survival, growth, migration and adhesion. These events are critical for corneal epithelial homeostasis as well as proper wound repair. Our proposed studies will also lead to a better understanding of the regulatory miRNAs that govern stem cell maintenance.
Information from this project will impact on our understanding of how microRNAs regulate vital cellular processes such as survival, growth, adhesion and migration in the corneal epithelium. A benefit from this research will be the use of microRNA inhibitors as innovative therapeutic strategies to promote corneal epithelial repair following injury. These studies will also lead to a better appreciation of the regulatory proteins that govern limbal epithelial stem cell behavior and thus ultimately contribute to our knowledge of how these progenitor cells maintain the corneal epithelium.
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