The long-term goals of this project are to: (i) characterize microRNAs (miRNAs) found in the stem cells of the corneal epithelium; and (ii) understand how miRNAs regulate stem cell proliferation and differentiation. MiRNAs are a family of endogenous, small RNAs that regulate the expression of complementary messenger RNAs by acting as post-translational repressors. MiRNAs have been shown to function in development, cell death, cell proliferation, differentiation, and the initiation and progression of cancer. Significantly, a subset of miRNAs has been identified that are expressed in stem cells, but are lost when these cells differentiate. Our hypothesis is that miRNAs help maintain stem cells by limiting their differentiation potential. Corneal epithelial stem cells are located in a distinct structure known as the limbal epithelium. Since the limbus is located millimeters away from its corneal epithelial progeny, it is possible to cleanly dissect the stem cell- enriched population from the more differentiated daughter cells. Thus this system provides an outstanding model for studying the role of miRNAs in stem cell control. Our preliminary results demonstrate the presence of several miRNAs that are restricted to the corneal epithelium of adult mice. One of these miRNAs is strongly expressed in corneal epithelial basal cells, but not in the stem cell-enriched limbal basal cells. We propose three specific aims: (1) to identify and validate the presence of miRNAs in the stem cell-enriched limbal epithelial basal cells and compare them with miRNAs in the TA cell-enriched corneal epithelial basal cells during limbal/corneal epithelial development, using a combination of laser capture microdissection (LCM), miRNA microarrays, Northern blots, and in situ hybridization; (2) to analyze the effects of overexpression and/or inhibition of selected limbal- and corneal-enriched miRNAs on their potential target mRNAS in cultured human limbal and corneal keratinocytes; and (3) to develop a mouse model to explore the biological function of miRNAs during corneal epithelial development using tissue specific Cre expression to disrupt the Dicer-1 gene, which is essential for miRNA biogenesis. Since miRNAs have the potential to regulate almost all aspects of cellular physiology, the proposed studies will provide: (i) new knowledge about the role that miRNAs play during the development of the limbal/corneal epithelium; and (ii) additional information on how this novel regulatory mechanism of gene control affects stem cell behavior. Information obtained from this proposal will have many practical applications. For example, a major challenge for ocular biologists is to maintain and expand limbal stem cells in vitro for use in corneal epithelial reconstruction. We believe that by modulating miRNAs it should be possible to restrict cell differentiation and maintain proliferation. Since miRNAs can function to prevent cell division, their identification and potential pharmacologic downregulation could be useful in promoting corneal epithelial wound repair. ? ?
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