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Julia Katsnelson, Jong Kook Park, Wending Yang, Han Peng, Robert Lavker; Unraveling the limbal epithelial stem cell niche: miRs-103/107 help maintain E-cadherin-mediated cell-cell contacts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):988.
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In an effort to understand the roles that microRNAs (miRNAs) play in regulating corneal epithelial stem cells and their transient amplifying (TA) progeny, we compared miRNA expression patterns in the stem cell-enriched limbal epithelium versus the TA cell-enriched corneal epithelium at several developmental stages.
Laser capture microdissection (LCM) was used to precisely isolate fresh populations of limbal and corneal epithelium from unfixed cryosections, thereby acquiring miRNA expression profiles from relatively “quiescent” cells. LCM was performed on mouse limbal and corneal epithelium from postnatal (PN) mice at days: 3, 7, 14 and 28. Total RNA was isolated from triplicate samples and miRNA expression profiles were obtained using qRT-PCR arrays (Exiqon). We conducted loss-of-function experiments with miR-103 and -107 in cultured human limbal keratinocytes (HLEKs), in conjunction with Northern, Western, and immunohistochemical analyses. We used luciferase reporter assays in HeLa cells to identify potential targets of miR-103 /107.
miR-103 /107 were preferentially expressed in the limbal epithelium at each of the developmental time points. Northern blot analysis revealed higher expression of miR-103 /107 in HLEKs versus human corneal epithelial keratinocytes. Treatment of HLEKs with antagomirs to either miR-103 or miR-107, resulted in an initial loss of cell-cell contacts, which yielded small, rounded cells compared to control-treated (antago-124) HLEKs. Consistent with a loss in cell-cell contact, E cadherin (E-cad) was no longer immunohistochemically detectable at the cell-cell junctions, 3hr post-antago treatment. A marked downregulation was noted in p120 catenin and E-cad protein, essential components of adherens junctions. NEDD9, a non-catalytic scaffolding protein that negatively regulates localization of E-cad and promotes its degradation, was shown to be a target of miR-103/107.
Maintenance of cell-cell contact and communication between stem cells and TA cells are crucial for stem cell niche homeostasis. The positive role that miR-103/107 plays in E-cadherin-mediated limbal epithelial cell-cell contacts via down-regulation of NEDD9 helps stabilize this aspect of the stem cell niche and maintain stem cell-TA cell integrity.
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