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Jong Kook Park, Han Peng, Julia V Katsnelson, Shuangni Yang, Wending Yang, Robert Lavker; The microRNA-103/107 family regulates the cell cycle to confer quiescence on stem cell-enriched limbal keratinocytes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6037.
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© ARVO (1962-2015); The Authors (2016-present)
It is well established that limbal epithelial stem cells cycle infrequently, which is a property that is shared with stem cells from other self-renewing tissues. Although progress has been made in our understanding of this important process, what regulates quiescence is less appreciated. We have established that the microRNA-103/107 (miRs-103/107) family is limbal epithelial-preferred. Thus we investigated whether this miRNA family affected the proliferative status of human limbal epithelial keratinocytes (HLEKs).
We analyzed the effects of miRs-103/107 on HLEK proliferation using a combination of submerged and 3-dimensional (3-D) raft cultures in conjunction with the kinetics of BrdU incorporation, WST-1 cell proliferation assay, cell cycle analysis (FACS), and epithelial progenitor competition assays. To validate potential targets of miRs-103/107 that regulate the cell cycle, we used in silico and cell-based luciferase reporter assays. Gain- and loss-of- function experiments with miRs-103/107 in HLEKs confirmed the targets. These investigations were complemented by siRNA studies against the targets to assess biological function.
Lentiviral over-expression of miRs-103/107 in HLEKs, resulted in a 50% increase in population doubling time and a 20% decrease in BrdU labeling compared with empty vector (EV) controls (p> .05). FACS analysis revealed that ectopic expression of miRs-103/107 lead to an arrest in the G1 phase of the cell cycle without an increase in apoptosis. Most basal cells in 3-D raft cultures formed from an equal mix of GFP-tagged miRs-103/107 or mCherry-tagged EV (control) keratinocytes were miRs-103/107-GFP-tagged. This indicates that miRs-103/107 overexpressing cells have more proliferative capacity than the mCherry cells and thus behave like progenitors. Luciferase and Western blot data indicated that HOXD13 and p90RSK2, two positive regulators of the G1/S transition, are direct targets of miRs-103/107. Silencing of HOXD13 induced a 20% reduction in cell proliferation compared with controls. Knockdown of p90RSK2 in HLEKs resulted in a 28% reduction of BrdU incorporation and a 15% increase in population doubling time.
Collectively, these observations demonstrate that miRs-103/107 contribute to a stem cell phenotype by negatively regulating HOXD13 and p90RSK2, which prevents the G1/S transition and thus maintains quiescence.
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