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Yasuo Ouchi, Hiroyuki Mano, Sumiko Watanabe; Analysis of the Role of Pax6 Targeting miRNA During Mouse Retinal Development. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6015.
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MicroRNAs represent a class of small (20-25 nucleotides), non-coding RNAs that are key regulators of many cellular events during tumorigenesis and organ development. Although recent studies provide an important role of miRNA in regulation of gene expression, the contributions of miRNAs to retinal development and function are largely unknown. To clarify the role of miRNAs in mouse retinal development, we used gain- and loss-of-function analyses.
Based on the solexa sequencing expression profile of miRNAs, we focused on five kinds of miRNAs which characteristically expressed in mouse retinal development. In order to elucidate the function of the miRNAs, we used retroviral vector based gain- and loss-of-function approach in E17 mouse retinal explant culture. To identify target gene(s) of miRNAs, we used bioinfomatic tools, such as Targetscan, miRTar and miRanda. The suppression of the target gene was confirmed by immunohistochemical analysis and luciferase reporter gene assay.
We found that forced expression of miR-7a significantly down-regulates proliferation of the retinal progenitor cells. In accordance with this result, inhibition of miR-7a using overexpression of decoy sequence slightly decreased proliferation-activity of the retinal progenitor cells. We next examined the expression pattern of miR-7a, and strong expression was detected in neuroblast and ganglion cells at E14 mouse retina. As the development proceeds, expression in neuroblast became weak, suggesting the role of miR-7a in retinal progenitor cells. To identify target gene(s) of miR-7a using bioinfomatic tools, we found evolutionary conserved 3’UTR region of Pax6 gene in human, mouse and zebrafish is a possible target of miR-7a. To determine whether Pax6 is a direct target of miR-7a, we cloned the 3' UTR of Pax6 into the 3' UTR of a SV40-driven luciferase reporter. In the presence of the Pax6 3' UTR, miR-7a repressed luciferase activity, and this repression was diminished by mutation of miR-7a binding sites. Furthermore, number of Pax6 positive cells was significantly decreased by miR-7a overexpression in retinal explant. As expected, inhibition of miR-7a in retinal progenitor cells increased Pax6 positive cells.
These results suggest that miR-7a regulates the Pax6 expression in retinal progenitor cells and regulates timing of differentiation of retinal progenitor cells during the retinal development.
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