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Hari Jayaram, Megan F Jones, Dan Frampton, Karen Eastlake, Silke Becker, Karl Matter, G Astrid Limb; MicroRNA Signatures Associated with the Differentiation of Human Müller Glia with Stem Cell Characteristics towards a Retinal Ganglion Cell Fate. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1383.
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MicroRNAs (miRNAs) are small, endogenous non-coding RNAs involved in the post-transcriptional silencing of gene expression during development and disease. The purpose of this study was to determine the miRNA signature associated with Notch pathway downregulation and in vitro differentiation of Human Müller Glia with Stem Cell Characteristics (hMSCs) towards a Retinal Ganglion Cell (RGC) fate.
hMSCs were differentiated in vitro towards a retinal ganglion cell fate using established protocols. Total cellular RNA was isolated from both differentiated and undifferentiated cells (Four replicates per condition) and the expression profiles were interrogated using the Agilent miRNA microarray platform. Partek Genomics Suite software was used to normalise the expression data. Subsequent analysis was performed using ANOVA in order to identify differentially expressed miRNAs using a 5% False Discovery Rate (adjusted P-values < 0.05). Validation of the microarray results was achieved by performing quantitative PCR.
Analysis of the microarray data identified nineteen differentially expressed miRNAs showing upregulation in differentiated cells when compared to undifferentiated controls (Fold Change >= 0.5, p<0.05). These included miR-199b, miR-151, miR-204 and let-7i whose target genes include components of the NOTCH pathway, and miR-222 that is known to be associated with the development of neurite outgrowth.
The miRNA signature associated with differentiation of hMSCs towards retinal ganglion cells is characterised by the upregulation of miRNAs known to be involved with the NOTCH pathway and neurite outgrowth. The identification of these targets and their future experimental modulation may provide opportunity for future research into the induction of in vivo retinal ganglion cell differentiation by hMSCs.
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