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L. J. Harwood, J. Guduric-Fuchs, A. O'Connor, D. A. Simpson; Next-Generation Sequencing Reveals Regulation of the microRNA Transcriptome During in vitro Differentiation of a Retinoblastoma Cell Line. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6443.
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MicroRNAs (miRNAs) are small non-coding RNAs which regulate mRNA expression at the post-transcriptional level. The potential functions of many miRNAs in retinal developmental are indicated by their unique stage-specific expression patterns. However, the specific roles of these miRNAs remain largely unknown. The purpose of this study was to optimise and characterise an in vitro model of retinal cell differentiation and to investigate changes in miRNA expression in this model.
Weri-Rb1 cells were treated with various combinations of laminin and all-trans retinoic acid (RA) on a poly-D-lysine/laminin substrate and assessed for morphological and molecular differentiation. Microarrays (Illumina HT-12) were performed to characterise induced changes in mRNA expression. Data were analysed using the Genomics Workbench (CLCbio) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) 2008. miRNA expression was analyzed using next generation sequencing (NGS) on an Illumina Genome Analyser. Reads were mapped to known miRNAs (mature, star and pre-miRNAs) using the miRAnalyser software. Selected findings were validated by real-time qPCR.
The optimal protocol to induce both morphological and molecular differentiation of Weri-Rb1 involved consecutive treatment with laminin and 20µM RA. Cells formed ramifying processes, positive for the neuronal marker Map2. Upregulated genes were significantly enriched for genes associated with visual perception and neurite morphogenesis. Upregulated retina-associated genes included ARR3, Crx, GUCA1A and Nhlh1. Quantitative analysis of miRNA expression with NGS revealed high levels of known neuronal and retinal-expressed miRNAs including miR-124 and miR-9. The expression of several miRNAs was altered during differentiation; most notably miR-9 and miR-181b were upregulated and miR-424 downregulated.
A combination of a laminin substrate and laminin and RA media supplementation over a 14 day period induces both morphological differentiation and expression of retina-associated genes in Weri-Rb1 cells. miRNAs potentially involved in retinal development were identified, to the best of our knowledge for the first time, using NGS. This model provides a platform for future studies of the interactions between miRNAs and mRNAs during retinal cell differentiation.
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