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Louise V. Wolf, Chun C. Gao, Karet Gueta, Nikhil Podduturi, Peggy S. Zelenka, Ruth Ashery-Padan, Jiri Zavadil, Ales Cvekl; Genome-wide Identification Of Genes And MicroRNAs Regulated By Fgf2 During In Vitro Lens Fiber Cell Differentiation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6908.
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© ARVO (1962-2015); The Authors (2016-present)
FGF signaling controls numerous processes during cell lineage specification, organogenesis and terminal differentiation. In lens, FGF signaling was implicated as the key pathway that controls lens fiber cell differentiation, but little is known about its nuclear components such as DNA-binding transcription factors and its cross-talk with other signaling pathways that function downstream and/or in parallel with FGF signaling.
We employed a rat lens epithelial explant system and performed integrated RNA and microRNA expression profiling in cells induced to differentiate by FGF2 (100 ng/ml). The RNAs were isolated at 2, 4, 12 and 24 hours following the treatment. RNA expression profiling was conducted using rat Affymetrix 230 2.0 arrays. MicroRNA profiling was conducted using the rodent TaqMan Low Density Array v2 ABI system. Candidate microRNA targets were identified through a combination of TargetScan 5.0 and integrated prediction databases in miRWalk and by inverse microRNA:mRNA correlation patterns. Role of microRNAs during lens fiber cell differentiation was assessed in vivo through conditional inactivation of Dicer using MLR10-cre.
Global analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on microRNAs. We identified a total number of 131 FGF2-regulated microRNAs. Forty-four of these microRNAs had at least two predicted and inversely regulated target RNA molecules. Analysis of RNA data revealed that activated FGF signaling influenced other major signaling pathways known to regulate lens differentiation including BMP/TGF-β, Notch, and Wnt signaling. In the "early" response phase (2-4 hours), microRNAs targeted expression of batteries of genes that control transcription, cell death, cell proliferation, cell junction and protein serine/threonine kinase activity. The genes regulated by multiple miRs included Dnmt3a, Maf, Map3k1/Mekk1, Mapk1/Erk2, Zeb2/Sip1 and Zfp148. In "late" stages (12-24 hours), the main miR targets included Nfat5, Mapk1/Erk2 and MafB. The functional requirement for microRNAs in lens is further demonstrated via disrupted lens fiber cell differentiation in lenses with inactivated Dicer.
These studies demonstrate a broad global impact of activated FGF signaling in stimulated lens and identified multiple novel cross-talks between FGF, BMP, TGF-β and Wnt signaling. In addition, microRNAs are important regulators of the lens fiber cell differentiation pathway.
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