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FGF2 stimulation experiment with NMRI mouse embryonic fibroblasts

ABSTRACT: Mouse embryonic fibroblasts stimulated with 40 ng/ml of FGF2 in conventional hES cell medium were analysed on whole-genome expression chips to reveal altered expression of genes encoding secreted proteins. Keywords: Growth factor stimulation experiment

ORGANISM(S): Mus musculus

PROVIDER: GSE5436 | GEO | 2006/10/03

SECONDARY ACCESSION(S): PRJNA96101

REPOSITORIES: GEO

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Project description:Background: 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 full range and spectrum of regulated genes. Results: Herein, we employed rat lens epithelial explant system and performed RNA and microRNA expression profiling in cells induced to differentiate by FGF2. The primary data were collected at explants grown overnight in the presence of 5 ng/ml of FGF2, followed by a treatment with 100 ng/ml of FGF2 and collection of samples at 2, 4, 12 and 24 hours. Global analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on microRNAs (miRNAs). We identified a total number of 131 FGF2-regulated miRNAs. Forty-four of these microRNAs had at least two predicted and inversely regulated target RNA molecules. The genes regulated by the highest number of miRs include Nfib, Nfat5, c-Maf, Ets1 and N-Myc, all encoding DNA-binding transcription factors. Analysis of RNA data revealed that activated FGF signaling influenced other major signaling pathways known to regulate lens differentiation including BMP/TGF-M-NM-2, Notch, and Wnt signaling. In the M-bM-^@M-^\earlyM-bM-^@M-^] response phase (2-4 hours), miRNAs targeted expression of batteries of genes that control transcription, cell death, cell proliferation, cell junction, and protein serine/threonine kinase activity. In M-bM-^@M-^\lateM-bM-^@M-^] stages (12-24 hours), the main miRNA targets included regulators of cell cycle arrest and cellular differentiation. Specific miRNA:mRNA interaction networks were identified for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Dicer1, Fbx33 and Wdr47 (RNA processing); Ash1l, Med1/PBP and Kdm5b (chromatin remodeling); and c-Maf, Ets1 and Stc1 (FGF signaling). MicroRNAs including miR-9, -143, -155, -455 and -543 downregulated expression of c-Maf in the 3M-bM-^@M-^Y-UTR luciferase reporter asssays. The functional requirement for miRNAs in lens was further demonstrated via disrupted lens fiber cell differentiation in lenses with inactivated Dicer1. Conclusions: These studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and identified novel gene regulatory networks (GRNs) connected by multiple miRNAs. Differential gene expression of FGF2-treated lens cell cultures was analyzed at 0, 2, 4, 12 and 24 hours following the treatment. Two biological replicate experiments were performed.

Project description:Background: 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 full range and spectrum of regulated genes. Results: Herein, we employed rat lens epithelial explant system and performed RNA and microRNA expression profiling in cells induced to differentiate by FGF2. The primary data were collected at explants grown overnight in the presence of 5 ng/ml of FGF2, followed by a treatment with 100 ng/ml of FGF2 and collection of samples at 2, 4, 12 and 24 hours. Global analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on microRNAs (miRNAs). We identified a total number of 131 FGF2-regulated miRNAs. Forty-four of these microRNAs had at least two predicted and inversely regulated target RNA molecules. The genes regulated by the highest number of miRs include Nfib, Nfat5, c-Maf, Ets1 and N-Myc, all encoding DNA-binding transcription factors. 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), miRNAs targeted expression of batteries of genes that control transcription, cell death, cell proliferation, cell junction, and protein serine/threonine kinase activity. In “late” stages (12-24 hours), the main miRNA targets included regulators of cell cycle arrest and cellular differentiation. Specific miRNA:mRNA interaction networks were identified for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Dicer1, Fbx33 and Wdr47 (RNA processing); Ash1l, Med1/PBP and Kdm5b (chromatin remodeling); and c-Maf, Ets1 and Stc1 (FGF signaling). MicroRNAs including miR-9, -143, -155, -455 and -543 downregulated expression of c-Maf in the 3’-UTR luciferase reporter asssays. The functional requirement for miRNAs in lens was further demonstrated via disrupted lens fiber cell differentiation in lenses with inactivated Dicer1. Conclusions: These studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and identified novel gene regulatory networks (GRNs) connected by multiple miRNAs. Differential gene expression of FGF2-treated lens cell cultures was analyzed at 0, 2, 4, 12 and 24 hours following the treatment.

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FGF2 stimulation experiment with NMRI mouse embryonic fibroblasts

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