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RNA-sequencing of non-senescent and senescent mouse skin fibroblast

ABSTRACT: Mouse skin fibroblasts (MSFs) were obtained from a FASST (Fibroblasts Accelerate Stromal-Supported Tumorigenesis) mouse. This mouse model allows for spatial and temporal control for senescence induction by using a stromal specific Cre-recombinase driven by the pro-collagen-alpha II promoter. The stromal specific Cre activates expression of the p27IRESGFP transgene that is expressed from the ROSA locus. We cultured the MSFs in vitro, induced senescence using 10uM tamoxifen added to the media. Non-senescent cells were treated with equal volume of vehicle alone (ethanol). Upon tamoxifen treatment, cells were moved to a modular incubation chamber and maintained at 3% oxygen at 37 degrees celcius for 12 days total before collection. At the time of collection, cells were trypsynized and pelleted by centrifugation. The cells were lysed using Trysol reagent and RNA was isolated using a RiboPure RNA isolation kit (Ambion).

ORGANISM(S): Mus musculus

PROVIDER: GSE78128 | GEO | 2017/02/10

SECONDARY ACCESSION(S): PRJNA312668

REPOSITORIES: GEO

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Project description:Alterations in the tissue microenvironment collaborate with cell autonomous genetic changes to contribute to neoplastic progression. The importance of the microenvironment in neoplastic progression is underscored by studies demonstrating that fibroblasts isolated from a tumor stimulate the growth of preneoplastic and neoplastic cells in xenograft models. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo. Because senescent cells accumulate with age, their presence is hypothesized to facilitate preneoplastic cell growth and tumor formation in older individuals. To identify senescent stromal factors directly responsible for stimulating preneoplastic cell growth, we carried out whole genome transcriptional profiling and compared senescent fibroblasts to their younger counterparts. We identified osteopontin (OPN) as one of the most highly elevated transcripts in senescent fibroblasts. Importantly, reduction of OPN protein levels by RNAi did not impact senescence induction in fibroblasts; however, it dramatically reduced the growth-promoting activities of senescent fibroblasts in vitro and in vivo, demonstrating that OPN is necessary for paracrine stimulation of preneoplastic cell growth. In addition, we found that recombinant OPN was sufficient to stimulate preneoplastic cell growth. Finally, we demonstrate that OPN is expressed in senescent stroma within preneoplastic lesions that arise following DMBA/TPA treatment of mice, suggesting that stromal-derived OPN-mediated signaling events impact neoplastic progression. Experiment Overall Design: Human foreskin BJ fibroblasts were mock or Bleomycin sulfate-treated (100ug/ml, Sigma, St. Louis, MO) for 24 hrs. Replicatively senescent fibroblasts were obtained by continuous passage. After 72 hr serum-starvation, RNA was collected using TRIzol (Invitrogen, Carlsbad, CA). Biotinylated cRNA was hybridized to Affymetrix Human Genome U133 Plus 2.0 GeneChips (Affymetrix, Santa Clara, CA) by the Washington University Microarray Facility. Experiment Overall Design: There were 4, 6, and 6 samples for SIPS, RS, and young respectively: Experiment Overall Design: - Stress-induced premature senescence (GSM336347..GSM336379) Experiment Overall Design: - Replicative Senescent (GSM336385..GSM336573) Experiment Overall Design: - Young (GSM336574..GSM336628) Experiment Overall Design: Microarray quality control, analysis, and clustering (UPGMA by Centroid) were performed using dChip (May, 2008 release). All GeneChip comparisons had a cut-off basis of a lower bound of 90% confidence of fold-change â?¥1.5 and expression intensity difference â?¥75.

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RNA-sequencing of non-senescent and senescent mouse skin fibroblast

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