MiRNA Expression in Senescent and Immortalized BJ fibroblasts
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ABSTRACT: Limited data exists regarding changes of microRNA (miRNA) expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ) fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT) that stably express the human telomerase reverse transcriptase subunit hTERT. The revelation that miRNA expression changes with extended passaging in BJ-hTERT cells will contribute to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.
Project description:Gene expression profiling was performed by use of serial analysis of gene expression (SAGE) on BJ normal human skin fibroblasts, A-T cells, and BJ and A-T cells transduced with hTERT cDNA and expressing telomerase activity. Keywords = telomere Keywords = telomerase Keywords = TERT Keywords = ataxia telangiectasia mutated Keywords = ATM Keywords = serial analysis of gene expression Keywords = SAGE Keywords = fibroblast Keywords: parallel sample
Project description:Gene expression profiling was performed by use of serial analysis of gene expression (SAGE) on BJ normal human skin fibroblasts, A-T cells, and BJ and A-T cells transduced with hTERT cDNA and expressing telomerase activity. Keywords = telomere Keywords = telomerase Keywords = TERT Keywords = ataxia telangiectasia mutated Keywords = ATM Keywords = serial analysis of gene expression Keywords = SAGE Keywords = fibroblast Keywords: parallel sample
Project description:We utilized whole genome sequencing of mRNA (RNA-seq) to understand the extent to which the senescence-associated secretory phenotype is regulated by p38MAPK Examination of replicates of young, senescent or p38MAPK-inhibited senescent BJ human foreskin fibroblasts.
Project description:We use RNA-seq to characterise the transcriptional programs of proliferating BJ-TERT fibroblasts versus those undergoing HRASV12-induced senescence (14 days)
Project description:We use RNA-seq to characterise the transcriptional programs of proliferating BJ-TERT fibroblasts versus those undergoing myrAKT1-induced senescence (6 days).
Project description:Overexpression myristoylated AKT1 in human BJ-TERT fibroblasts induces senescence (AIS). We depleted CBS, Tp53 or NF1 gene by shRNA in AIS cells and performed RNA-seq analysis to investigate the regulatory effect of these genes in AIS maintenance.
Project description:Aim: identification of differentially expressed genes after LIN-9 depletion.<br> <br> hTERT immortalized human BJ fibroblasts were infected with pMSCV-Blasticidin based retroviruses encoding a shRNA which targets human LIN-9 and the empty vector respectively.<br> After 4 days of selection total RNA was isolated. Cy3 and Cy5 labelled cDNA probes were generated using the CyScribe Post-labelling Kit (Amersham Biosiences).
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.
Project description:Comparison of gene expression of human BJ fibroblasts at different population doublings (PD). RNA-seq data comprises 2 groups: 34 and 70 population doublings of BJ fibroblasts. Jena Centre for Systems Biology of Ageing - JenAge (www.jenage.de)