Transcription profiling of human coronary smooth muscle cells (hcaSMC) treated with two different beta-blockers to study their effects on gene expression
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Bernhard Brehm
PROVIDER: E-GEOD-3356 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions, including smooth muscle contraction, cell proliferation, cell adhesion, apoptosis, cell migration and inflammation. Many aspects of regulation via ROCK and ZIPK, however, remain unclear. In this study, we utilized an siRNA approach to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells. Microarray analysis was performed, using a whole-transcript expression chip, to identify changes in gene expression profiles induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes (355 down-regulated and 198 up-regulated), while ZIPK knockdown affected the expression of 390 genes (219 down-regulated and 171 up-regulated). A high incidence of up- and down-regulation of transcription regulator genes was observed in both ROCK1 and ZIPK knockdowns. Other markedly affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Three microRNAs (mir-145, mir-199 and mir-622) were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown had no effect on microRNA expression. 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown, of which 41 were down-regulated and 26 up-regulated by both treatments, while the other 9 genes were differentially up/down-regulated. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns, which are mainly involved in cell cycle regulation. Marked differences in the effects of ROCK1 and ZIPK knockdown on the genes involved in cell cycle regulation suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown significantly reduced the viability of vascular SMC. ROCK1 knockdown also affected several cytokine signaling pathways with up-regulation of 5 and down-regulation of 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Finally, ROCK1 but not ZIPK knockdown inhibited proliferation of vascular smooth muscle cells. We conclude that ROCK1 and ZIPK have diverse, but predominantly distinct regulatory functions in vascular smooth muscle cells. Human coronary artery smooth muscle cells were transfected with siRNA targeting ROCK1 or ZIPK or with negative control siRNA that does not target any gene product. 48 h later, total RNA was isolated, reverse transcribed, amplified, labeled with the Ambion WT Express kit and hybridized to Human Gene 1.0 ST arrays (Affymetrix) at 45 oC for 16 h. The probe arrays were washed and stained on an Affymetrix GeneChip Fluidics-450 and scanned on an Affymetrix GeneChip Scanner 3000 7G System. Triplicates were prepared under all three conditions for microarray analysis.
2015-05-15 | E-GEOD-56819 | biostudies-arrayexpress
Project description:Recent meta-analyses of genome wide association studies (GWAS) have identified approximately 150 loci that are associated with coronary artery disease (CAD). To link the causal genes in these loci to functional transcriptional networks, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with human coronary artery smooth muscle cells (HCASMC) to investigate the cellular and molecular program downstream of one CAD associated transcription factor, TCF21. Analysis of the TCF21 downstream target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including growth factor binding (PDGF, VEGF), matrix interactions (“integrin binding,” “cell adhesion”), and smooth muscle contraction (“actin filament-based processes,” “actin cytoskeleton”). Motif searches of peak sequences confirmed the canonical E-box CAGCTG as the likely binding sequence for TCF21, but also identified bZip motifs that coordinate binding of AP1 family transcription factors. Follow-up ChIP-Seq studies verified enriched binding of bZip factors JUN and JUND to TCF21 target loci. Importantly, analysis of the representation of CAD genes among TCF21 target loci showed highly significant enrichment. Further, expression quantitative trait variation mapped to target genes of TCF21 were significantly enriched among variants with low P-values in the GWAS analyses, and single nucleotide polymorphisms within TCF21 peaks were shown to be in linkage disequilibrium with CAD-associated SNPs, suggesting a functional interaction between TCF21 binding and causal variants in other CAD disease loci. Thus, data and analyses presented here provide evidence for a transcriptional regulatory network that links TCF21 function in smooth muscle cells with a number of other CAD-associated genes, and suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology. We did ChIP-Seq on human coronary artery smooth muscle cells grown in SmGM-2 Smooth Muscle Growth Medium-2 including hEGF, insulin, hFGF-B and FBS, but without antibiotics (Lonza, #CC-3182. We did immunoprecipitations with two antibodies (Sigma HPA013189 and Abcam 49475). We conducted two biological replicates for each antibody, and also did an IgG control for these studies. For these experiments, sequencing was done on an Illumina GAII, single end reads. In separate set of experiments we performed ChIP-Seq for JUN (Ab sc-1694) and JUND (Ab sc-74) with the same HCASMC cells under the same culture conditions. For these studies we did one replicate for each antibody, and also included an IgG control. Sequencing for the JUN and JUND studies was paired ends, on an Illumina HiSeq machine.
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2022-10-14 | PXD032353 | Pride
Project description:Coronary artery disease (CAD) is the leading cause of mortality and morbidity driven by both genetic and environmental risk factors. Meta-analyses of genome-wide association studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with CAD and myocardial infarction (MI) susceptibility in multi-ethnic populations. The majority of these variants reside in non-coding regulatory regions and are co-inherited with hundreds of candidate regulatory SNPs. Herein, we use integrative genomic, epigenomic, and transcriptomic fine-mapping in human coronary artery smooth muscle cells (HCASMC) and tissues to identify causal regulatory variation and mechanisms responsible for CAD associations. Using these genome-wide maps we prioritize 65 candidate variants and perform allele-specific binding and expression analyses on 7 top candidates. We validate our findings in two independent cohorts of diseased human arterial expression quantitative trait loci (eQTL), which together demonstrate fundamental links between CAD associations and regulatory function in the appropriate disease context. We performed ATAC-seq, ChIP-seq, and RNA-seq on human coronary artery smooth muscle cells grown in SmGM-2 Smooth Muscle Growth Medium-2 including hEGF, insulin, hFGF-B and FBS, but without antibiotics (Lonza, #CC-3182). For ATAC-seq and RNA-seq we performed stimulations with growth factors (TGF-B1, PDGF-BB, PDGF-DD) versus serum-free control. We conducted two biological replicates for each condition using independent donors. For ATAC-seq experiments, sequencing was completed on an Illumina Hiseq 2500, paired-end 50bp reads. For ChIP-seq we performed immunoprecipitations using H3K27ac (Abcam ab4729). We conducted two biological replicates using HCASMC from independent donors, and also did an IgG control for these studies. For RNA-seq we also conducted two replicates using HCASMC from independent donors. For both ChIP-seq and RNA-seq experiments, sequencing was completed on an Illumina HiSeq 2500, paired-end 100bp reads. We also performed ex-vivo ATAC-seq on frozen tissues (isolated media) from normal and atherosclerotic human coronary arteries, using three independent donors for each. Sequencing was also completed on an Illumina HiSeq 2500, paired end 50bp reads.
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