Unknown,Transcriptomics,Genomics,Proteomics

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SiRNA profiling of human aortic vascular smooth muscle cells knocked down for FoxM1

ABSTRACT: Transcription factor FoxM1 is expressed in proliferating cells, and its expression is critical for cell proliferation in embryos and tumors. FoxM1 regulates a multi-gene transcriptional network for cell cycle regulation. We used microarrays to detail the global program of gene expression either directly or indirectly regulated by FoxM1, and distinct classes of up- and down-regulated genes. Experiment Overall Design: Human aortic vascular smooth muscle cells (HAVSMC) were transfected with FoxM1 siRNA and control siRNA for 48 hours. Total RNA was extracted, purified, and prepared for hybridization on Affymetrix microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: I-Ching Wang

PROVIDER: E-GEOD-17543 | 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.

Project description:The transcription factor FOXM1 coordinates the expression of cell cycle-related genes and plays a pivotal role in tumorigenesis and cancer progression. We have previously shown that FOXM1 acts downstream of 14-3-3ζ signaling, which correlates with a more aggressive tumor phenotype. However, the role that FOXM1 might play in engendering the resistance to endocrine treatments in estrogen receptor-positive (ER+) patients when tumor FOXM1 is high, has not been clearly defined. To understand the role of FOXM1 in endocrine resistance and cancer aggressiveness, we analyzed FOXM1 protein expression by IHC in 501 ER-positive breast cancers; this revealed high FOXM1 expression in 20% of the tumors and a significantly reduced survival in these patients (p=0.003, log rank Mantel-Cox). We also mapped genome-wide FOXM1 binding events by chromatin immunoprecipitation, followed by high-throughput sequencing (ChIP-seq), in hormone-sensitive and resistant breast cancer cells after tamoxifen treatment. FOXM1 binding sites were enriched at the transcription start site of genes involved in cell cycle progression, maintenance of stem cell properties, and invasion and metastasis, all of which are correlated with the worst prognosis in ERα-positive patients treated with tamoxifen. Binding profiles were also integrated with gene expression data from cells before and after FOXM1 knockdown to highlight specific FOXM1 transcriptional networks. By modulating the levels of FOXM1 and newly discovered FOXM1-regulated genes [in breast cancer cells], we demonstrate that increased expression of FOXM1 was associated with an expansion of the cancer stem-like cell population and with increased cell invasiveness and resistance to endocrine treatments. Use of a selective FOXM1 inhibitor proved very effective in restoring endocrine therapy sensitivity and decreasing breast cancer aggressiveness. Collectively, our findings uncover novel roles for FOXM1 and FOXM1-regulated genes in promoting cancer stem-like cell properties and therapy resistance, and highlight the relevance of FOXM1 as a therapeutic target to be considered for reducing invasiveness and enhancing breast cancer response to endocrine treatments. MCF-7 human breast adenocarcinoma cells were tranfected with control, and FOXM1 siRNA for 72 hours and treated with 0.1% EtOH (Vehicle) or 1 uM TOT for 24 hours, and cDNA microarray analyses were carried out using Affymetrix [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array.

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SiRNA profiling of human aortic vascular smooth muscle cells knocked down for FoxM1

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