Retinoic acid and Azakenpoullone treatment in SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN cell line in high and low MYCN levels
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina Genome Analyzer IIx
ORGANISM(S): Homo sapiens
SUBMITTER: Thomas Schwarzl
PROVIDER: E-MTAB-2689 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:MYCN overexpression in the doxycline MYCN inducible cell line SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN) using the dynamic transcriptome analysis (DTA) protocol. Samples at different time-points after MYCN over-expression and uninduced controls (0h) were sequenced in duplicates. The experimental setup consists of [A] total mRNA at 0h, 1h, 4h, 24h, which corresponds to the standard mRNA-seq protocol, [B] 4-thioUridine (4sU) labelled mRNA 30 min before extraction at time-points 0h, 1h, 4h, which is the freshly transcribed mRNA within the last 30 min before the time-point and [C] the counter-part, 4sU unlabelled mRNA 30 min before extraction which corresponds to the mRNA from before 30 min of extraction. The samples were sequenced on an Illumina GA IIx using the Illumina protocols.
2014-12-30 | E-MTAB-2691 | biostudies-arrayexpress
Project description:miRNAs which are differentially expressed upon MYCN overexpression (1h, 4h and 24h after induction) in a neuroblastoma line were detected using deep sequencing.
2015-07-24 | E-MTAB-2787 | biostudies-arrayexpress
Project description:The aim of the experiment is to identify genome wide binding sites for the transcription factor MYCN in MYCN non-amplified and MYCN amplified human neuroblastoma cell lines. Datasets are presented for the ChIP-seq analysis in the tetracycline inducible cell line SH-SY5Y-MYCN (SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN), derivative of the parental cell line SH-SY5Y; for noninduced cells and for 24 and 48 hours of Tet induction. Analysis for patinet matched MYCN amplified cell lines SMS-KCN and SMS-KCNR is also included.
2016-10-25 | E-MTAB-4100 | biostudies-arrayexpress
Project description:Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signaling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is not sufficiently understood. To shed new light on the mechanism, we comprehensively compared the gene expression profiles between SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which showed a different phenotype during RA-mediated differentiation. Results: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. In combination with perturbation using a PI3K inhibitor, LY294002, we identified 386 genes and categorized them into two clusters dependent on the PI3K signaling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster was greatly reduced in SK-N-SH cells or partially impaired in SH-SY5Y-E cells in coincidence with a defect in the neuronal phenotype of these cell lines. Additional stimulation with BDNF induced a set of neural genes which were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in the differentiated SH-SY5Y-A cells. Conclusions: We identified the gene clusters controlled by PI3K- and TRKB-mediated signaling pathways during differentiation in two subtypes of SH-SY5Y cells. TRKB-mediated bypass pathway compensates for the impaired neural functions generated by defects in several signaling pathways including PI3K in SH-SY5Y-E cells. The expression profiling data are useful for further studies to elucidate the signal transduction-transcriptional network including PI3K and/or TRKB. Experiment Overall Design: Human neuroblastomas, SK-N-SH (HTB-11) and SH-SY5Y-A cells (CRL-2266) were obtained from the American Type Culture Collection (ATCC). We also obtained SH-SY5Y-E cells (EC94030304) from the European Collection of Cell Cultures (ECACC). Tissue culture cells were maintained in D-MEM/F12 1:1 mixture supplemented with 15% FBS (Fetal Bovine Serum) and 1% NEAA (Non-essential amino acid) in a 5% CO2 humidified incubator at 37oC. The culture medium was changed twice a week. For the RA-inducible experiment, random culture cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded in laminin coated culture dishes (BioCoat Laminin Cellware; BD Biosciences, Billerica, MA, USA) for 1 day and then transferred to a medium containing 10 μM of RA in the presence or the absence of LY294002 (10μM) for five days. For BDNF-induced sequential differentiation of the SH-SY5Y-E strain, cells were washed with D-MEM/F12 twice after five days in the presence of RA and then incubated with 50 ng/ml of BDNF in D-MEM/F12 without serum for three days.
2009-09-04 | E-GEOD-9169 | biostudies-arrayexpress
Project description:Presence of BM-infiltrating neuroblastoma (NB) cells occurs in about 50% of patients affected by this peculiar pediatric cancer. Children aged more than 18 months at diagnosis with metastatic NB are high-risk patients with 25-30% overall survival at 5 years, despite multimodal aggressive therapy. Little is known about the metastatic process in NB patients. Hereby, we focused on miRNA expression profiles of BM-infiltrating as compared to those of primary NB tumors. For this purpose, we analyzed 22 BM-infiltrating NB cells, 22 primary NB tumors, and 4 paired BM-infiltrating and primary tumor specimens, all from patients with metastatic disease and aged > 18 months at diagnosis. Statistical analysis indicated that in human BM-infiltrating cells miR-659-3p was down-modulated as compared to primary tumors, in all sets of samples. Functional studies in HTLA and SH-SY5Y NB cell lines by miR-659-3p mimic and inhibitors demonstrated that miR-659-3p regulates the expression of the transcription factor CNOT1, that in turn regulates the expression of AU-rich element (ARE)-containing target genes AKT3, BCL2, THSB2 and CYR61, all belonging to the focal adhesion pathway. Indeed, CNOT1 expression was found significantly higher, whereas that of the ARE-containing target genes was significantly lower in BM-infiltrating cells than in primary tumors. Our findings about a role of the focal adhesion pathway, regulated by miR-659-3p through CNOT1, in NB metastatic process are intriguing for the development of new therapeutic strategies aimed to interrupt the metastatic process. To evaluate the effect of miR-659-3p up- and down-regulation on gene expression, HTLA-230 and SH-SY5Y cells were transfected with specific miR-659-3p mirVana™ mimic and inhibitor, respectively (Ambion, Life Technologies, Carlsbad, CA, USA, catalog# MC and MH 11582). Samples transfected with mirVana™ miRNA Mimic Negative Control #1 and mirVana™ miRNA Inhibitor Negative Control #1 were used as reference conditions. Transfection was performed at 20 nM miRNA concentration in OptiMEM© medium (Sigma-Aldrich), using Lipofectamin RNAiMAX© (Life Technologies), according to manufacturer’s protocol. Cells were then cultured for 48 hours, checked for viability and processed for total mRNA and miRNA extraction, as described below. Transfection experiments were performed twice with similar results. HTLA-230 (kindly donated by Dr E. Bogenman, Los Angeles, USA) and SH-SY5Y (purchased from Banca Biologica and Cell Factory, Genoa, Italy) NB cell lines were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FCS, 50 mg/ml streptomycin, 50 mg/ml penicillin and 2 mM glutamine (Sigma-Aldrich, Milan, Italy). The HTLA-230 cells present MYCN amplification and SH-SY5Y cells have normal MYCN status. Both cell lines were checked for MYCN amplification, morphology, proliferation rate and mycoplasma contamination, after thawing and within four passages in culture. To evaluate the effect of miR-659-3p up- and down-regulation on gene expression, 1x106 HTLA-230 and SH-SY5Y cells were transfected with specific miR-659-3p mirVana™ mimic and inhibitor, respectively (Ambion, Life Technologies, Carlsbad, CA, USA, catalog# MC and MH 11582). Samples transfected with mirVana™ miRNA Mimic Negative Control #1 and mirVana™ miRNA Inhibitor Negative Control #1 were used as reference conditions. Transfection was performed at 20 nM miRNA concentration in OptiMEM© medium (Sigma-Aldrich), using Lipofectamin RNAiMAX© (Life Technologies), according to manufacturer’s protocol. Cells were then cultured for 48 hours, checked for viability and processed for total mRNA and miRNA extraction, as described below. Transfection experiments were performed twice with similar results.
2015-04-20 | E-GEOD-65153 | biostudies-arrayexpress
Project description:The proteomes of undifferentiated and differentiated SH-SY5Y cells are characterised and compared. For this, neuronal differentiation using retinoic acid (RA) or a combination of RA and phorbol-12-myristat-13-acetate (RA/PMA) was explored. An MS-based label-free quantification approach is applied to identify changes in the protein expression, the as proteins’ subcellular localisation abundance as well as their association with enriched KEGG pathways. By employing formaldehyde cross-linking insights into protein interaction networks of undifferentiated as well as RA- and RA/PMA-differentiated neurons are obtained. The analyses provided insights into the proteomes of undifferentiated and differentiated SH-SY5Y cells and suggest structural rearrangements, for instance, of the actin network during neuronal differentiation.
2022-04-08 | PXD031054 | Pride
Project description:Multiple genetic and environmental factors play a role in the development and progression of ParkinsonM-bM-^@M-^Ys disease (PD). The main neuropathological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SN). To study genetic and molecular contributors to the disease process, there is a great need for readily accessible cells with prominent DAergic features that can be used for reproducible in vitro cellular screening. Here, we investigated the molecular phenotype of retinoic acid (RA) differentiated SH-SY5Y cells using genome wide transcriptional profiling combined with gene ontology, transcription factor and molecular pathway analysis. We demonstrated that RA induces a general neuronal differentiation program in SH-SY5Y cells and that these cells develop a predominantly mature DAergic-like neurotransmitter phenotype. This phenotype is characterized by increased dopamine levels together with substantial suppression of other neurotransmitter phenotypes, such as those for noradrenaline, acetylcholine, glutamate, serotonin and histamine. In addition, we show that RA differentiated SH-SY5Y cells express dopamine and noradrenalin neurotransmitter transporters that are responsible for uptake of MPP(+), a well known DAergic cell toxicant, and that MPP(+) treatment alters mitochondrial activity according to its proposed cytotoxic effect in DAergic neurons. Taken together, RA differentiated SH-SY5Y cells have a DAergic-like phenotype, and provide a good cellular screening tool to find novel genes or compounds that affect cytotoxic processes that are associated with PD. SH-SY5Y cell differentiation process was assessed by comparing RA differentiated and noRA differentiated cells in 8 days of culture. Cells were compared at 6 different time points.
2013-06-01 | E-GEOD-43368 | biostudies-arrayexpress
Project description:In neuroblastoma, amplification of the oncogenic basic helix-loop-helix (bHLH) transcription factor (TF) MYCN is the defining prognosticator of high-risk disease, occurs in one-third of neuroblastoma, and drastically reduces overall survival rates. As a proto-oncogene, targeted MYCN overexpression in peripheral neural crest is sufficient to initiate disease in mouse models. In MYCN amplified neuroblastoma, elevated expression of the factor is crucial to maintain tumor stemness and is associated with increased proliferation and aberrant cell cycle progression, as these tumors lack the ability to arrest in G1 in response to irradiation. MYCN down-regulation broadly reverses these oncogenic phenotypes in a variety of neuroblastoma models and recent thereapeutic strategies to indirectly target MYCN production or protein stability have reduced tumor growth in vivo. These observations motivate an investigation of MYCN binding in MYCN amplified tumors as it remains fundamentally unclear how elevated levels of the factor occupy the genome and alter transcriptional programs in neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of direct MYCN perturbation in neuroblastoma. We find that at oncogenic levels, MYCN associates with E-box (CANNTG) binding motifs in an affinity dependent manner across most active cis-regulatory promoters and enhancers. MYCN shutdown globally reduces histone acetylation and transcription, consistent with prior descriptions of MYC proteins as non-linear amplifiers of gene expression. We establish that MYCN load at the promoter and proximal enhancers predicts transcriptional responsiveness to MYCN shutdown and that MYCN enhancer binding occurs prominently at the most strongly occupied and down-regulated genes, suggesting a role for these tissue specific elements in predicating MYCN responsive âtargetâ genes. At these invaded enhancers, we identify the lineage specific bHLH TWIST1 as a key collaborator and dependency of oncogenic MYCN. These data suggest that MYCN enhancer invasion helps shape transcriptional amplification of the neuroblastoma gene expression program to promote tumorigenesis. ChIP-Seq in SHEP21, BE2C, KELLY, and NGP neuroblastoma cell lines for H3K27ac, H3K4me3, RNA PolII, MYCN, BRD4, or TWIST1
2016-09-01 | E-GEOD-80151 | biostudies-arrayexpress
Project description:During brain development, histone-modifying enzymes play an important role by orchestrating transcriptional programs that regulate neuronal maturation. Lysine-Specific Demethylase 1 (LSD1, also named as KDM1A) functions as a transcriptional repressor by removing methyl groups at lysine 4 of histone H3 (H3K4). In neurons, alternative splicing can include an additional exon (exon E8a) within LSD1 transcripts, generating a LSD1+8a neuro-specific isoform. We here report that LSD1+8a isoform does not have the intrinsic ability to demethylate H3K4. LSD1+8a functions as a co-activator on its target genes by removing H3K9 repressive histone marks. We identify the supervillin protein (SVIL) as a LSD1+8a interacting partner and demonstrate that SVIL protein regulates neuronal maturation by controlling LSD1+8a mediated H3K9 demethylation. Thus, our results show that alternative splicing provides a genius mechanism by which LSD1 isoforms can acquire a dual specificity (H3K9 vs H3K4) and therefore differentially control specific gene expression patterns during brain development. In order to find some LSD1+8a regulated genes at differentiated SH-SY5Y cell lines, we infected SH-SY5Y with control or LSD1+8a shRNA, then induced differentiation with RA and BDNF, (Retinoic acid (RA) (Sigma) was added at a final concentration of 10 μM the next day after plating. After 4 days, the cells were washed three times with PBS and incubated with 50 ng/mL of Brain Derived Neural Factor (BDNF) (Millipore) in serum-free medium for 3 days), we extracted RNA from BDNF induced SH-SY5Y cells for expression analysis.Duplicates were included for Affymetrix Human transcriptome version 2 array.
2015-06-05 | E-GEOD-63097 | biostudies-arrayexpress
Project description:As part of functional characterization of neuroblastoma assocated lncRNA, we performed its knock-down in neuroblastoma cell line SH-SY5Y, which resulted in modulation of expression levels of a set of genes involved in angiogenesis and inflammation, the hallmarks of metastatic cancer. SH-SY5Y cells were transfected with non-targeting siRNA control and two siRNAs targeting lncRNA BEHOT. Two days after transfection total RNA was isolated and hybridized to microarray, each sample was done in four replicas.
2013-04-01 | E-GEOD-40680 | biostudies-arrayexpress