Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human neuroblastoma cell lines under normoxic and hypoxic conditions

ABSTRACT: Hypoxia is a low oxygen condition that occurs in the developing tumor mass and that is associated with poor prognosis and resistance to chemo- and radio-therapy. The definition of the hypoxia gene signature is fundamental for the understanding of tumor biology, as in the case of neuroblastoma, the most common pediatric solid tumor. The issue of identifying a significant group of variables in microarray gene expression experiments is particularly difficult due to the typical high dimensional nature of the data and great effort has been spent in the development of feature selection techniques. Our main goal is to define a robust hypoxia gene signature in neuroblastoma cell lines. A set of 9 neuroblastoma cell lines were cultured under normoxic and hypoxic conditions for 18 hours, and their gene expression profiles were measured with Affymetrix GeneChip HG-U133 Plus 2.0. The clustering analysis of the expression profiles based on different clustering methods consistently revealed that hypoxia was not the major factor characterizing the data set. T-test analysis with multiple testing correction fails to identify significantly differentially expressed genes. Conversely the l1-l2 regularization selects 11 significant probesets while building an effective classification rule. The algorithm is cast within a cross-validation framework in order to achieve an unbiased analysis. The estimated cross-validation error is 17% (3 out of 18). We show that the use of l1-l2 regularization allowed us to model the effect of hypoxia, which was not detected by conventional t-test based approaches and we find a panel of genes able to properly discriminate the normoxic versus the hypoxic status of neuroblastoma cell lines. Experiment Overall Design: The expression profile of 9 neuroblastoma cell lines under normoxia vs hypoxia was studied

ORGANISM(S): Homo sapiens

SUBMITTER: Paolo Fardin

PROVIDER: E-GEOD-15583 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This SuperSeries is composed of the following subset Series: GSE21298: Profiling wt SOD versus ALS SOD1(G93A) mutant GSE21305: Profiling neuroblastoma SH-SY5Y with Paraquat treatment Mitochondrial dysfunction has been directly or indirectly implicated in the pathogenesis of a number of neurodegenerative disorders including Parkinson's disease, Alzheimer's disease and Amyotrophic Lateral Sclerosis (ALS). We used exon-sentive microarrays to characterize the responses to different mitochondrial perturbations in cellular models. We examined human SH-SY5Y neuroblastoma cells treated with paraquat, a neurotoxic herbicide which both catalyzes the formation of reactive oxygen species (ROS) and induces mitochondrial damage in animal models, and SH-SY5Y cells stably expressing the mutant SOD1(G93A) protein, one of the genetic causes of ALS. We identified a common set of genes that have a deregulated transcription and alternative splicing in both models. Noticeably, pathway analysis revealed that the expression of a subset of genes involved in neuritogenesis and axon guidance is perturbed, suggesting that alterations of axonal function may descend directly from mitochondrial damage and be responsible for neurodegenerative conditions. Paraquat treatment on cell lines derived from human neuroblastoma SH-SY5Y and the same cell line stably transfected with cDNAs coding for wild type SOD1 or ALS mutant SOD1 G93A was carried out as described in (Maracchioni et al. J Neurochem 2007, 100, 142-153). Human neuroblastoma SH-SY5Y untransfected or stably transfected with cDNAs coding for wild type SOD1 or the mutant SOD1(G93A) , were cultured in D-MEM/F-12 media with GlutaMAX™ (Gibco, Invitrogen, UK), 10% FBS, 100 Units/ml penicillin G, 100µg/ml streptomycin/ penicillin (Euroclone, Milano, Italy). Stably transfected cells were also maintained in the presence of 400µg/ml Geneticin (G418 sulphate, Euroclone). Cells were fed every 2–3 days and passed once a week. For the microarray experiment, after an initial amplification of each cell type [(SH-SY5Y; SOD1(WT), SOD1(G93A)], cells were aliquoted and cryopreservated in liquid nitrogen. For every experiment an aliquot of each cell line was thawed and seeded. After having reached confluence, cells were reseeded at 3x106cells in 100mm dish. Paraquat (N,N’-dimethyl-4,4’-bipyridinium dichloride, Sigma-Aldrich) treatment was carried out as described in (Maracchioni et al. J Neurochem 2007, 100, 142-153). Total RNA was extracted using TRIzol® Reagent (Invitrogen), and subsequently purified using silica membrane spin columns from RNeasy Mini kit (Qiagen). RNA quantity and purity were assessed using a NanoDrop® instrument (Thermo Fisher Scientific Inc.). Total RNA integrity was assessed by using a 2100 Bioanalyzer (Agilent Technologies) and the RNA Integrity Number (RIN) was calculated. Each condition was replicated 5 times. After extraction and quality check 1.5 µg of total RNA was subjected to removal of ribosomal RNA following the procedure suggested by the manufacturer (Affymetrix). The resulting total RNA was then used to created the biotin-labeled library to be hybridized on GeneChip® Exon 1.0 ST human microarrays following the procedure described by the manufacturer (Affymetrix). The CEL files resulting from the hybridization were analyzed using oneChannelGUI 1.6.5 (Sanges et al. Bioinformatics 2007, 23, 3406-3408). Exon and gene-level probeset summarization was done by mean of RMA and sketch quantile normalization. Gene-level differential expression: to assess differential expression at gene-level, we used an empirical Bayes method together with a false discovery rate (FDR) correction of the p-value Thus, the list of differentially expressed genes was generated using an FDR ≤ 0.05 together with an absolute log2(fold-change) threshold of 1. Exon-level analysis: an intensity filter was subsequently applied at gene-level to remove not expressed and low expressed genes, i.e. genes were retained for exon-level analysis if in all biological replication gene-level signal was greater than 5. Subsequently, only genes characterized to have at least two RNA isoforms annotated in Ensembl database were retained for further analysis. The Splicing Index value was calculated by taking the log2 ratio of the normalized exon intensity (NI) in Sample 1 and the NI in Sample 2. The normalized exon intensity (NI) is the ratio of the probe set intensity to the gene intensity. Alternative splicing events (ASEs) were detected as described in (Della Beffa et al. BMC Genomics 2008, 9, 571).

Project description:In multiple myeloma (MM), hypoxia-inducible transcription factor-1 (HIF-1) is overexpressed in the MM cells of the hypoxic bone marrow (BM) microenvironment. Herein, we explored in MM cells the in vitro and in vivo effects of persistent HIF-1 inhibition by expression of a lentivirus shRNA pool on proliferation, survival and transcriptional and pro-angiogenic profiles. Among the significantly modulated genes (326 and 361 genes in hypoxic and normoxic condition, respectively), we found that HIF-1 inhibition in the human myeloma cell line JJN3 downregulates the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, and MMP9. Interestingly, several pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1. The effect of HIF-1 inhibition was assessed in vivo in NOD/SCID mice both in subcutaneous and intratibial models, indicating in either case a dramatic reduction of weight and volume of the tumor burden as a consequence of HIF-1 knockdown. Moreover, a significant reduction of the number of vessels per field and VEGF immunostaining were observed. Finally, in the intra-tibial experiments, HIF-1 inhibition significantly blocks JJN3-induced bone destruction. Overall, our data indicate that HIF-1 suppression in MM cells significantly blocks MM-induced angiogenesis and reduces both tumor burden and bone destruction in vivo, strongly indicating HIF-1 as an emerging therapeutic target in MM. The transcriptional profiles on JJN3 transduced with shRNA anti-HIF-1 (JJN3-anti-HIF-1), as compared to those infected with the control vector pLKO.1 (JJN3-pLKO.1), have been analyzed either in hypoxic or normoxic conditions. To perform gene expression profiles, total RNA was purified using the RNeasy Total RNA Isolation Kit (Qiagen, Valencia, CA). Preparation of biotin-labeled cRNA, hybridization to GeneChip Human Genome U133 Plus 2.0 Arrays and scanning (GeneChip¨ Scanner 3000 7G, Affymetrix Inc.) were performed according to manufacturer's protocols.

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Transcription profiling of human neuroblastoma cell lines under normoxic and hypoxic conditions

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