Comment[ArrayExpressAccession] E-GEOD-44841 MAGE-TAB Version 1.1 Public Release Date 2013-03-05 Investigation Title Microarray analysis of differentiation of human glioblastoma neurospheres Comment[Submitted Name] Microarray analysis of differentiation of human glioblastoma neurospheres Experiment Description Brain tumor neurospheres (BTCSs) are cancer cells with neural stem cell-like properties found in the fatal brain tumor glioblastoma multiforme (GBM). These cells account for less than 1% of total tumor cells, are poorly differentiated and are believed to be involved in tumor induction, progression, treatment resistance and relapse. Specific miRNAs play important roles in modulating the proliferation and differentiation of neural stem cells, therefore, we aimed to identify miRNAs controlling differentiation in GBM-BTSCs through high throughput screening miRNA array profiling. We compared the miRNA expression profiles at the neurosphere state and upon 4 and 14days of differentiation by using LIMMA, finding 21 differentially expressed miRNAs : hsa-miR-103, hsa-miR-106a, hsa-miR-106b, hsa-miR-15b, hsa-miR-17, hsa-miR-19a, hsa-miR-20a, hsa-miR-25, hsa-miR-301a and hsa-miR-93 were found up-regulated upon differentiation, while hsa-miR-100, hsa-miR-1259, hsa-miR-21, hsa-miR-22, hsa-miR-221, hsa-miR-222, hsa-miR-23b, hsa-miR-27a, hsa-miR-27b, hsa-miR-29a and hsa-miR-29b were down-regulated. Expression of 11 of the 21 miRNAs was examined by qPCR and 7 of them were validated: hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222 increased their expression upon differentiation, while hsa-miR-93 and hsa-miR-106a were inhibited. Functional studies demonstrated that miR-21 over-expression induced the expression of glial and/or neuronal cell markers in the neurospheres, possibly due to SPRY1 targeting by miR-21 in these cells, while miR-221 and miR-222 inhibition at the differentiated state reduced the expression of those differentiation markers. On the other hand, miR-29a and miR-29b targeted MCL1 in the GBM neurospheres and increased apoptotic cell death. Gene expression in differentiated cells relative to neurospheres in four different glioblastoma cultures Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Malumbres Aldaz Malumbres Luna Martinez-Climent Guruceaga Segura Person First Name Raquel Beatriz Raquel Jose Jose Elizabeth Victor Person Mid Initials L A Person Email rmalumbres@yahoo.com Person Affiliation University of Navarra/ Center for Applied Medical Research Person Phone +34 948194700 Person Address Oncology, University of Navarra/ Center for Applied Medical Research, Avenida Pio XII 55, Pamplona, Navarra, Spain Person Roles submitter Protocol Name P-GSE44841-1 P-GSE44841-6 P-GSE44841-3 P-GSE44841-8 P-GSE44841-7 P-GSE44841-2 P-GSE44841-4 P-GSE44841-5 Protocol Description ID_REF = VALUE = RMA normalized signal intensity Following fragmentation, 15 microg of cRNA were hybridized to the Affymetrix HG-U133A 2.0 chip according to the protocols provided by the manufacturer. Tumor cells were resuspended in serum-free DMEM/F12 medium (Invitrogen, Carlsbad, CA) containing human recombinant EGF (20 ng/ml; Sigma, St. Louis, MO), bFGF (20 ng/ml; Sigma), B-27 (20 ml per ml of medium; Invitrogen) and heparin (2 mg/ml), and seeded at a density of 3x106 live cells/60-mm plate Data analysis performed using R (2.15.0) and Bioconductor packages. The function rma of package affy (1.36.0) was used for normalization GeneChips were scanned using the GeneChip Scanner 3000 from Affymetrix. Glioblastoma neurospheres were differentiated in the presence of 10% fetal calf serum and in absence of B-27 supplement for 4days The cells were harvested with TRIzol Reagent (Invitrogen) and the RNA was extracted according to the manufacturer's instructions. As a last step of the extraction procedure, the RNA was purified with the RNeasy Mini-kit (Qiagen, Hilden, Germany). The cDNA was prepared from 5 microg total RNA using the One –Cycle cDNA Synthesis kit from Affymetrix. This cDNA was used for synthezising the cRNA using the IVT labeling kit (Affimetrix). The synthezised cRNA was purified with the GeneChip Sample Cleanup Module (Affimetrix). Protocol Type bioassay_data_transformation hybridization grow feature_extraction image_aquisition specified_biomaterial_action nucleic_acid_extraction labeling Experimental Factor Name PATIENT CELL TYPE Experimental Factor Type patient cell type Comment[SecondaryAccession] GSE44841 Comment[GEOReleaseDate] 2013-03-05 Comment[ArrayExpressSubmissionDate] 2013-03-04 Comment[GEOLastUpdateDate] 2013-03-07 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-44841.sdrf.txt