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Series GSE35320 Query DataSets for GSE35320
Status Public on Sep 11, 2013
Title microRNAs distinguish cytogenetic subgroups in pediatric AML and contribute to complex regulatory networks in AML-relevant pathways
Platform organisms Homo sapiens; Mus musculus; Rattus norvegicus
Sample organisms Homo sapiens; synthetic construct
Experiment type Other
Non-coding RNA profiling by array
Expression profiling by array
Summary microRNAs, important regulators of cell proliferation and apoptosis, have been shown to be involved in the pathogenesis of acute myeloid leukemia in adulthood AML. However, comprehensive studies in AML of children and adolescents are missing so far. We investigated the miRNA expression profiles of different AML subtypes from 102 pediatric patients in comparison to CD34+ cells from healthy donors and adult AML patients, in order to identify differentially expressed miRNAs. Pediatric samples with core factor binding acute myeloid leukemia and promyelocytic leukemia could be distinguished from each other and MLL rearranged AML subtypes by 9 and 18 miRNAs, respectively. miR-126, -146a, -181a/b, -100, and miR-125b were identified as highest differentially expressed with marked difference of expression between pediatric and adulthood samples of the same cytogenetic subgroup. We next isolated the miRNA targeting complex from t(8;21) and t(15;17) cell line models and comprehensively identified bound miRNAs and targeted mRNAs by a newly devised immunoprecipitation assay followed by rapid microarray detection. Our findings indicate separate binding preferences for the four human Argonaute proteins. Subsequent bioinformatic analysis revealed a concerted action of different Ago proteins in the regulation of AML-relevant pathways, providing an experimental based database of miRNA-mRNA target interaction in Argonaute proteins.
 
Overall design Ago-associated microRNAs: Co-immunoprecipitation in the acute myeloid leukemia cell line models, KASUMI-1 and NB4, of the four human Argonaute complexes using monoclonal antibodies and stringent washing conditions. We performed photo-activated UV cross-linking using 4’-thioruidine before cell lysis. Unspecific binding to the bead matrix and to the Fc part of the monoclonal rat antibody were recorded and corrected by empty bead controls and an antibody isotype control. Argonaute-associated miRNAs/mRNAs and unspecific bound miRNAs/mRNAs of the isotype control were identified by microarray hybridization. These experiments were performed in triplicates.
Ago-associated mRNAs: Co-immunoprecipitation in the acute myeloid leukemia cell line models, KASUMI-1 and NB4, of the four human Argonaute complexes using monoclonal antibodies and stringent washing conditions. We performed photo-activated UV cross-linking using 4’-thioruidine before cell lysis. Unspecific binding to the bead matrix and to the Fc part of the monoclonal rat antibody were recorded and corrected by empty bead controls and an antibody isotype control. Argonaute-associated miRNAs/mRNAs and unspecific bound miRNAs/mRNAs of the isotype control were identified by microarray hybridization. These experiments were performed in triplicates.
AML-patient microRNAs: RNA extraction using the TRIzol protocol (Invitrogen), labeling of RNA with Cy3- (universal reference) and Cy5-Dyes (samples) using a truncated and mutated RNA ligase, hybridization of miRNAs over night at 42°C using miRXplore microarrays and the a-Hyb Hybridization Station (MACS molecular Miltenyi Biotec)
 
Contributor(s) Daschkey S, Landgraf P, Borkhardt A, Röttgers S, Bradtke J, Teigler-Schlegel A, Meister G
Citation(s) 23418555
Submission date Jan 24, 2012
Last update date Dec 06, 2018
Contact name Pablo Landgraf
E-mail(s) pablo.landgraf@gmx.de
Organization name Heinrich-Heine University Düsseldorf
Department Clinic of Pediatric Oncology, Hematology and Clinical Immunology
Street address Moorenstraße 5
City Düsseldorf
ZIP/Postal code 40225
Country Germany
 
Platforms (2)
GPL571 [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array
GPL10993 miRXplore TM Microarray (922)
Samples (182)
GSM865746 KASUMI-1, Ago1-associated miRNAs, biological rep1
GSM865747 KASUMI-1, Ago1-associated miRNAs, biological rep2
GSM865748 KASUMI-1, Ago1-associated miRNAs, biological rep3
Relations
BioProject PRJNA150757

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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE35320_RAW.tar 135.8 Mb (http)(custom) TAR (of CEL, GPR)
Processed data included within Sample table
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