CPG stimulation for Gene Expression of mutated B-CLL
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: daniela marconi
PROVIDER: E-GEOD-31360 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The B-cell receptor (BCR) plays an important role in pathogenesis and progression of chronic lymphocytic leukemia (CLL). We investigated the BCR triggering-dependent mRNA modulation by stimulating CLL cells with immobilized anti-IgM. miRome of immobilized anti-IgM stimulated CLL cells (n=16) identified a substantial upregulation of miR-132 in both unmutated (UM) and mutated (M) IGHV subgroups. A parallel gene expression profile and an in-silico analysis to identify miR-132 target genes¸ allowed us to focus on SIRT1, that encodes for a histone deacetylase targeting several proteins including TP53. We defined a reduction of SIRT1 protein levels upon immobilized anti-IgM stimulation (P=0.001), and a concomitant increase in TP53 acetylation (P=0.0072). The TP53 target gene CDKN1A was consistently up-regulated in immobilized anti-IgM stimulated CLL cells. Of note, the miR-132 constitutive expression levels in CLL cases (n=134) were of similar magnitude of those obtained in in vitro immobilized anti-IgM stimulated CLL cells. Additionally, high miR-132 expression levels retained a favorable prognostic impact in M (P=0.005), but not in UM CLL patients (P=0.968). The described miR-132/SIRT1/TP53 axis, sequentially characterized by BCR triggering, miR-132 up-regulation, SIRT1 down-regulation and TP53 acetylation, should be considered in the light of emerging drugs targeting the BCR pathway in CLL. Investigated the BCR triggering-dependent gene expression modulation by stimulating CLL cells with immobilized anti-IgM.
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Project description:The B-cell receptor (BCR) plays an important role in pathogenesis and progression of chronic lymphocytic leukemia (CLL). We investigated the BCR triggering-dependent microRNA modulation by stimulating CLL cells with immobilized anti-IgM. miRome of immobilized anti-IgM stimulated CLL cells (n=16) identified a substantial upregulation of miR-132 in both unmutated (UM) and mutated (M) IGHV subgroups. A parallel gene expression profile and an in-silico analysis to identify miR-132 target genes¸ allowed us to focus on SIRT1, that encodes for a histone deacetylase targeting several proteins including TP53. We defined a reduction of SIRT1 protein levels upon immobilized anti-IgM stimulation (P=0.001), and a concomitant increase in TP53 acetylation (P=0.0072). The TP53 target gene CDKN1A was consistently up-regulated in immobilized anti-IgM stimulated CLL cells. Of note, the miR-132 constitutive expression levels in CLL cases (n=134) were of similar magnitude of those obtained in in vitro immobilized anti-IgM stimulated CLL cells. Additionally, high miR-132 expression levels retained a favorable prognostic impact in M (P=0.005), but not in UM CLL patients (P=0.968). The described miR-132/SIRT1/TP53 axis, sequentially characterized by BCR triggering, miR-132 up-regulation, SIRT1 down-regulation and TP53 acetylation, should be considered in the light of emerging drugs targeting the BCR pathway in CLL. investigated the BCR triggering-dependent gene expression modulation by stimulating CLL cells with immobilized anti-IgM.
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Project description:Changes in gene expression profile of CLL cells in response to an antigen-specific Th cell clone that is specific for a Ckappa peptide of mouse Abs. Mouse anti human BCR mAbs were used to ligate BCR and deliver antigen to CLL cells. 32-45 x10e6 PBMC were incubated overnight with or without 2 μg/ml of Ms κ+IgG anti-kappa and anti-lambda mAbs. The PBMC were then cultured in presence or absence of 12.5 x10e6 T18 cells. On day 3, CLL cells were purified by negative selection using CD3 and CD14 Dynabeads (Invitrogen). RNA from CLL cells was controlled and quantified on a NanoDrop1000 (Thermo Scientific, Wilmington, DE, USA). 290ng per sample was amplified and labeled with the TotalPrep™-96 RNA Amplification Kit (Illumina, San Diego, CA, USA). Sample quality was further tested by Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA). 1500ng of biotin labeled cRNA was used to hybridize onto Illumina HumanWG-6 v3 Expression BeadChips. After scanning, the results were imported into Illumina GenomeStudio v. 2010.1, Gene Expression module v. 1.6.0 for quality control and data export. Analysis was performed utilizing GeneSpring GX v11 software (Agilent, Santa Clara, CA, USA). In GeneSpring GX, Logbase 2-transformed data were normalized to the 75 percentile with baseline transformation to median of all samples. Flag Information: Lower cutoff for 'Present' call: 0.8, Upper cutoff for 'Absent' call: 0.6 Testing antigen dependent Th cell - CLL cell collaboration, effects on gene expression of CLL cells
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Project description:Three different cell populations (6 healthy B-lymphocytes, 6 leukemic CLL B-lymphocyte of indolent form and 5 leukemic CLL B-lymphocyte of aggressive form) were stimulated in vitro with an anti-IgM antibody, activating the B-cell receptor (BCR). We analyzed the gene expression at 4 time points (60, 90, 210 and 390 minutes). Each gene expression measurement is performed both in stimulated cells and in control unstimulated cells. For one aggressive CLL case, we silenced expression of DUSP1 by transfecting DUSP1-specific RNAi and, as a control, transfected cells with a non-targeting RNAi. We then stimulated the BCR of these cells and analyzed the gene expression at the same time points in stimulated cells and in control unstimulated cells. B-cells were negatively selected from healthy donors and previously untreated CLL patients. BCR stimulated and unstimulated control B-cells were treated at four time points after stimulation for total RNA extraction and hybridization on Affymetrix microarrays.
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Project description:The aim is to identify the differential gene expression profile of mutated B-CLL stimulated with CPG
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Project description:Prospective series of 136 clinical monoclonal B lymphocytosis (cMBL) and 216 chronic lymphocytic leukemia (CLL) Rai 0 patients, were investigated in this study. While the distribution of CD38 and ZAP-70 positivity was similar, IGHV-mutated cases were more frequent among cMBL (P = 0.005). A Cox multivariate analysis on the whole patient cohort showed that cMBL condition was predictive of longer PFS, while CD38 expression and IGHV-unmutated status and CD38 expression correlated significantly with a shorter PFS in cMBL and Rai0-CLL, respectively. Trisomy 12, 11q- and 17p- abnormalities were scanty and of no predictive value in both conditions. Notably, gene and miRNA expression profiling showed no significant differences between cMBL and Rai0-CLL. Furthermore, similar gene and miRNA expression signatures were found in cMBL and Rai0-CLL according to the IGHV gene mutational status: that is, unmutated cases had different signatures from mutated cases, irrespectively of the cMBL or CLL condition. Overall, our study based on a prospective series of patients indicates that no major biological differences exist in cMBL compared to Rai0-CLL, suggesting that this two entities mainly differ for the initial size of the monoclonal cell population which may reflect in the longer time for clonal expansion. This series of microarray experiments contains the microRNA profiles of purified B-cell chronic lymphocytic leukemia (B-CLL) cells obtained from 150 patients (Binet stage A) including 39 cMBL and 111 Rai0-CLL. Peripheral blood mononuclear cells from CLL patients were isolated by Ficoll-Hypaque (Seromed, Biochrom KG, Berlin, Germany) density-gradient centrifugation. For gene and miRNA expression profiling experiments CLL cells were enriched by negative selection with the EasySep-Human B cell enrichment kit without CD43 depletion (Stem Cell Technologies) using the fully automated protocol of immunomagnetic cell separation with RoboSepTM (Stem Cell Technologies). The proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence with antibodies for CD19 FITC, CD23 PE and CD5 PC5. 500 nanograms of total RNA was processed in accordance with the manufacturer's protocols (Agilent Technologies) to generate Cy3-labelled RNA, which were purified on chromatography columns (Micro Biospin 6, Bio-Rad, Hercules, CA) and then hybridized on an Agilent microarray (G4470B) at 55C for 17 hr in a rotating oven. Images at 5 um resolution were generated using an Agilent scanner G2505B. The Feature Extraction 10.7.3.1 software (Agilent Technologies) was used to obtain the microarray raw-data. The raw gTotalGeneSignal has been recalculated using the procedures described in Agilent Feature Extraction Software version 10.1 manual. Non-human probes, miRNAs flagged as 'absent' (i.e. expressed below background levels) throughout the whole dataset and miRNAs expired according to Sanger miRBase Release 15 (April 2010) were discarded, and a quantile normalization was applied on raw data using the aroma.light package for Bioconducor. The data were then converted to obtain positive values throughout the dataset, at a minimum value of 1, and log2 transformed.
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