Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiles of the EBV-induced lymphomas against ‘normal’ human PBL

ABSTRACT: Peripheral blood lymphocytes (PBLs) were isolated from the fresh blood samples of six healthy donors and intraperitoneally grafted into SCID mice at 8~10 × 107 PBLs/mouse. PBLs from each donor were grafted into 3~4 SCID mice. EBV was isolated from the supernatant of B95-8 cells cultured in vitro under starvation conditions. EBV infection was not required for SCID mice that were grafted with PBLs from donors who were positive for EBV viral capsid antigen (VCA)-IgG and IgA. On the other hand, 0.4 mL of EBV suspension was intraperitoneally injected into SCID mice that were grafted with PBLs from donors who were VCA-IgA negative on day 3 post-grafting. All surviving mice were euthanized on day 135 post-PBL grafting. Total RNA was extracted from the six EBV-induced lymphomas (tumor group) and normal human lymphocytes (normal group) using TRIZOL reagent, and labelled as per the instructions of the Agilent Quick Amp Labeling Kit. First-strand cDNA was synthesized from purified total RNA (template) using the T7 promoter primer and Moloney murine leukemia virus reverse transcriptase, followed by the synthesis of the second-strand cDNA. Both cDNA strands were then synthesized into cRNA using transcription master mix and labelled with cyanine-3-cytidine-5'-triphosphate. Gene expression profiles of the normal and tumor groups were detected using Agilent whole genome microarray containing 41,000 known human gene transcripts. The microarrays were scanned with the Agilent Microarray Scanner and the raw data were recorded and normalized using Agilent Feature Extraction Software to calculate the signal fold change between the tumor and normal groups. A ≥2-fold change in gene expression, as determined by the t-test, was considered as up-regulated, and a ≤0.5-fold change in gene expression was considered as down-regulated.

ORGANISM(S): Homo sapiens

SUBMITTER: Chengkun Wang

PROVIDER: E-MTAB-11857 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Macrophage activation during the innate immune response is tightly regulated to prevent tissue damage while activating the defense to cellular attack. Using a mouse model where Trim33 is specifically deleted in mature myeloid cells, we show that TRIM33 is essential for two aspects of the inflammatory response in vivo. Loss of TRIM33 attenuates the initiation of macrophage activation by lipopolysaccharide (LPS) and TRIM33 is necessary to switch off transcription of inflammatory genes during late stages of LPS activation. Using chromatin immunoprecipitation coupled to deep sequencing, we provide a link between TRIM33 binding, RNA Polymerase II occupancy and H3K4me3 spreading on inflammatory genes in macrophages and reveal novel insights concerning the transcriptional regulation of Ifn-beta where TRIM33 exerts a repressive function via a distal regulatory region during late stages of LPS activation of macrophages. These findings pinpoint TRIM33 as a major regulator of the resolution of inflammation and indicate that transcriptional regulators can fine-tune H3K4me3 spreading. To study the role of TRIM33 in the transcriptional response induced by pathogen receptors, we analyzed whether lack of TRIM33 in macrophages affected the TLR-mediated regulation of proinflammatory and antimicrobial genes. To study this role, we bred TRIM33fl/fl mice with Lyz-Cre mice (obtained from The Jackson Laboratory, Bar Harbor, Maine, USA) where the Cre recombinase gene is under the regulatory sequences of the Lyz gene that is expressed only in mature myeloid cells. Bone marrow cells from 2 LyzCre/Trim33+/+ mice and 2 LyzCre/Trim33flox/flox mice were then differentiated in macrophages and treated during 0h, 4h, 12h and 24h with LPS. Total RNA was extracted from macrophages and analysed using cDNA microarrays. The set of gene expression consists of 16 samples of RNA of bone marrow derived macrophages activated with 100ng/ml of LPS during 0h, 4h, 12h, 24h, 8 samples from 2 LyzCre/Trim33+/+ mice and 8 samples from 2 LyzCre/Trim33flox/flox mice.

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Gene expression profiles of the EBV-induced lymphomas against ‘normal’ human PBL

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