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MiRNA profiles of diffuse large B cell lymphoma (DLBCL) derived cell lines

ABSTRACT: miRNA profiling of human Diffuse Large B Cell Lymphoma (DLBCL) cell lines derived from the two main subtypes of this disease: Activated B Cell like (ABC-DLBCL) and Germinal Center B cell like (GCB-DLBCL), analyzing the 711 human miRNAs present in miRBase V10.0. Five ABC-like DLBCL cell lines (RIVA, Oci-Ly3, Oci-Ly10, HBL1 and U2932) and three GCB-like DLBCL cell lines (Oci-Ly7, Oci-Ly19 and SUDHL-6) were cultured in IMDM (Cellgro) with 20% human plasma, 1% penicillin/streptomycin/L-glutamine (Cellgro,) and 0,2% beta mercaptoethanol (Invitrogen). Total RNA was extracted from cell pellets using the mirVana™ miRNA Isolation Kit (Ambion), and sent to LC Sciences facility for microarray hybridization using microfluidics technology. FirstChoice® Human Skeletal Muscle Total RNA (Ambion) was used as common reference for all hybridizations. Background substracted and normalized data from each channel was used to calculate log ratios sample/reference. Keywords: miRNA profiling

ORGANISM(S): Homo sapiens

PROVIDER: GSE12933 | GEO | 2009/01/28

SECONDARY ACCESSION(S): PRJNA110857

REPOSITORIES: GEO

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Project description:miRNA profiling of human Diffuse Large B Cell Lymphoma (DLBCL) cell lines derived from the two main subtypes of this disease: Activated B Cell like (ABC-DLBCL) and Germinal Center B cell like (GCB-DLBCL), analyzing the 711 human miRNAs present in miRBase V10.0. Five ABC-like DLBCL cell lines (RIVA, Oci-Ly3, Oci-Ly10, HBL1 and U2932) and three GCB-like DLBCL cell lines (Oci-Ly7, Oci-Ly19 and SUDHL-6) were cultured in IMDM (Cellgro) with 20% human plasma, 1% penicillin/streptomycin/L-glutamine (Cellgro,) and 0,2% beta mercaptoethanol (Invitrogen). Total RNA was extracted from cell pellets using the mirVana™ miRNA Isolation Kit (Ambion), and sent to LC Sciences facility for microarray hybridization using microfluidics technology. FirstChoice® Human Skeletal Muscle Total RNA (Ambion) was used as common reference for all hybridizations. Background substracted and normalized data from each channel was used to calculate log ratios sample/reference. Keywords: miRNA profiling Dual channel experiments using FirstChoice® Human Skeletal Muscle Total RNA (Ambion) as common reference for all hybridizations. Reference was labeled with Cy5 and the cell line extracted RNA with Cy3. Biological replicates: 5 ABC-DLBCL cell lines and 3 GCB-DLBCL cell lines. Microarray hybridization was performed at LC Sciences, using slides containing in situ synthesized oligonucleotides to detect the 711 miRNAs present in miRBase V10.0. Probes hsa-miR-337 and hsa-miR-542-5p were excluded due to systematic dye bias, according to the manufacturer intructions. Background substracted and normalized detectable data was used to calculate log ratios sample/reference. Background is determined using a regression-based background mapping method. The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots. Raw data matrix is then subtracted by the background matrix. Normalization was carried out using a LOWESS (Locally-weighted Regression) method on the background-subtracted data. A transcript to be listed as detectable must meets at least two conditions: signal intensity higher than 3×(background standard deviation) and spot CV < 0.5. CV is calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level.

Project description:The activated B cell-like (ABC) subgroup of diffuse large B cell lymphoma (DLBCL) is characterized by constitutive activation of the NF-ÃªB pathway. Here we show that the NF- ÃªB pathway induces the expression of the cytokines IL-6 and IL-10 in ABC DLBCL cell lines, which also have high levels of total and phosphorylated STAT3 protein, suggesting autocrine signaling. Using RNA interference for STAT3, we defined a gene expression signature of IL-6 and IL-10 signaling through STAT3. Based on this signature, we constructed a molecular predictor of STAT3 signaling that defined a subset of ABC DLBCL tumors with high expression of STAT3, IL-6 and/or IL-10, and their downstream targets. Although the STAT3-high and STAT3-low subsets had equivalent expression of genes that distinguish ABC DLBCL from GCB DLBCL, STAT3-high ABC DLBCLs had higher expression of signatures that reflected NF-kB activity, proliferation, and glycolysis. A smallmolecule inhibitor of JAK signaling, which blocked STAT3 signature expression, was toxic only for ABC DLBCL lines, and synergized with an inhibitor of NF-kB signaling. These findings suggest that the biological interplay between the STAT3 and NF-kB pathways may be exploited for the treatments of a subset of ABC DLBCLs. Activated B cell-like (ABC) subgroup of diffuse large B cell lymphoma (DLBCL) cell lines were used as model systems to study the cytokine pathways in these cells. We expressed inducible IkB super-repressor for 1 to 24 hours to identify NF-kB target genes in OCI-Ly3 and OCI-Ly10 cells for a total of 13 arrays with replicates of each time point. We treated OCI-Ly3 cells with IL-10 for 1 to 96 hours for a total of 10 arrays with replicates of each time point. We treated OCI-Ly10 cells with IL-6 for 30 minutes to 24 hours for a total of 8 arrays with replicates of each time point. OCI-Ly10 cells transfected with no siRNA versus STAT3-siRNA for 8, 24, or 48 hours for a total of 5 arrays with replicates for 24 and 48 hours. We treated OCI-Ly10 cells with JAK inhibitor I for 3 and 6 hours for a total of 4 arrays with replicates of each time point.

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MiRNA profiles of diffuse large B cell lymphoma (DLBCL) derived cell lines

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