Microarray expression profile of long noncoding RNAs in germinal center-like diffuse large B-cell lymphoma cell lines.
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE97336 | GEO | 2019/01/01
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
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Project description:Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category with distinct molecular subtypes that can be defined by gene expression profiling. However, even within these defined subtypes, heterogeneity prevails. To further elucidate the pathogenesis of these entities, we determined the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in 248 primary DLBCL patient samples. These analyses revealed that loss of PTEN was detectable in 55% of germinal center B-cell-like (GCB) DLBCLs, whereas this abnormality was found in only 14% of non-GCB DLBCL patient samples. In GCB DLBCL, the PTEN status was inversely correlated with activation of the oncogenic PI3K/ protein kinase B (AKT) pathway in both DLBCL cell lines and primary patient samples. Re-expression of PTEN induced cytotoxicity in PTEN-deficient GCB DLBCL cell line models by inhibiting PI3K/AKT signaling, indicating an addiction to this pathway in this subset of GCB DLBCLs. PI3K/AKT inhibition induced down-regulation of the transcription factor MYC. Re-expression of MYC rescued GCB DLBCL cells from PTEN-induced toxicity, identifying a regulatory mechanism of MYC expression in DLBCL. Finally, pharmacologic PI3K inhibition resulted in toxicity selectively in PTEN-deficient GCB DLBCL lines. Collectively, our results indicate that PTEN loss defines a PI3K/ AKT-dependent GCB DLBCL subtype that is addicted to PI3K and MYC signaling and suggest that pharmacologic inhibition of PI3K might represent a promising therapeutic approach in these lymphomas.
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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.
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