Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiles of RPMI8226 cells after knockdown of KDM3A and KLF2

ABSTRACT: Recent studies have implicated KDM3A, which catalyzes removal of H3K9 methylation, is associated with tumorigenesis. However, the biological role of KDM3A in multiple myeloma, has not been delineated. Here we identify KDM3A-KLF2-IRF4 axis dependence in multiple myeloma. We demonstrate that knockdown of KDM3A leads to apoptosis and significant growth inhibition in myeloma cells. Mechanistically, KDM3A directly regulates myeloma cell survival factor IRF4 expression through H3K9 demethylation at its promoter. We further show that KDM3A directly regulates KLF2 expression and that knockdown of KLF2 leads to growth inhibition in myeloma cells. The goal of this analysis is to identify genes whose expression changes after shRNA-mediated knockdown of KDM3A and KLF2 using the human U133 plus 2.0 Affymetrix GeneChip in myeloma cell line (RPMI8226). Two independent experiments were performed: 1. Myeloma cell line (RPMI8226) was transduced with either shRNAs targeting KDM3A (duplicate hairpins) or luciferase (control) in duplicate. The gene expression profiles of KDM3A knockdown cells were compared with that of control cells. A total of 6 RNA samples (4 KDM3A knockdown and 2 control) were analyzed. 2. Myeloma cell line (RPMI8226) was transduced with either shRNAs targeting KLF2 (duplicate hairpins) or luciferase (control) in duplicate. The gene expression profiles of KLF2 knockdown cells were compared with that of control cells. A total of 6 RNA samples (4 KLF2 knockdown and 2 control) were analyzed.

ORGANISM(S): Homo sapiens

SUBMITTER: Manoj Bhasin

PROVIDER: E-GEOD-55667 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In multiple myeloma (MM), abnormal plasma cells interact with bone marrow (BM) stromal cells and vascular cells among others. A part of the BM milieu is considered highly hypoxic, and myeloma cells in situ may be influenced by circumstances other than normoxia in vitro. Hence, we attempted to confirm the role of hypoxic MM-derived exosomes in the BM milieu. We established a novel hypoxia-resistant cell line, RPMI8226HR, derived from RPMI8226 cells cultured for >4 months under hypoxia (1% O2), as a model of MM cells localizing in an extensively hypoxic milieu. We used RPMI8226 cells and RPMI8226HR cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from RPMI8226 cells (normoxia or hypoxia) and exosomes derived from RPMI8226HR cells (hypoxia-resistant sub-line) were used for validation of angiogeneic activity, such as tube formation assay. Exosomes derived from the RPMI8226HR cells significantly increased tube formation of HUVECs than those from RPMI8226 cells. To identify intercellular and exosomal miRNAs specifically expressed in hypoxia-resistant cells, we assess the expression profiles of intercellular and extracellular miRNAs in RPMI8226 cells and RPMI8226HR cells using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). RPMI8226 cells were cultured for 24 hours under hypoxic conditions (1% O2) or normoxic conditions (20% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).

Project description:The c-MET signaling axis is increasingly implicated in tumorigenesis and chemioresistance. In this study, we investigated gene expression modifications induced by SU11274 (Selleck Chemicals, Boston, USA), a novel selective c-MET inhibitor, in a pair of isogenic multiple myeloma (MM) cell lines either sensitive (RPMI8226) or multi-resistant, highly c-MET expressing (RPMI8226/R5) cells. On the whole, RPMI8226/R5 cells after SU11274 were characterised by wider and diverse gene expression modifications than RPMI8226, indicating that c-MET over-expression, and its inhibition, is an important aspect of the adaptive response associated to drug resistance. RPMI8226 and RPMI8226/R5 cells are cultured in RPMI 1640 supplemented with10% fetal calf serum (FCS), 100U/ml penicillin and streptomicin (Euroclone UK) and 1mM Hepes buffer at 37 Â°C in a humified 5% CO2 atmosphere. The effect of SU11274 on gene expression of both cell lines was evaluated in three distinct experiments. 500,000 cells/ml in T25 flasks were treated with SU11274 (10 ÂµM final concentration) or vehicle, for 6 h at 37 Â°C. At the end of the incubation time, treated and control untreated cells were washed and resuspended for RNA extraction. Linear mRNA amplification and labeling of fluorescent cDNA targets (Cy5, treated sample, Cy3, untreated sample) were performed using the Amino Allyl aRNA Amplification MessageAmp II Kit (Ambion), and examined Phalanx Human OneArray HOA (Phalanx Biotech Group's ) microarrays. Array scanning was carried out using a ChipReader VersArray Â® 5 micron dual confocal laser scanner with VersArray ChipReader v3.1 analysis software, while the images were analyzed by scanner row VersArray Analyzer v4.5 software (Bio-Rad Laboratories) with intensity average pixels for each spot. Global Fund has been removed from biquadratic polynomial and the normalization of multiple channels was carried out by local regression (loess), the logarithmic transformation was then performed for each level of expression.

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Gene expression profiles of RPMI8226 cells after knockdown of KDM3A and KLF2

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