Quantitative Analysis by Next Generation Sequencing (NGS) of KBM7 and K562 chronic myeloid leukemia (CML) cells transcriptomes upon USP15 siRNAs-mediated knockdown.
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PROVIDER: PRJNA673445 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: the goal of this study is to investigate the consequences of ubiquitin specific protease 15 (USP15) knockdown on gene expression in KBM7 and K562 leukemia cells. Methods: KBM7 or K562 cells were transfected with USP15 ( siGENOME Human USP15 (9958) siRNA-SMART pool M-006066-01) or control (Ctrl) siRNAs (siGENOME Non-Targeting siRNA Control Pool#2 D-001206-14-05, Dharmacon). Cells were transfected using Lipofectamine RNAiMAX Reagent from Life Technologies following the manufacturer’s instructions and assayed at 72 hrs after transfection in Western blotting, RNA-seq and qRT-PCR. Western blotting confirmed knokdown of USP15 protein. mRNA profiles were generated by deep sequencing using Illumina Hiseq2500. Alignments of the sequence reads that passed quality filters were performed using TopHat2.1, Genome build 38 and Ensembl gtf version 77 and genecounts have been generated using Itreecount. https://github.com/NKI-GCF/itreecount. qRT–PCR validation was performed using SYBR Green assays.The amount of target, normalized to an endogenous reference (HPRT), was calculated by 2-DDCT. Results: We assigned about 30 million reads per sample uniquely to a gene of the human reference genome (hg38). We identified 26,227 genes in KBM7 and 28,329 genes in K562 cells using TopHat2.1 in combination with Itreecount (https://github.com/NKI-GCF/itreecount) workflow. Comparison of normalized gene expression data resulted in a total of 657 and 330 differential expressed genes between USP15 KD vs Ctrl in KBM7 and K562 cells (in two separate experiments), respectively, with a treshold of logFC_C1 ≥1. and adj.PVal_C1 value <0.05. Downregulation of USP15 was confirmed by Western blotting and by qRT-PCR. Ingenuity pathway analysis of USP15-dependent genes in the KBM7 and K562 datasets (cut-off logFC > 1; adjusted P value <0.05) uncovered activation of inflammation-related pathways, which involve JAK/STAT and PI3K signal transduction. In K562, we also measured significant down-modulation of TGF-beta signaling. Expression of a set of 17 genes was assessed by RT-qPCR in three independent experiments per cell line and altered expression was validated. Overlap between Top 50 significantly enriched Ingenuity Upstream Regulators in each cell line RNAseq datasets was found. The Upstream Regulators were calculated based on differential gene expression between USP15 and control siRNAs. The nine overlapping terms include: Ifnar, IRF3, STAT1, Interferon alpha, IFNG, CD40LG, IFNB1, TNF and poly rI:rC-RNA. Conclusions: Our results represent the first detailed analyis of the consequences of USP15 depletion on gene expression in malignant hematopoietic populations such as KBM7 and K562 chronic myeloid leukemia cells by genome wide expression profiling in USP15 KD vs Ctrl cells. We confirmed downregulation of USP15 and validated altered expression of a set of 17 genes by qRT-PCR. Ingenuity pathway analysis identified 657 and 330 differentially regulated genes in KBM7 and K562, respectively. The changes in gene expression, though not extensive, indicate that RNAi of USP15 led to activation of inflammation-related pathways, which involve JAK/STAT and PI3K signal transduction in both cell lines. In K562, we also measured significant down-modulation of TGF-beta signaling, which is required for USP15 pro-oncogenic role in human glioma cells (Eichhorn et al., 2012). Regulation of inflammatory signals and TGF-beta are relevant both in normal HSC and in malignant development (Blank and Karlsson, 2015).
2020-10-31 | GSE160524 | GEO
Project description:The Bmi1 Polycomb protein is involved in the epigenetic repressive control of self renewal and survival of cancer initiating cells. In Chronic Myeloid Leukemia (CML), bmi1 expression increases gradually as the disease progresses from a chronic latent phase to a deadly blast crisis. We developped an inducible shRNA system to silence Bmi1 in the human K562 chronic myeloid leukemia (CML) cell line in order to identify new Bmi1-target genes. Gene profiling was performed on inducible shBmi1-K562 cells incubated without (P3-K562+shBMI1) or with doxycycline for 96h (P4-K562+shBMI1+doxycycline) using HG-U133 Plus2 Affymetrix Arrays.
2015-05-12 | E-GEOD-54262 | biostudies-arrayexpress
Project description:The series were performed to study the changes in gene expression upon diploidization of KBM7 cancer (CML) cell line. The line can exist either as a clone with 24 chromosomes (nearly haploid) or with 48 chromosomes (nearly diploid). Gene expression patterns are largely ploidy-independent, as suggested by this experiment Single cell derived clones of KBM7 cell line were grown. Included 7 haploid and 10 diploid clones, each with 2 independent total RNA extraction/microarray run. Also included peripheral blood mononuclear cells (PBMC) samples for comparison purposes.
2014-04-10 | E-GEOD-56465 | biostudies-arrayexpress
Project description:The series were performed to study the changes in gene expression upon diploidization of KBM7 cancer (CML) cell line. The line can exist either as a clone with 24 chromosomes (nearly haploid) or with 48 chromosomes (nearly diploid). Gene expression patterns are largely ploidy-independent, as suggested by this experiment
2014-04-10 | GSE56465 | GEO
Project description:The Bmi1 Polycomb protein is involved in the epigenetic repressive control of self renewal and survival of cancer initiating cells. In Chronic Myeloid Leukemia (CML), bmi1 expression increases gradually as the disease progresses from a chronic latent phase to a deadly blast crisis. We developped an inducible shRNA system to silence Bmi1 in the human K562 chronic myeloid leukemia (CML) cell line in order to identify new Bmi1-target genes.
2015-05-12 | GSE54262 | GEO
Project description:We performed ChIP-seq to measure H3K9me3 levels in KBM7 cells
2015-05-28 | GSE60056 | GEO
Project description:Our objective is to decipher a miRNA expression signature associated with Chronic myeloid leukemia (CML), with or without Imatinib or Dasatinib and compared to normal blood leukocytes, and to determine potential target genes and signaling pathways affected by these signature miRNAs. Background/Aims: MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. However, little is known about the role of miRNAs in chronic myeloid leukemia (CML). Our objective is to decipher a miRNA expression signature associated with CML and to determine potential target genes and signaling pathways affected by these signature miRNAs. Results: Using miRNA microarrays we characterized the miRNAs expression profile of K562 cells in reference to healthy blood. We also looked into the expression profile of K562 cells treated with imatinib or dasatinib. We identified a miRNA signature that distinguishes K562 cells from healthy blood that included downregulation of miRNAs such as miR-31, miR-34a, miR-143, miR-145, miR-155, miR-196b and miR-564 and upregulation of miR-128. We also identified a miRNA signature that distinguishes untreated K562 cells from the treated ones that included downregulation of miRNAs such as miR-128 and upregulation of miR-564. Results: Using miRNA microarrays we characterized the miRNAs expression profile of K562 cells in reference to healthy blood. We also looked into the expression profile of K562 cells treated with imatinib or dasatinib. We identified a miRNA signature that distinguishes K562 cells from healthy blood that included downregulation of miRNAs such as miR-31, miR-34a, miR-143, miR-145, miR-155, miR-196b and miR-564 and upregulation of miR-128. We also identified a miRNA signature that distinguishes untreated K562 cells from the treated ones that included downregulation of miRNAs such as miR-128 and upregulation of miR-564. We next analyzed predicted targets and affected pathways of the deregulated miRNAs. Reassuringly, the analysis identified CML as the main disease associated with these miRNAs. MAPK, TGF-beta and Wnt were the main molecular pathways related with these expression patterns. Utilizing Venn diagrams we found appreciable overlap between the CML-related miRNAs and the MAPK and TGF-beta signaling pathways and focal adhesion-related miRNAs. Conclusions: The miRNAs identified in this study might offer a pivotal role in CML. Nevertheless, while these data point to a central disease, the precise molecular pathway/s targeted by these miRNAs is variable implying a high level of complexity of miRNA target selection and regulation. These deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment. MiRNA profiling of CML: With the objective of deciphering a potential miRNA expression signature associated with CML, we analyzed the miRNAs expression profile of K562 in reference to a pool of 3 healthy blood samples using an miRNA-based microarray chip assay. In addition, to search for Bcr-Abl-dependent or Src-dependant miRNA expression patterns, K562 cells were treated or not with imatinib or with dasatinib to inhibit Bcr-Abl tyrosine kinase activity or Bcr-Abl and Src tyrosine kinase activity, respectively. In order to validate the microarray data and to ensure that the variability observed among samples was not technical, we carried out Taqman miRNA quantitative real-time PCR analysis (Applied Biosystems, USA) on miRNAs that we find to be most relevant, according to bioinformatics analysis (see below) and published data.
2011-10-20 | E-GEOD-28825 | biostudies-arrayexpress
Project description:Increased FTO expression has been connected to resistance to tyrosine kinase inhibitors in CML. To explore the therapeutic potential of targeting FTO in CML, we tested the FTO catalytic inhibitor in the K562 CML cell line. The RNA-seq was performed to identify relevant regulated genes.
2023-12-31 | E-MTAB-12774 | biostudies-arrayexpress
Project description:Transcriptomic analysis of SRSF1 overexpression and imatinib treatment in K562 CML cell lines
| PRJNA589295 | ENA
Project description:Chronic myeloid leukemia (CML) epitomizes successful targeted therapy, with 86% of patients in the chronic phase treated with tyrosine kinase inhibitors (TKIs) attaining remission. However, resistance to TKIs occurs during treatment, and patients with resistance to TKIs progress to the acute phase called Blast Crisis (BC), wherein the survival is restricted to 7-11 months. About 80 % of patients in BC are unresponsive to TKIs. This issue can be addressed by identifying a molecular signature which can predict resistance in CML-CP prior to treatment as well as by delineating the molecular mechanism underlying resistance. Herein, we report genomic analysis of CML patients and imatinib-resistant K562 cell line to achieve the same. WGS was performed on imatinib-sensitive and -resistant K562 cells. Library preparation was done by 30x WGS KAPA PCR-Free v2.1 kit, and Illumina HiSeq X sequencer was used for 2 x 150 bp paired-end sequencing. Our study identified accumulation of aberrations on chromosomes 1, 3, 7, 16 and 22 as predictive of occurrence of resistance. Further, recurrent amplification in chromosomal region 8q11.2-12.1 was detected in highly resistant K562 cells as well as CML patients. The genes present in this region were analyzed to understand molecular mechanism of imatinib resistance.
2024-01-01 | E-MTAB-11896 | biostudies-arrayexpress