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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ABSTRACT:
PROVIDER: PRJNA673445 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12953604.fastq.gz | Fastqsanger.gz | |||
| SRR12953605.fastq.gz | Fastqsanger.gz | |||
| SRR12953606.fastq.gz | Fastqsanger.gz | |||
| SRR12953607.fastq.gz | Fastqsanger.gz | |||
| SRR12953608.fastq.gz | Fastqsanger.gz |
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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).
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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
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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:Transcriptome studies of brain resections from mesial temporal lobe epilepsy (mTLE) patients revealed a dysregulation of transforming growth factor (TGF)-β, interferon (IFN)-α/β and nuclear factor erythroid 2-related factor 2 (NRF2) pathways among other neuroinflammatory mechanisms. Since ubiquitin-specific proteases (USP), in particular USP15, have been shown to regulate these pathways, we hypothesized that the blockade of USP15 may provide therapeutic relief in treatment-resistant epilepsies. The intrahippocampal kainate mouse model for mTLE, an established model for pharmacoresistant epilepsy was used for validation of USP15 as a therapeutic target. Transgenic mice which constitutively lack USP15 underwent intrahippocampal kainate injections to investigate the impact of USP15 inactivation at the transcriptomic level.
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Project description:Functionally characterizing the genetic alterations that drive pancreatic cancer progression is a prerequisite for precision pedicine. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to assess the transforming potential of 125 recurrently mutated ‘long-tail’ pancreatic cancer genes, which revealed USP15 and SCAF1 as novel and potent pancreatic ductal adenocarcinoma PDAC tumor suppressors, with USP15 functioning in a haplo-insufficient manner. Mechanistically, we found that loss of USP15 leads to reduced inflammatory responses associated with TNFa, TGF-b and IL6 signaling and sensitizes pancreatic cancer cells to PARP inhibition and Gemcitabine. Similarly, genetic ablation of SCAF1 reduced inflammatory responses linked to TNFa, TGF-b and mTOR signaling and increased sensitivity to PARP inhibition. Furthermore, we identified that loss of SCAF1 resulted in the formation of a truncated inactive USP15 isoform at the expense of full length USP15, functionally coupling SCAF1 and USP15. Notably, USP15 and SCAF1 mutations or copy number losses are observed in 31% of PDAC patients. Together, our results demonstrate the utility of in vivo CRISPR screens to integrate human cancer genomics with mouse modeling for selective discovery of novel cancer driver genes such as USP15 and SCAF1 with potential prognostic and therapeutic implications.
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Project description:Transcriptome studies of brain resections from mesial temporal lobe epilepsy (mTLE) patients revealed a dysregulation of transforming growth factor (TGF)-β, interferon (IFN)-α/β and nuclear factor erythroid 2-related factor 2 (NRF2) pathways among other neuroinflammatory mechanisms. Since ubiquitin-specific proteases (USP), in particular USP15, have been shown to regulate these pathways, we hypothesized that the blockade of USP15 may provide therapeutic relief in treatment-resistant epilepsies. The intrahippocampal kainate mouse model for mTLE, an established model for pharmacoresistant epilepsy was used for validation of USP15 as a therapeutic target. Transgenic mice which inducibly lack USP15 underwent intrahippocampal kainate injections to investigate the impact of USP15 downregulation at the transcriptomic level.
2023-10-12 | E-MTAB-12634 | biostudies-arrayexpress
Project description:Purpose: the goal of this study is to investigate the consequences of ubiquitin specific protease 15 (USP15) deletion on gene expression in mouse LSK hematopoietic progenitors. Methods: mRNA profiles of 8 weeks-old wild-type (WT) and ubiquitin specific protease 15 knockout (Usp15−/−) mice were generated by deep sequencing using Illumina Hiseq2500. The sequence reads that passed quality filters 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 Results: We assigned about 30 million reads per sample uniquely to a gene of the mouse reference genome (mm10) We identified 21,219 genes in the LSKs of WT and Usp15−/− mice using TopHat2.1 in combination with Itreecount. https://github.com/NKI-GCF/itreecount. Distinct LSK-specific expressed genes (such as Eng, Tek, the MlI receptor and the Kit receptor) are identified. Comparison of normalized gene expression data for Usp15-/- versus WT LSK confirmed the loss of Usp15. Apart from Usp15, almost no other significantly deregurated genes were detected using a treshold of fold change ≥1.5 and p value <0.05, suggesting the maintenance of an overall stable identity of the cellular compartment in Usp15-/- mice. Conclusions: Our results represent the first detailed analyis of the consequences of USP15 deletion on gene expression in hematopoietic populations such as LSKs progenitors by genome wide expression profiling in WT and Usp15-/- mice. RNAseq of two freshly isolated biological replicas of sorted LSKs from 8 weeks old Usp15-/- animals confirmed the loss of Usp15, while showing almost no significant other up or down regulated genes among the 21,219 genes identified in Usp15-/- LSKs. We conclude that young adult hematopoietic stem and progenitor cells (LSKs) perpetuated a stable gene expression program regardless of the homozygous deletion of USP15.
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