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Profiles of gene expression and alternative splicing in K562 cells exposed to 2 mM sodium valproate for 12 hours assessed using exon array

ABSTRACT: To investigate the effect of valproate (VPA) treatment on K562 cell line, the growth and survival of the K562 cell line were investigated using the Annexin V/PI dual staining method, and global profiles of gene expression and alternative splicing in K562 cells were assessed using exon microarray. A significant increase in cell apoptosis was observed in valproate exposed K562 cells using flow cytometry. A total of 628 transcripts were identified as being significantly differentially expressed. The number of genes demonstrating increased expression levels was greater than the number of genes demonstrating decreased expression levels (445 genes vs. 183 genes, respectively). The significant enrichment analysis of GO terms for the differentially expressed genes revealed that these genes are involved in many important biological processes such as apoptosis. Six of observed differentially expressed genes that might be involved in apoptosis were selected to undergo qRT-PCR validation. In total, 198 candidates of alternative splicing variants were identified. Among them, three alternative splicing events were selected for validation, and CBLC and TBX1 were confirmed as alternative splicing by semi-nested PCR. In conclusion, valproate exposure facilitated cell apoptosis, altered mRNA expression and alternative splicing events in the K562 cell line. Gene expression/alternative splicing in K562 cell line were measured after exposure to 2 mM valproate for 12 hours or left untreated as a control using exon array.

ORGANISM(S): Homo sapiens

SUBMITTER: Hong Weiguo

PROVIDER: E-GEOD-32047 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Profiles of gene expression and alternative splicing in K562 cells exposed to 2 mM sodium valproate for 12 hours assessed using exon array

Project description:To investigate the effect of valproate (VPA) treatment on K562 cell line, the growth and survival of the K562 cell line were investigated using the Annexin V/PI dual staining method, and global profiles of gene expression and alternative splicing in K562 cells were assessed using exon microarray. A significant increase in cell apoptosis was observed in valproate exposed K562 cells using flow cytometry. A total of 628 transcripts were identified as being significantly differentially expressed. The number of genes demonstrating increased expression levels was greater than the number of genes demonstrating decreased expression levels (445 genes vs. 183 genes, respectively). The significant enrichment analysis of GO terms for the differentially expressed genes revealed that these genes are involved in many important biological processes such as apoptosis. Six of observed differentially expressed genes that might be involved in apoptosis were selected to undergo qRT-PCR validation. In total, 198 candidates of alternative splicing variants were identified. Among them, three alternative splicing events were selected for validation, and CBLC and TBX1 were confirmed as alternative splicing by semi-nested PCR. In conclusion, valproate exposure facilitated cell apoptosis, altered mRNA expression and alternative splicing events in the K562 cell line.

2011-11-11 | GSE32047 | GEO

Profile of gene expression in K562 cells exposed to 2 mM sodium valproate for 12 hours using Agilent SurePrint G3 Human GE 8x60K Microarray.

Project description:To explore the mechanism underlying anti-leukaemia effect of sodium valproate, the growth and survival of the K562 cell line were investigated using the Annexin V/PI dual staining method. Global profiles of gene expression in K562 cells exposed to sodium valproate were assessed using gene expression microarray and validated by qRT-PCR. Gene ontology analysis was performed to establish the function of differentially expressed genes. The differentially expressed genes identified by using gene expression array were further used to query the CMAP database to retrieve a ranked list of compounds that act on the same intracellular targets as sodium valproate. A significant increase in cell apoptosis was observed in valproate exposed K562 cells using flow cytometry. A total of 706 transcripts were identified as being significantly differentially expressed. Eight differentially expressed genes that might be involved in apoptosis were verified by qRT-PCR. The significant enrichment analysis of gene ontology terms for the differentially expressed genes revealed that these genes are involved in many important biological processes such as apoptosis. The connectivity map analysis showed gene expression profile in K562 cells exposed to sodium valproate observed in this study was most similar to that of HDACi and PI3K inhibitor in the connectivity map database, suggesting that sodium valproate might exert anti-leukaemic action by inhibiting HDAC as well as inhibiting PI3K pathway. In conclusion, the data obtained in this study might provide the ground for further studies to elucidate the molecular and therapeutic potential of VPA in leukaemia treatment. Gene expression in K562 cell line were measured after exposure to 2 mM valproate for 12 hours or left untreated as a control using Agilent SurePrint G3 Human GE 8x60K Microarray.

2011-11-11 | E-GEOD-32189 | biostudies-arrayexpress

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR) [RNA-seq]

Project description:Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. HepG2 and K562 cell lines were stably transfected with plasmids containing siRNA designed to specifically knock down ADAR expression (ADAR KD). This in order to examine how ADAR affects alternative splicing globally.

2013-06-17 | E-GEOD-47997 | biostudies-arrayexpress

Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR) [expression]

2013-06-15 | E-GEOD-47945 | biostudies-arrayexpress

Knockdown of PTBP3 in human K562 cells

Project description:PTBP1 is a widely expressed RNA binding protein known to play roles in regulation of alternative pre-mRNA splicing and other post-transcriptional processes. The PTBP2 paralog is expressed in neurons and switching from expression of PTBP1 to PTBP2 regulates a subset of alternative splicing events during neuronal differentiation. The PTBP3 paralog is conserved across vertebrates and is expressed highly in cells of the hematopoietic system. However, the unique non-redundant roles of PTBP3 are currently unclear. We therefore tested the transcriptomic consequences of knocking down PTBP3 in human K562 erythroleukemia cells. K562 cells were treated with two separate siRNAs (Rd5 and Rd9) targeted against all isoforms of PTBP3 or with a control non-specific siRNA (C2). Triplicate samples were used for each of the three experimental conditions. Transcriptome analysis used the Affymetrix HJAY array, allowing detection of changes in alternative splicing and in transcript levels. Despite at least 75% PTBP3 depletion, as monitored by western blot, only modest changes at the transcriptome level were observed.

2016-08-11 | E-MTAB-4847 | biostudies-arrayexpress

Profile of gene expression in K562 cells exposed to 2 mM sodium valproate for 12 hours using Agilent SurePrint G3 Human GE 8x60K Microarray.

2011-11-11 | GSE32189 | GEO

Interactions of aCPs with Cytosine-rich Polypyrimidine Tracts Enhance Splicing of Cassette Exons

Project description:Alternative splicing comprises a robust generator of mammalian transcriptome complexity. Splice site specification and activity are controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. A major subset of these interactions comprises interactions localized to the intronic U-rich polypyrimidine tract located immediately 5â to the majority of splice acceptors. alphaCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNP Es) comprise a subset of KH-domain proteins with high specificity and affinity for C-rich polypyrimidine motifs. Prior studies have revealed that binding of alphaCPs to C-rich motifs can modulate splicing and 3â processing of the human alpha-globin mRNA transcript in the nucleus as well as stabilization of the halpha-globin mRNA in the cytoplasm. In the current report, we demonstrate that alphaCPs have a positive impact on the activity of splice acceptor sites in a defined subset of mammalian transcripts via binding to polypyrimidine tracts that are predominantly C-rich. These findings lead us to conclude that the alphaCPs play a global role in determining the splicing activity and levels of cassette exon inclusion within the mammalian transcriptome. To test the impact of aCP proteins on alternative splicing, aCP proteins were knockdown from K562 cells by siRNA. Since aCP1 and aCP2 have redundent function, we therefore designed siRNAs capable of knockdown both isoform at the same time. 3 aCP1/2 combined knockdown and 3 control siRNA knockdown were performed in K562 cells. RNA-seq were then performed to identify alternative splicing pattern mediated by aCP proteins.

2016-03-01 | E-GEOD-71569 | biostudies-arrayexpress

Protocol for performing and analyzing TPP-TR and TPP-CCR experiments

Project description:Explore the effect of Panobinostat on the thermal stability of the proteins of the K562 cell line proteome.

2019-05-24 | PXD013774 | Pride

human leukemia cell line K562 offspring acquring imatinib resistance(K562-r) : Control vs. Drug treated

Project description:Transcriptional profiling of K562-r comparing control untreated human leukemia cells with human leukemia cells treated with AMN107 and ATO individually or combined.Four timepoints included are 3h,12h,24h,48h, covering the whole time window of K562-r cells responsing to the drug treatment.At the combination of 1.5uM ATO and 8uM AMN107, K562-r cells have the most significant coordinated effects (the apoptosis rate at 72h was 56.41% compared to 12.23% in ATO alone and 29.8% in AMN107 alone.). We studied gene expression series in K562-r cells with or without drug treatments by cDNA microarray analysis. Many genes involved in endoplasmic reticulum (ER) stress signaling, including ATF3, DDIT3, HERPUD1, HSPA5, XBP1 and TXNDC12, were highly up-regulated within 12 h of co-treatment, suggesting that the combination of ATO with AMN107 induced an ER stress response leading to apoptosis later on. Other ER-stress markers like the JNK pathway, which bridges the ER-stress and apoptosis, have been activated. Knock down the initiator of JNK partially alleviate the effects of apoptosis (p<0.05). Co-administering AMN107 and arsenic trioxide, inducing apoptosis via the ER-stress, indicates a novel therapy alternative for the imatinib induced secondary resistance. 17-condition experiment, untreated K562-r vs. Drug-treated K562-r cells, including 4 time points, for each point the untreated and 1.5uM ATO treated,8uM AMN107 treated and both 1.5uM ATO and 8uM AMN107 treated, independently grown and harvested. One replicate per array. Untreated K562-r_0h used to counteracting the background.

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