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.
Project description:To test the function and regulated genes of ARID3A gene, we conducted lentivirus-mediated short hairpin RNA (shRNA) against ARID3A in K562 cell line.
Project description:Summary: Ribonuclease Inhibitor (RI also known as Rnh1) is a 50 kDa, ubiquitously expressed leucine-rich repeat (LRR) protein. It is localized in cytosol and binds to pancreatic-type ribonucleases and inhibit their function. However, the entire biological role for Rnh1 is unknown. We generated RNH1 knock out K562 cells by CRISPR/Cas9 method. Here we studied differential gene expression from wild type and RNH1 knock out K562 cells by RNA-Seq analysis. Overall design: Total RNA was isolated from wild type and RNH1 deficient K562 cells.
Project description:We previously characterized zinc finger protein gene HZF1 (ZNF16) and demonstrated its important roles in erythroid and megakaryocytic differentiation of K562 cells by loss-function assay. However its effect in erythroid and megakaryocytic differentiation of hematopoietic stem/progenitor cells (HSPCs) and the mechanisms by which it functions have not been understood. In this study, we detected up-regulation of ZNF16 during erythroid and megakaryocytic differentiation of K562 cells and normal CD34+ HSPCs, and demonstrated that ZNF16 promotes erythroid and megakaryocytic differentiation by gain-of-function and loss-of-function experiments. Gene expression profiling by mRNA array and PCR validation in the K562 transforments with ZNF16 over-expression suggested that cell division cycle-associated 7-like gene (JPO2) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog gene (c-KIT) were among the genes regulated possibly by ZNF16. Luciferase reporter assay and Chromatin Immunoprecipitation demonstrated that ZNF16 binds to JPO2 and c-KIT promoters and inhibits their expression in K562 cells. A significant decrease of JPO2 and c-KIT levels was observed during erythroid and megakaryocytic differentiation of K562 and CD34+ cells. The knockdown of either JPO2 or c-KIT partially rescued the differentiation inhibition caused by ZNF16 knockdown. We also found that ZNF16 inhibits c-KIT/c-Raf/MEK/ERK/c-Jun/HEY1 signal pathways, which finally up-regulated expression of GATA1, a central regulator of erthroid and megakaryocyte differentiation. By lentivirus-mediated gene transfer, we demonstrated that enforced expression and knockdown of ZNF16 in HSPCs down-regulated and up-regulated expression of its targets respectively. Our data collectively demonstrate that ZNF16 promotes erythropoiesis and megakaryocytopoiesis via its regulation on JPO2 and c-KIT. 4 samples are analyzed, H4-1 and H4-2 are two stable K562 transductants with ZNF16 over-expression, PC1 and PC2 are stable control K562 transductants. Stable K562 transductants were obtained for RNA extraction and hybridization on an Illumina HumanHT-12 V4.0 expression beadchipe xpression Array platform.
Project description:microRNA profiling of human K562 cells comparing control or ETO2-overexpressed cells Two-condition experiment, K562-pcDNA vs. K562-pcDNA-ETO2, Biological replicates: 1, 1 pcDNA, 1 pcDNA-ETO2, independently.
Project description:microRNA profiling of comparing control or ETO2 siRNA-treated human K562 cells Two-condition experiment, K562-control siRNA vs. K562-ETO2 siRNA, Biological replicates: 1, 1 control siRNA, 1 ETO2 siRNA, independently.
Project description:K562 cells untreated (S) and treated (S+IM) with Imatinib as well as sensitive (S+IM) and resistant (R) to imatinib were subjected to labelled quantification by iTRAQ to identify Bcr-Abl downstream signaling components and proteins modulated in resistance respectively
Project description:miRNA-Sequencing of RUNX1 overexpressing K562 cells were compared to K562 cells transduced with an empty vector control to analyse the differential expression of miRNAs in hematopoiesis. K562 cells were transduced either with an empty lentiviral vector as control or a lentiviral vector overexpressing human RUNX1. The samples were biological triplicates and the results were validated by qRT-PCR using SYBR green.
Project description:Genomic profiling of K562 cells resistant to imatinib as well as imatinib resistant CML patients harboured a recurrent chromosomal amplification in 8q11.2-12.1. Gene encoding PLAG1, a transcription factor associated with cancers and chemo resistance, resides on this locus and was found to be amplified in resistant cells. Upon PLAG1 knockdown we observed a decrease in imatinib resistance, and to understand the molecular events underlying PLAG1-mediated imatinib resistance, RNA sequencing was carried out. Imatinib-resistant K562 cells were transfected with lentiviral particles containing shRNA against PLAG1 and lentiviral particles containing pLKO.1-empty vectors. Monoclonal populations were established. Total RNA sequencing was performed. For QC, RNA samples were quantified using the Qubit RNA BR Assay kit (Invitrogen, Cat# Q10211). RNA purity was checked using QIAxpert, and RNA integrity was assessed on TapeStation using High Sensitivity RNA ScreenTape® (Agilent, Cat# 5067-5579). QC passed RNA samples were subjected to the library preparation, and NEB Ultra RNA-Seq Library Prep kit protocol (Cat# E7530L) was used and sequenced on Illumina NovaSeq 6000 instrument. Transcriptomic analysis identified several genes down-regulated upon PLAG1 knockdown which were further investigated.