Project description:Purpose:RNA sequencing (RNA-Seq) was performed to analyze the differential expression of genes in control and lncRNA-up91 knockdown K562 cells
Project description:K562 cells were infected with none specific or Drosha CRISPR lenti-virus to silence the gene and subjected to iTRAQ-TMT mass spectrometry
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:miR-34a is strongly induced upon TPA-induced megakaryocyte differentiation of K562 cells. To investigate the gene networks regulated by this miRNA during the process of differentiation we performed gene microarray analysis in K562 cells overexpressing miR-34a or a control sequence.
Project description:Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells. Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells.
Project description:Purpose: The aim of this study was to evaluate the difference of mRNA and lncRNA in K562 cells treated with vandetanib (1.56 μM) and control (DMSO) by high-throughput sequencing results. Methods: Total RNA was extracted using the RNAfast200 kit according to the manufacturer’s protocol. Then, 200 ng RNA was sequenced with high throughput, and then pathway and GO analysis were used for comprehensive analysis. Finally, RT-PCR method was used to validate in large sample population. Results: High-throughput sequencing results showed that there were significant differences in mRNA and lncRNA in K562 cells treated with vandetanib (1.56 μM) and control (DMSO). Conclusions: It is the first time that we have used high-throughput sequencing to study the difference of mRNA and lncRNA in K562 cells treated with vandetanib (1.56 μM) and control (DMSO). The results of sequencing show that the expression of mRNA and lncRNA is quite different in K562 cells treated with vandetanib (1.56 μM) and control (DMSO).