Effect on Proliferation and Erythroid Differentiation of K562 Cells by IER3IP1-Knockdown

Objective： To investigate the effect on erythroid differentiation and proliferation of K562 cells by IER3IP1-knockdown with RNA interference targeting at IER3IP1 gene. Methods： The shRNA eukaryotic expression vectors targeting at IER3IP1 gene were designed and constructed. Inhibitory effect was detected by semiquantitative RT-PCR. The impacts on K562 cells by RNAi were studied by MTT assay, benzidine staining, light microscope and electron microscopy observation, cell cycles analysis, colony formation assay and RT-PCR. The expressions of erythroid differentiation correlated genes Gfi-lB, GPA and 7-globin were studied after being exposed to 0.2μmol/L imatinib for two days. Results： The shRNA eukaryotic expression vectors were successfully constructed. The expression of IER3IP1 gene was significantly inhibited with an inhibition efficiency of 76% （P〈0.01）. Compared with the control groups, bcr/abl mRNA level was increased in K562/shRNA-IER3IP1 group （P〈0.01）. The proliferation ability was enhanced （P〈0.01） and the proportion of cells at G0/G1 phase decreased but S phase increased （P〈0.05） in K562/shRNA-IER3IP1 group. Under electron microscopy, the amount of euchromatin increased but heterochromatin decreased. There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular. The percentage of benzidine staining positive cells and mRNA expression levels of Gfi-1B, GPA and γ-globin were all decreased after being exposed to 0.2 μmol/L STI571 for two days in K562/shRNA-IER3IP1 group （P〈0.01）. Conclusion： IER3IP1-knockdown can hinder the erythroid differentiation and elevate the proliferation level of K562 cells. IER3IP1 may play a role in erythroid differentiation and proliferation of K562 cells.

Global transcriptome analysis for identification of interactions between coding and noncoding RNAs during human erythroid differentiation

Studies on coding genes, miRNAs, and lncRNAs during erythroid development have been performed in recent years. However, analysis focusing on the integration of the three RNA types has yet to be done. In the present study, we compared the dynamics of coding genes, miRNA, and IncRNA expression profiles. To explore dynamic changes in erythropoiesis and potential mechanisms that control these changes in the transcriptome level, we took advantage of high throughput sequencing technologies to obtain transcriptome data from cord blood hematopoietic stem cells and the following four erythroid differentiation stages, as well as from mature red blood cells. Results indicated that lncRNAs were promising cell marker candidates for erythroid differentiation. Clustering analysis classified the differentially expressed genes into four subtypes that corresponded to dynamic changes during sternness maintenance, mid-differentiation, and maturation. Integrated analysis revealed that noncoding RNAs potentially participated in controlling blood cell maturation, and especially associated with heine metabolism and responses to oxygen species and DNA damage. These regulatory interactions were displayed in a comprehensive network, thereby inferring correlations between RNAs and their associated functions. These data provided a substantial resource for the study of normal erythropoiesis, which will permit further investigation and understanding of erythroid development and acquired erythroid disorders.

Long noncoding RNA PCED1B-AS1 promotes erythroid differentiation coordinating with GATA1 and chromatin remodeling

Erythropoiesis is a complex and sophisticated multi-stage process regulated by a variety of factors,including the transcription factor GATA1 and non-coding RNA.GATA1 is regarded as an essential transcriptional regulator promoting transcription of erythroidspecific genes—such as long non-coding RNAs(lncRNA).Here,we comprehensively screened lncRNAs that were potentially regulated by GATA1 in erythroid cells.We identified a novel lncRNA—PCED1B-AS1—and verified its role in promoting erythroid differentiation of K562 erythroid cells.We also predicted a model in which PCED1B-AS1 participates in erythroid differentiation via dynamic chromatin remodeling involving GATA1.The relationship between lncRNA and chromatin in the process of erythroid differentiation remains to be revealed,and in our study we have carried out preliminary explorations.

CGI-100 Specific shRNA Inhibits Proliferation and Induces Differentiation in Leukemia K562 Cells

Objective： To investigate the characteristics of CGI-100- knockdown K562 cells and the effect of CGI-100 RNA interference （RNAi） on matrine-treated K562 cells. Methods： Three oligonucleotides targeting CGI-100 gene and a pair of negative control containing the same nucleotide composition with a different sequence were devised and chemically synthesized. The inhibition efficiency of CGI-100 expression by shRNA-CGI-100 in K562 cells was determined using semiquantitative RT-PCR and dot blot hybridization. The effect of CGI-100 RNAi on the growth of K562 cells was examined using MTT assay and cell differentiation was measured by distinct approaches including flow cytometry, benzidine staining and electron microscope. After CGI-100-konckdown K562 cells were incubated with 0.2 mg/ml of matrine or 30 Ixmol/L of hemin for 48 h, the expression levels of Glycophorin A（GPA）（CD235a） and Growth factor independence-lB mRNA（Gfi-IB mRNA） were measured by RT-PCR and the protein levels of GPA, CD14 and CD15 were detected by flow cytometry. Results： The eukaryotic expression vectors of CGI-100 RNAi were successfully constructed. The K562/shRNA-CGI-100 cell line was established in which the inhibition efficiency of CGI-100 gene expression by shRNA-CGI-100 was 54%. CGI-100-knockdown inhibited the proliferation and induced erythroid differentiation in K562 cells. Compared with the control K562 cells, the K562/shRNA-CGI-100 cells showed decreased absorbance value detected by MTT assay, decreased enchromation, increased heterochromation, increased percentage of G0/Gx phase cells, decreased population of S phase cells, decreased PI （proliferation index of cells）, and elevated percentage of benzidine-positive cells. Moreover, the sensitivity of K562/shRNA-CGI-100 cells to either matrine or hemin was enhanced and the sensitivity of these cells to matrine was higher than that to hemin. Compared with the control K562 cells, matrine treatment in K562/shRNA-CGI-100 cells resulted in increased inhibitory rate of proliferation, elevated percentage of be nzidine-positive cells, obviously up-regulated mRNA expressions of GPA and Gfi-IB, and increased mean fluorescence intensity （MFI） of GPA. No CD14 expression was detected and no statistical significance was found for the detected CD15. Finally, the MFI of GPA increased in K562/shRNA-CGI-100 cells treated with hemin and was 1.7 times less than that in cells exposed to matrine. Conclusion： These results suggest that the function of CGI-100 gene is correlated with the deregulated proliferation and the block of erythroid differentiation in K562 cells and may also be involved in matrine-induced erythroid differentiation in K562 ceils.