| Research purpose:Erythropoiesis is a process by which mature red cells are generated from hematopoietic stem cells.This continuum can be subdivided into 3 stages:early erythropoiesis,terminal erythroid differentiation,and reticulocyte maturation.The entire development process is accompanied by a series of unique changes,including decrease in cell size,increase in hemoglobinization,increased chromatin condensation,and enucleation.Thus,erythropoiesis is a complex process that is tightly regulated by multiple mechanisms.Previous studies have firmly established essential role for Erythropoietin(EPO)and its receptor(EPOR),glucocorticoid and its receptor,transcription factors GATA1,GATA2,KLF1 and genetic modification such as small RNA(miRNA),long non-coding RNA(lncRNA),DNA methylation and acetylation modification.However,studies on protein kinases in human erythropoiesis remains rare.PIM2(Proviral Integrations of Moloney virus 2)is a serine/threonine protein kinase that plays an important role in the progression of various tumors by promoting proliferation and inhibiting apoptosis.However,the function of PIM2 in human erythropoiesis is still unclear.To investigate the function of PIM2,we used shRNA knockdown strategy to knock down PIM2 expression in human CD34~+hematopoietic stem cells and observed the function of PIM2 during human erythropoiesis.At the same time,we applied the same method to knock down the expression of PIM2,use Hemin to induce its differentiation into erythroid cells,and detect the function of PIM2in the process of erythroid differentiation.The research of this subject will help to explain the role and potential molecular mechanisms of PIM2 in normal physiologic processes and disease.Research methods:RNA-seq was used to analyze the expression profile of PIM2 in erythroid cells differentiated from fetal liver,peripheral blood and umbilical cord blood-derived CD34~+cells.qRT-PCR and Western Blot were applied to confirm PIM2 expression in umbilical cord blood CD34~+cells derived erythroid cells.Umbilical cord blood CD34~+hematopoietic stem cells were also used to culture human macrophages,granulocytes and so on.Then we detected the mRNA expression of PIM2 in these cell types by qRT-PCR.In order to study the role of PIM2 in human erythropoiesis,shRNA was designed to knock down PIM2 expression in human umbilical cord blood CD34~+cells.Knock down efficiency was verified by qRT-PCR and Western Blot.Flow cytometry was used to detect early erythroid differentiation,terminal differentiation,proliferation,cell cycle,apoptosis and enucleation.Cell counting was applied to assess the proliferation of erythroid cells,and Cytospins were used to observe the morphology of erythroid cells in different culture days.During terminal erythroid differentiation,obvious apoptosis was observed and the apoptosis was further confirmed by AO-EB staining.Based on the increased apoptosis,P53 protein levels were detected by flow cytometry.And then,qRT-PCR was performed to detect the P53 downstream genes(BAX,P21,NOXA and BAK)expression as well as P53 mRNA levels.Finally,we examined the mRNA expression of MDM2 by qRT-PCR and protein levels of P53,P21 and BAX by Western blot.By using the same method,PIM2 was knocked down in three hematological malignant cell lines K562,HEL and TF1.The effects of PIM2 on proliferation,differentiation and apoptosis of these three cell lines were observed by flow cytometry,cell counting and benzidine staining.Results and conclusions:Our RNA-seq data showed that PIM2 expression was gradually increased during human erythropoiesis from fetal liver,peripheral blood and umbilical cord blood-derived CD34~+hematopoietic stem cell.At the stage of Ortho-E phase,PIM2has the highest expression level compared with other stages.The increased expression of PIM2 was further confirmed by qRT-PCR and Western Blot analysis.Finally,we found that the expression of PIM2 in Ortho-E phase was higher than other common cell types by qRT-PCR.Cell counting and flow cytometry results indicated that PIM2 knock down had no effects on early stage erythropoiesis but significantly affected human terminal erythroid differentiation.Compared with the control group,the terminal erythroid cell numbers in the knock down group reduced by more than 2 times,the proportion of cells in the G0/G1 phase increased by about 15%,and S phase decreased by about 10%,the apoptotic cells increased more than 2.5 times.In addition,knockdown of PIM2 also lead to a significant decrease of enucleation in the final step of human erythropoiesis.By using flow cytometry,we found that P53 protein increased more than 2 times.Furthermore,qRT-PCR was used to detect the expression of P53 mRNA and the expression of P53 downstream genes BAX,P21,BAK and NOXA.We oberved that PIM2 knock down resulted in more than 3 times increase in P53 downstream genes expression,but not P53 mRNA expression.MDM2 could degrade P53 protein.Finally,we identified that the expression of MDM2 decreased more than 50%by qRT-PCR and the protein levels of P53,P21 and BAX significantly increased by Westernblot under PIM2 knock down.PIM2 knock down had no significant effects on proliferation,differentiation and apoptosis of K562 cells.It had no effects on apoptosis and proliferation of HEL cells by PIM2 knock down.But the differentiation of HEL cells was inhibited.Proliferation and apoptosis of TF1 cells had no effect by PIM2 knock down.While differentiation of TF1 cells was promoted.PIM2 knock down has a stage-specific regulation in normal human erythropoiesis,it has no effects on early stage erythropoiesis but significantly affects human terminal erythroid differentiation,including impaired proliferation,increased apoptosis,delayed differenytiation and decreased enucleation.Mechanistically,PIM2 knock down resulted in activation of P53-mediated endogenous apoptotic signaling pathway,which could contribute to increased apoptosis and cell cycle arrest.In addition,PIM2knock down has no obvious effect on proliferation and apoptosis in the differentiation of malignant hematological tumor cell lines into erythroid cells. |
