The Functional Study Of STAG2 In Erythropoiesis

Research purposes:Erythropoiesis is a complex process during which hematopoietic stem cells(HSCs)proliferate and differentiate to mature red cells.There are three stages of erythropoiesis:early erythropoiesis,terminal erythroid differentiation,and reticulocyte maturation.Any defects during the process will lead to erythroid disorders.Myelodysplastic syndromes(MDS)are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by cytopenia including anemia,myelodysplastic changes in the bone marrow and increased risk of progression to acute myeloid leukemia.It is well documented that STAG2 is one of most frequently mutated genes in MDS patients and may play an important role in the pathogenesis and malignant progression of MDS.In order to study the function of STAG2 during erythropoiesis,we first performed STAG2 knockdown in K562,HEL and cord blood derived CD34~+cells.Next,we induced erythroid differentiation in these cells to identify the role of STAG2 in erythropoiesis.This study greatly expands our knowledge about the mechanism of normal erythropoiesis,and improves our understanding of the pathogenesis of MDS.Methods:First,we performed bioinformatics analysis of the STAG2 gene family,including analysis of the evolutionary relationships,protein structure,and gene information.Next,shRNA-mediated approach was used to knockdown STAG2 in K562,HEL and CD34~+cells.Then,K562 and HEL cells were induced with hemin,which could promote the cells differentiate into erythroid lineage.On days 1-4 after induction,the experiments were conducted as follows:the proliferation was measured by cell counting,hemoglobin was detected by benzidine staining,apoptosis was detected by Annexin V combined with PI staining,and PI staining was used to detect cell cycle by flow cytometry.In the CD34~+cells induced with EPO,the differentiation of erythroid progenitor cells was detected by flow cytometry on day 6,which based on cell surface markers of IL-3R,GPA,CD34,and CD36.Band3,?4-integrin and GPA were then used as cell surface markers,and the terminal differentiation of cells was detected every other day from day 7 to day 15.Finally,the enucleation of cells was detected with syto16 fluorescent dye on day 15 and day17.Results:First,STAG2 and its family genes are located on different chromosomes.Their proteins all contain STAG and SCD domains,and their amino acid sequences are conserved.However,from the results of phylogenetic tree analysis,they appear to be isolated in the evolution of vertebrates.Next,K562,HEL and CD34~+cells with STAG2 knockdown were established,and knockdown efficiency was verified by qRT-PCR.During the erythroid differentiation of K562 and HEL cells,we found that STAG2 knockdown caused slow cell growth,delayed differentiation and increased apoptosis.In the CD34~+erythroid differentiation experiment,BFU-E increased,CFU-E and GPA positive population decreased in the STAG2 knockdown group on day 6.At day 7 and day 9,GPA expression level of STAG2 knockdown group was still lower.However,from day 11 to day 15,STAG2 knockdown group became nearly indistinguishable from the control group with GPA,band3 and?4-integrin.These results show that STAG2 knockdown delays the differentiation of erythroid progenitor cells.Finally,syto16 negative cells were significantly decreased in the STAG2 knockdown group,which indicated that STAG2 knockdown led to a decrease in the enucleation rate.Conclusion:1.STAG2 and its family proteins are conserved in structure which all contain STAG and SCD domains.Their amino acid sequences are highly conserved,but its family members are separated in the evolution of vertebrates.2.STAG2 knockdown inhibits cell proliferation,promotes cell apoptosis,delays erythroid progenitor cells differentiation and is unable to generate mature red blood cells during erythropoiesis.Therefore,STAG2 plays a key role in the process of HSCs differentiation and erythropoiesis.