Explore The Effects Of RUNX1/HOXA9/HOXC4 On Human Hematopoiesis And Corresponding Mechanism With In Vitro System

Background:RUNX1-205 is a novel splicing isoform of the human RUNX1 gene.Compared with RUNX1b,RUNX1-205 lacks exon 6 due to its alternative splicing,and plays a key role in the initiation of ovarian cancer.However,its function on human hematopoiesis is still unclear.Different toRUNX1a/b/c that has been studied in depth,RUNX1-205 may be the final virginal field in the study of human RUNX1 gene splice isoforms.RUNX1b overexpression strongly blocked the transition of mesoderm to hematopoiesis in the early stage,and at the same time strongly down-regulates many members of the HOX gene family,including HOXA9 and HOXC4.These genes play a widely regulatory role in hematopoiesis and leukemogenesis.Methods:In this experiment,the unique co-culture system of hESC and AGM-S3 and PB-tet-on-OE vector(an inducible expression system based on the transposon PiggyBac)were used to explore the role of RUNX1-205,HOXA9,and HOXC4 in the process of hematopoietic development,Doxycycline(DOX)was added to induce the overexpression of target genes at different stages of hematopoietic development to investigate their effects on hematopoiesis.Cellular biology techniques(flow cytometry,immunofluorescence staining,MGG staining,colony culture assay,etc.)and molecular biology techniques(western blotting,qRT-PCR,RNA-seq,etc.)were utilized together to elucidate their roles in hematopoietic development.Results:1.The overexpression of RUNX1-205 induced from DO did not significantly affect induction of mesoderm,but severely blocked the transition from mesoderm to hematopoiesis;its overexpression at late stages(later than D6)did not negatively affect the hematopoietic development,and even promotes hematopoiesis in some degree.The protein stability of RUNX1-205 is higher than RUNX1b.2.The overexpression of HOXA9 induced from DO could severely block the production of CD34+ cells at the early stage,and their derived definitive hematopoiesis-related populations,such as CD34+CD43+,CD34+CD45+ cells.Induced HOXA9 overexpression from D4(or later)significantly promoted hematopoietic development.Among them,the promotion effects on CD34-CD45+cells were the most obvious,indicating that it may strongly promote the production of myeloid progenitor cells.When HOXA9 overexpression was induced from D4,the proportion of S-phase cells was significantly increased in CD45+cells at D14,while the NF-?B signaling pathway was also up-regulated.All these effects can be eliminated by adding a NF-?B signaling inhibitor(QNZ).3.HOXC4 overexpression induced from D0 had no significant effects on the generation of CD34+cells,but blocked the generation of subsequent hematopoiesis-related populations,such as CD34+CD43+and CD34+CD45+cells.Induction of HOXC4 from D6 or later(especially D10)could significantly promote hematopoietic development,and promote the proliferation of most hematopoietic progenitor cells(including myeloid progenitors and erythroid progenitors),especially CD34-CD43+cells.CD34-CD43+cells can be clearly divided into CD34-CD43low and CD34-CD43high sub-populations.Function assay of differentiational potentials show that CD34-CD43low cells had higher myeloid differentiation potential,and CD34-CD43high cells had higher differentiation potentials of megakaryocyte and erythroid.At the same time.When HOXC4 overexpression was induced from D10,the S-phase proportion of CD43+cells were significantly increased at D14,while the NF-?B signaling pathway was up-regulated.All these effects can be eliminated by adding a NF-?B signaling inhibitor(QNZ)and NF-?B1 siRNA.Conclusion:1.Induction of RUNX1-205 at the early stage,such as D0(or D2),could block the transition of mesoderm to hematopoiesis,and the induction of RUNX1-205 during mid and late stages,such as D6 or later,had no negative effects on hematopoiesis,and even promoted hematopoiesis.The protein stability of RUNX1-205 is higher than RUNX1b.2.Induction of HOXA9 from D4 or later could effectively promote hematopoiesis and production of myeloid progenitor cells,which might be caused by up-regulating NF-?B signaling and changing the status of the cell cycle.3.Induction of HOXC4 from D6 or later(especially D10)could effectively and widely promote hematopoiesis,especially for the CD34-CD43high cells(containing erythrocyte and megakaryocytes progenitor cells),which might be caused by up-regulating NF-?B signaling and changing the status of the cell cycle.