Beclin1 Is Essential For Adult Hematopoiesis

ObjectiveTo determine whether Beclinl plays an essential role in adult hematopoiesis,and explore the mechanism by which Beclinl regulates hematopoiesis.Methods1)To detect the role of Beclinl in adult hemaopoiesis,we crossedBeclin1f/f mice to Mx1-Cre mice,which can finally generateBeclin1f/f;Mx1-cre mice.Injection of pIpC can induce the deletion of Beclinl in adult hematopoiesis.Q-PCR and western blot were used to validate the efficiency of the gene knockout.2)To confirm the phenotype of Beclin1-/-mice,we conducted complete blood counting.Spleens were taken from Beclin1+/+and Beclinl-/-mice,fixed by paraformaldehyde,and then were stained by hematoxylin and eosin.The component of cells in spleen and thymus were measured by flow cytometry.3)To detect whether Beclin1-/-bone marrow was impaired,bones were fixed for H&E slices.Total bone marrow cells were stained with Ter119 antibody to analyze red blood cells in bone marrow.4)To detect whether Beclin1-/-HSPCs can function normally,we conducted colony forming unit assay to measure the function of HSPCs in vitro.Total bone marrow cells were cultured in methylcellulose,and number of colonies was counted;competitive transplantation experiment was used to analyze the function of Beclinl-/-HSPCsin vivo.5)To clarify how deletion of Beclinl damages the function of HSPCs,flow cytometry was used to analyze the number and percentage of HSPCs in bone marrow.Different phases of cell cycle in LSK were measured by Ki67/DAPI staining.6)To explore the mechanism by which beclinl regulates hematopoiesis,LSK cells were sorted by flow cytometry.Total RNA were extracted from LSK,and then were used to conduct RNA-seq.KEGG pathway enrichment,GSEA analysis were used to analyze results of RNA-seq.Q-PCR and ELISAwas used to validate the results of RNA-seq.Results1)6-8 weeks Beclin1f/f and Beclin1f/f;Mx1-cre mice were injected with pIpC.Results of Q-PCR and western blot indicated that Beclin1 were deleted efficiently in adult hematopoietic system.The lifespan of Beclin1-/-mice was shorter compared to control.Number of WBC and PLT in peripheral blood was decreased.Number of RBC was similar to control,but HGB,MCV and MCH were decreased.Splenomegaly was exhibited in Beclinl-/-mice,which suggests increased extramedullary hematopoiesis in spleen.Hematoxylin and eosin staining of spleen and thymus shows disordered structure due to deletion of Beclin1.Cells of spleen and thymus were detected by flow cytometry,which shows abnormal myeloid lineage and lymphoid lineage.2)Beclin1-/-bone marrow was paler than Beclinl+/+ mice.Terl19+ cells were decreased in Beclin1-/-bone marrow.H&E staining shows that deletion of Beclinl causes the increase of megakaryocytes and the decreased of red blood cells in bone marrow.3)Colony forming unit assay was conducted using total bone marrow cells.Beclin1-/-bone marrow cells produced fewer and smaller colonies than control.Results of competitive transplantation experiment indicated thatBeclin1-/-HSPCs failed to contribute to hematopoietic system reconstitution.4)Bone structure of Beclinl-/-mice was abnormal.Percentage and number of LSK in Beclin1-/-mice were increased,as mature cells in peripheral blood decreased and colony forming ability was damaged,which indicated that differentiation ability of Beclin1-/-LSK is impaired.Ki67/DAPI staining shows deletion of beclin1 disturbed the quiescence of LSK.5)Beclin1-/-LSK shows aberrant genetic network.Sphk1,which can phosphorylate sphingosine to SIP,increased almost 64 times.Downstream signaling pathways of S1P were upregulated in Beclin1-/-LSK.Conclusion1)Deletion of Beclinl in adult hematopoiesis disturbs cell cycle of LSK,which causes increased percentage and number of LSK,and destroys the differentiation ability of HSPCs.Beclin1-/-HSPCs fail to reconstitute damaged hematopoietic system.Therefore,Beclinl is essential for maintaining adult hematopoiesis.2)Deletion of Beclinl strikingly increases the expression of Sphk1,which is an upstream regulator of S1P.