Restarting Acute Myeloid Leukemia Stem Cells Senescence And Preventing Them From Inducing Bone Marrow Mesenchymal Cells Senescence By Inhibiting RAB27B

Objective:The aims of the project were to analyse the expression of RAB27B within bone marrow mononuclear cells from patients with acute myeloid leukemia（AML）or normal control and its relationship with overall survival rate of AML patients,to investigate the expression of RAB27B mRNA of bone marrow CD34⁺CD38^-cells from the patients with acute myeloid leukemia and normal volunteers,to study the effect of down-regulation of RAB27B expression on leukemia stem cells in vitro,and research the effect of inhibiting the secretion of exosomes from leukemia stem cells on bone marrow mesenchymal cells via down-regulation of RAB27B expression in vitro.Methods:The differential expression of RAB27B of bone marrow mononuclear cells from patients with AML or normal volunteers and its relationship with overall survival rate of patients with AML were analyzed by online databases,such as GEPIA,Oncomine,UALCAN and LinkedOmics;Bone marrow mononuclear cells from patients with AML,healthy volunteers and three leukemia cell lines were collected,then the CD34⁺CD38^-cell population was separated by magnetic bead sorting and the expression of their RAB27B mRNA was detected by qPCR.The expression of RAB27B in KG1a cell line rich in the CD34⁺CD38^-cell population was down-regulated by RAB27B siRNA via electroporation transfection.At 24 hours after transfection,the mRNA level of RAB27B was detected by qPCR,and the transfection efficiency of siRNA was verified.On day 1,2,and 3 after transfection,CCK8 was used to detect cell proliferation;At 72 hours after transfection,cell proliferation,cell cycle,β-galactosidase activity and cell apoptosis were detected.Cell culture supernatants from KG1a cell line were collected to separate exosomes by ultracentrifugation;Simultaneously,BMSCs from bone marrow of normal volunteers were enriched by adherence culture and passaged.Their morphological characteristics were observed by microscopy.The immunophenotype markers of the 3^rd passage BMSCs including CD73、CD90、CD34 and CD45 were detected by flow cytometry.Then the BMSCs were co-cultured with exosomes derived from KG1a cell line transfected with siRNA by electroporation.Next,theβ-galactosidase activity and the expression of RAB27B mRNA of these BMSCs were detected byβ-galactosidase staining or qPCR.Results:Based on database analysis,the expression of RAB27B in patients with AML was higher than that in normal controls and negatively correlated with overall survival rate of the patients.RAB27B had a higher expression in CD34⁺CD38^-cells from patients with AML than that in normal controls.Down-regulation of RAB27B induced proliferation inhibition,cell cycle arrest,increasedβ-galactosidase activity and no cell apoptosis change of KG1a cells.KG1a cell-derived exosomes induced an increased human BMSCsβ-galactosidase activity and RAB27B mRNA expression,but the decreased exosomes of KG1a cells by down-regulated RAB27B induced a decreasedβ-galactosidase activity of human BMSCs.Conclusions:AML stem cells have an increased RAB27B expression.The decreased exocytosis of AML stem cells exosomes by down-regulation of RAB27B in vitro can restart the senescence of AML stem cells and prevent BMSCs from senescence induction.Therefore,RAB27B could be used as a new molecular target for the treatment of AML.