CML Cell-Derived Exosomal MiR-711 Weaken Adhesive Abilities Of BM-MSCs By Silencing The Expression Of The Adhesion Molecule CD44

Background:Chronic myeloid leukemia(CML)is a kind of malignant myeloproliferative blood disease caused by abnormal proliferation of hematopoietic stem cells.The incidence of CML accounts for about 15%of all leukemias,which is characterized by the fact that in most patients,a new bcr-abl fusion gene is formed by the specific Ph chromosome generated by the translocation of t(9;22)(q34;q11),and the fusion protein P~21010 encoded by the gene has strong tyrosine kinase activity can affect the proliferation,differentiation,and apoptosis of cells,and inhibition of normal hematopoiesis by activating oncogenes.Tyrosine kinase inhibitors(TKIs)have shown superior efficacy in the treatment of CML and can induce deep and sustained disease remission in CML patients.However,about 10%of CML patients still do not respond to any available TKI unfortunately.For those patients,allogeneic hematopoietic stem cell transplantation(allo-HSCT)is the only treatment option for which they are expected to be cured.However,clinical studies have shown that some patients with CML still face leukemia recurrence after received allo-HSCT,which may affect the efficacy of treatment.With recent studies,it has been found that normal hematopoietic stem cells have insufficient ability to homing,adhering,and colonizing the bone marrow microenvironment after implantation in CML patients,which is an important cause of failure in transplantation therapy.At present,the mechanism leading to adhesion defects in the bone marrow microenvironment of CML patients remains unclear.Exosomes are a kind of vesicles with a diameter of30-150 nm that can be transported between cells.They are found in almost all body fluids.The exosomes have a function of transmitting biological substances such as proteins,mRNA,microRNA(miRNA),and cytokines derived from the parental cell to the recipient cells.In our previous work,we found that the expression level of miR-711 in CML cell-derived exosomes was significantly higher than that in CML cells,and we found that miR-711 has a binding site on the 3'-untranslated region(3'-UTR)of CD44 mRNA through further online bioinformatics,suggesting that miR-711 may bind to CD44.CD44 is a type of transmembrane glycoprotein belonging to the family of adhesion molecules.It is involved in the function such as activation of lymphocytes in vivo,mediating cell-to-cell signaling,and promoting cell adhesion.CD44 abnormally expressed are closely related to tumor invasion and transfer.Therefore,this study mainly explores that CML exosomes transferred miR-711 by targeting CD44 participating in the regulation of the adhesion function of the bone marrow microenvironment,providing new idea for the mechanism of CML disease progression and therapy.Objective:The sProvide new ideas for the study of allogeneic hematopoietic stem cell transplantation failure.tudy was to explore K562 cell-derived exosomal miR-711 can be transferred to BM-MSCs,thereby weakening the adhesive abilities of the BM-MSCs by deregulating expression of the adhesion molecule CD44,and to provide a new understanding of the mechanism of CML allogeneic hematopoietic stem cell transplantation failure and improve the efficacy of CML.Methods:K562 cell derived exosomes were isolated by ultracentrifugation.The morphology of exosomes and the size distribution of exosomes were observed by transmission electron microscopy and nanometer particle size analyse machine.Western Blot was used to detect expression of exosome surface marker proteins Alix and CD63;dynamic amount changes of exosome uptake by BM-MSCs after co-culture with exosomes were monitored by fluorescence confocal microscopy;the expression of miR-711 in K562 cells and exosomes was detected by qRT-PCR;the BM-MSCs and K562 cell exosomes were co-cultured for 24 h,and BM-MSCs without exosomes treatment as control group,the expression of miR-711 in different group was detected by qRT-PCR;the targeting of miR-711 and CD44 was verified by dual luciferase reporter assay;qRT-PCR and Western Blot were used to detected expression levels of miR-711 and CD44 after transfection of BM-MSCs with miR-711 mimics and inhibitor.Results:(1)K562 cell derived exosomes were successfully extracted by ultracentrifugation,the shape of exosomes were observed by transmission electron microscopy,the exosome particle size distribution was concentrated at about 100 nm,and the results of Western Blot showed that the exosomes expression proteins Alix and CD63;(2)BM-MSCs cells were absorbed K562 cells derived exosomes in a time-dependent manner.Exosomes entered the BM-MSCs cells after co-culture for 3h,reached a peak after 9 h,and then the amount of exosomes entering BM-MSCs decreased with time changes;(3)The expression of miR-711 in exosomes was significantly higher than that in K562 cells(P<0.001),and the expression of miR-711in BM-MSCs after treatment with exosomes for 24 h was higher than that of the control group(P<0.05).(4)Luciferase assay results showed that miR-711 could bind to CD44 3'-UTR;(5)Compared with the control group,the expression level of miR-711 in BM-MSCs was increased in the miR-711 mimics transfected group,CD44 mRNA and protein expression levels were down-regulated;in contrast,the expression level of miR-711 was down-regulated in BM-MSCs transfected with miR-711 inhibitor,and the mRNA and protein expression level of CD44 was up-regulated.Conclusion:Our study reveals that K562 cell-derived exosomal miR-711 can be transferred to BM-MSCs and weaken adhesive abilities by silencing the expression of the adhesion molecule CD44,which may be provides new ideas for the basic mechanism research and clinical treatment of CML hematopoietic stem cell transplantation failure.