Effects Of AML Exosomal MiR-155 On Tight Junctions Of Brain Microvascular Endothelial Cells And Its Preliminary Mechanism

Objective:AML(acute myeloid leukemia)is a kind of malignant clone disease originating from hematopoietic stem cells.Most of the patients with AML have low rate of long-term survival and prone to relapse.The main causes of recurrence are closely related to the chemotherapeutic drugs that are not easily infiltrated into extramedullary leukemic lesions.The central nervous system is a common part of extramedxullary infiltration of leukemia.Blood-brain barrier is a structure that leukemic cells must penetrate through the process of central nervous system invasion.The tight junction between brain microvascular endothelial cells is the principal component of blood-brain barrier.The mechanism of leukemic cell infiltration into the central nervous system is unclear.Exosome is one kinds of vesicle structure contains of many components,such as RNAs,microRNAs(miRNA,miR)and so on.The exosomes secretion from tumor cells is fused with the target cell,and the gene expression of the target cell can be regulated by miRNA in exosomes.The study found that the exosomes secretion of miRNA can promote tumor metastasis,and the high expression of mir-155 in brain inflamnation can damage the tight junction between the microvascular endothelial cells and increase the permeability of blood brain barrier.Our previous studies have found that miR-155 in plasma exosomes of patients with AML were increased,but whether the leukemia cells exosomal miR-155 can destroy the tight junctions between brain microvascular endothelial cells,thereby promoting leukemia cell infiltration of the central nervous system?It is not clear now.Therefore,this study discusses of the effects of AML cell lines HL-60 exosomal miR-155 on tight junctions of brain microvascular endothelial cells,and carries on the preliminary research on its mechanism.It has important and far-reaching significance for clarifying the molecular mechanism of leukemia cell infiltration of the central nervous system,looking for a new effective method of treatment for AML,and improving the patients survival rate.In addition,it may provide new ideas for the molecular mechanism and treatment of other types of leukemic cells infiltrating the central nervous system.Methods:1.Exosomes extraction and identification:Total Exosome Isolation Reagent exocrine extraction kit was used to isolate exosomes from the culture supernatant of HL-60 and THP-1 cells.The morphology and size of exosomes were observed by transmission electron microscope,and the size of exosomes was detected by ZETASIZER Nano series-Nano-ZS.The expression of CD63 and CD81 on the surface of exosomes was identified by flow cytometry.2.Analysis of the expression of mir-155 in AML cell line and its exosomes:RT-qPCR was used to detect the expression of miR-155 in HL-60 THP-1 cells and exosomes.3.Lentivirus packaging:miR-155 overexpression and miR-155 anti expression lentivirus were constructed and packaged through enzyme digestion,transformation,plasmid extraction,plasmid transfection,concentration and purification,then the virus quality was detected by physical detection,aseptic detection,titer detection and so on.4.Construction of mir-155 overexpression and mir-155 inhibited expression cell lines in HL-60 cells:transfection of miR-155 overexpression and miR-155 inhibited expression lentivirus in HL-60 cell line.5.Analysis the expression of miR-155 in HL-60 cell lines and exosomes:the expression of miR-155 in cells and exosomes was detected by RT-qPCR method after lentivirus transfected HL-60 cells.6.Human brain microvascular endothelial cells(HBMEC)absorb the exosomes:exosomes extracted from miR-155 overexpression and miR-155 inhibits expression of HL-60 cell culture supernatant marked with CM-Dil after training with HBMEC cells,use immunofluorescence microscopy observe the uptake of exosomes by HBMEC cells.7.Analysis of the effects of exosmal miR-155 on tight junctions integrity of HBMEC:use the cell transmembrane impedance value(TEER)and endothelial cell tight junction integrity were detected by chemoresistive assay and immunofluorescence assay respectively.8.Analysis the effect of AML exosomal miR-155 on the gene level of ZO-1 in endothelial cells:RT-qPCR method was usecd to detect the effect of AML exosomal miR-155 on the gene level of ZO-1 of endothelial cells.9.Analysis the effect of AML exosomal miR-155 on the protein level of ZO-1 in endothelial cells:Westen Blot method was used to detect the effect of AML exosomal miR-155 on the protein level of ZO-1 in endothelial cells.Results:1.The characteristics of exosomes:round and oval vesicles with a diameter of 30?150 nm were isolated from leukemia cell line HL-60 and THP-1,with intact capsule,clear edges and small particles with low electron density.The positive expression rates of CD63 and CD81 were 86.1%and 92.1%respectively.2.Analysis of expression of miR-155 in AML cell line and exosomes:by RT-qPCR assay,it was found that miR-155 was expressed in both HL-60 and THP-1 cells as well as in exosomes.Compared with THP-1 cells and exosomes,the expression of miR-155 in HL-60 cells and exosomes was increased 8.12 fold and 6.73 fold respectively,the difference is statistically significant(P<0.05).3.Analysis of expression of miR-155 in AML cell line and exosomes after lentivirus transfection:The transfection efficiency of overexpression of miR-155 and the antiexpression miR-155 were successfully more than 80%,the expression of miR-155 in HL-60 cells and exosomes which transfected with overexpression lentivirus increased 10.03 fold and 13.75 fold respectively,and the expression of miR-155 in HL-60 cells and exosomes which transfected with inhibit expression lentivirus decreased 5.55 fold and 2.53 fold respectively,the differences are statistically significant(P<0.05).4.HBMEC cells uptake exosomes:immunofluorescence assay showed that after co-culture with HBMEC the exosomes from HL-60 cells with overexpression and antiexpression miR-155 can be ingested by brain microvascular endothelial cells.5.The effects of exosomal miR-155 on TEER value and tight junctions integrity of HBMEC cells:compared with control and inhibit expression of exosomal miR-155 groups,overexpression of exosomal miR-155 could decrease the transmembrane impedance value(P<0.05)and destroy tight junctions integrity of HBMEC cells.6.The effects of exosomal miR-155 on ZO-1 which is the key factor of tight junctions of HBMEC cells:compared with control and inhibit expression of exosomal miR-155 groups,overexpression of exosomal miR-155 could inhibit the expression of ZO-1 in gene and protein level(P<0.05).Conclusions:1.AML exosomal mir-155 can enter vascular endothelial cells;2.AML exosomal miR-155 reduces the electrical impedance value of brain microvascular endothelial cells and destroys the integrity of tight junctions.3.AML exosomal miR-155 has a negative regulatory effect on tight junction key molecule ZO-1.