| Backgrouds: Leukemia is a type of highly heterogeneous hematologic malignancy originating from hematopoietic stem and progenitor cells.Currently,the complete response(CR)rate of patients with leukemia was obvious improvement by comprehensive treatment based on chemotherapy,but the percentage of disease free survival(DFS)was only about 20%.In addition,it was few that the specific molecular markers of diagnosis and treatment in leukemia.Therefore,to explore treatment strategies and search for novel molecular markers in leukemia had important significance for improvement of treatment efficacy and survival rate.Microvesicles(MVs)are nanosized bilayer-lipid membrane macrovesicles which is shed from almost all living cell types.Mvs with diameter ranged 100-1000 nm contain microparticles,exosomes and apoptotic bodies,which carried bioactive molecules including nucleic acid(DNA,mRNA,miRNA,etc.),protein and lipids derived from the source cells.MVs may mediate inter long distance cells communications as well as regulated the pathophysiological process via body fluids,which is a breakthrough point to investigate the mechanism of leukemia and find biomarkers.Cytokine induced killer(CIK)cells are a heterogeneous immune cell population containing CD3+CD56+ double positive cells with cytotolytic specificity against the minimal residual disease(MRD)of leukemia.However,the biological characteristics of CIK cells derived exosomes still has no evidence,and its promoting tumor proliferation or anti-tumor in leukemia bearing environment remains to be elucidated.Objective: To investigate the effect of CIK cell-derived microvesicles on the apoptosis and migration of myeloid leukemia K562 cells,and to explore the miRNA expression profiles of serum microvesicles derived from AML patients obtaining specific miRNA markers.It had important significance for clarifying the biological characteristics of CIK cell-derived microvesicles and further studying the function of miRNA loaded serum microvesicles of AML patients.Methods: Firstly,CIK cells could be stimulation with various activators from peripheral blood mononuclear cells(PBMCs)of myeloid leukemia patients.The expressions of CD3 and CD56 were detected by flow cytometry.The killing effect of CIK cells on K562 cells was tested by A Cell Counting Kit-8(CCK-8)assays.Culture supernatants of CIK cells were collected,the microparticles and exosomes were separated using gradient centrifugation,ExoQuick Precipitation,respectively.MVs were identified with a transmission electron microscope,qPCR and Western blot.Secondly,CCK-8 assay was performed to detect the effect of the culture superman from CIK cells on proliferation of K562 cells.The impaction of CIK cells derived MVs on the apoptosis of K562 cells using Annexin V FITC was detected by flow cytometry,and cells migration was analyzed by Transwell assays.In addition,The exosomes were separated from serum of 3 patients with primary AML and 3 health volunteers using ExoQuick Precipitation.The miRNA expression profiles were examined using NGS(next generation sequencing)instrument Illumina HiSeq-X10,and analyzed the differentially expressed miRNAs.Then the targets of the differentially expressed miRNAs were predicted by software,and the possible significant functions and involvement in signaling pathways of gene ontology(GO)and KEGG pathways were analyzed.Results:(1)CIK cells were generated from peripheral blood mononuclear cells(PBMCs)isolated from myeloid leukemia patients,and by addition of rhIFN,rhIL-2 and anti-CD3 antibodies.After cultured for 14 d,CIK cells have strong proliferative ability and the CD3+CD56+double positive cells contents of CIK cells were(21.50±6.20)%.CIK cells revealed a mean cytotoxicity of(31.31±5.20%),(56.63±8.88%)and(81.87±5.86%)against K562 cells when the effector to-target ratio was 5:1,10:1 and 20:1,respectively.(2)We performed SEM to observe the morphology of MVs.SEM clearly revealed that MVs exhibited round-shaped morphology with a size of 30-1000 nm.qPCR analysis of differentially expressed transcription factors including HSP-70,CD2,TCR-α,GZM-B and perforin mRNA identified in the CIK cells derived MVs.Western blotting confirmed the expression of CD9,HSP-70 and GZM-B of MVs markers,CD2 as well as TCR-α in MVs from source cells.(3)The culture supernatants of CIK cells didn’t stimulate the proliferation of K562 cells(p>0.01).The migration ability of K562 cells incubated with MVs were increased at 5 μg/ml,15 μg/ml,20 μg/ml,50 μg/ml and 100 μg/ml(p<0.01),and concentrationdependent effects were observed.The apoptosis of K562 cells were decreased under the action of CIK cells derived MVs at 20 μg/m L as well as 50ug/mL by 48 h,but 100 μg/mL MVs were not influence the susceptibility to the apoptosis.The expression of IDO-1 and MMP-9 was increased in response to 50ug/mL MVs derived from CIK cells incubation with K562 cells at 48 h,whereas VEGF was decreased(p<0.01).(4)NGS instrument Illumina HiSeq-X10 was performed to identify mi RNAs profile of serum exosomes with primary AML patients by comparing the health volunteers,and 3,194 miRNAs are identified containing 1,742 known miRNAs and 1,452 new miRNAs.Compared with the control group,15 different miRNAs with statistically different expression levels were up-regulated and 11 miRNAs were down-regulated in the HLL group;in the NHLL group,11 miRNAs were found to be up-regulated,and 8 miRNAs were down-regulated.Bioinformatics analysis reveals that serum exosomal microRNAs control pathways involved in the tumor,especially in the MAPK,Wnt,NF-κB and hippo signaling pathways.Conclusion: CIK cells derived MVs induced the K562 cells migration,upregulated the expression of IDO-1,MMP-9,whereas VEGF was down-regulated.In the HLL group,the statistically different expression levels of 15 miRNAs derived from serum exosomes were increased and 11 miRNAs were decreased;11 different miRNAs were found to be up-regulated,and 8 miRNAs were down-regulated in the NHLL group.These results improve the understanding of the CIK cells derived MVs,and provide important clues for further exploration of biological markers in the diagnosis and treatment of myeloid leukemia. |
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