Omics Analysis And Functional Study Of Exosome Proteins In Acute Myeloid Leukemia

Objective:Acute myeloid leukemia(AML)is a common type of malignant hematological disease in which myeloid hematopoietic cells clonally proliferate.Drug resistance during chemotherapy and high recurrence after treatment are the main reasons for the high recurrence and mortality of AML.At present,the lack of method of diagnosis and evaluation of AML,which is not conducive to early diagnosis and accurate evaluation of the disease.Moreover,the pathogenesis of AML is complicated and has not been clarified.Studies have shown that AML cells release exosomes.These exosomes can mediate cell-to-cell communication,acusing cross-interference between target cell environments and influencing tumor-related processes..This topic started from disease samples,collected serum samples from AML patients and healthy control samples,extracted exosomes,and screened specific and highly enriched exosomal proteins in the serum of AML patients by quantitative mass spectrometry,and as an entry point to study the molecular mechanism of AML cell-derived exosomes and disease occurrence and metastasis,verify the expression of the selected exosomal proteins in clinical samples,and conduct functional studies on the identified differential proteins.It provides a theoretical basis for the early diagnosis and prognosis evaluation of AML,as well as the research of clinical drug resistance and treatment strategies.Methods:1.Collect 80 mL of serum from patients diagnosed with AML in the outpatient or inpatient department of the Department of Hematology of the Second Affiliated Hospital of Kunming Medical University(onset period),and 100 mL of serum from healthy individuals during the same period.Extracted Serum exosomes by ultracentrifugation and identified by transmission electron microscopy and western-blot.TMT-labeled quantitative mass spectrometry analyze the protein composition of the extracted exosomes,Gene Ontology(GO)analysis and KEGG signal pathway analysis were performed for differentially expressed proteins;2.ExoQuick kit extracts serum exosomes from AML patients and healthy subjects,and and identified by Scanning electron microscopy,NAT,western-blot.The expression levels of differentially expressed proteins screened were detected by western-blot,and combined with clinical data to analyze;the western-blot was used to detect the location of the differentially expressed proteins obtained by screening in the sample;3.Culture OCI-AML3,THP-1,HL60 leukemia cell lines and HUVEC cell lines in vitro,extract AML serum exosomes by ExoQuick kit,and co-culture the exosomes with different cell lines,and use CCK8,Annexin V/PI,cell cycle experiment and tube formation experiment to study the effect of serum exosomes with the biology phenotype of cell lines;lentivirus interferes with the expression of FN in OCI-AML3 cell line,extracts the exosomes of the cell line that knockdown FN,and identifies exosomes by transmission electron microscopy,NAT,western-blot,and the extracted exosomes co-culture with cell lines.And CCK-8,Annexin V/PI,cell cycle experiments to explore the effect of exosomal FN protein on the biological functions of tumor cells.Results:1.Exosomes identification:Transmission electron microscopy showed that exosomes were round vesicles,Western-blot results showed that exosomes were positive for CD63,which was consistent with the classical identification results of exosomes.Protein quantitative mass spectrometry:A total of 146 differentially expressed proteins were detected.Protein quantitative mass spectrometry:A total of 146 differentially expressed proteins were detected.In AML serum exosomes,89 proteins were up-regulated and 57 proteins were down-regulated..bioinformatics analysis showed that the differentially expressed protein is closely related to the immune response of the body,and were involved in the growth,proliferation,differentiation,degranulation and the whole cell cycle of blood cells,as well as biological processes such as hematopoiesis and anticoagulation.Differentially expressed proteins were significantly enriched in 25 biological pathways,which were related to cell proliferation,migration and apoptosis.2.Exosomes identification:Scanning electron microscopy showed that exosomes were round vesicles,Western-blot results showed that exosomes were positive for CD63 and TSG101.NTA showed that average serum exosome concentration of 1.2x107/mL,and a peak exosome diameter of 145.5nm,which was consistent with the classical exosome identification results.The clinical sample verification results show that the differentially expressed proteins Clu,FN,and ANG expressed in the serum of AML patients are higher than those of healthy subjects(p<0.001),and the expression of ACTN4 in serum exosomes of AML patients is lower than those of healthy subjects(p<0.001),the results were consistent with mass spectrometry results.The exosomal proteins FN and ANG are differentially expressed in serum exosomes of patients with different type of AML(M1-6,M7).Clu,FN,and ANG were significantly expressed in the serum and exosomes of the same AML patient,but were not detected in the supernatant after the exosomes were extracted,indicating that Clu,FN,and ANG were almost all packaged in exosomes.indicating that Clu,FN,and ANG are almost all packaged in exosomes.3.Using AML patient serum exosomes to stimulate AML cell lines OCI-AML3,THP,HL60 cell lines and umbilical vein endothelial cells HUVEC.CCK-8 experimental results show that AML serum exosomes can promote the proliferation of AML cells(p<0.05),and the proliferation effect of exosomes at a concentration of 200ug/ml is stronger than that of 100ug/ml.It also has a proliferation effect on HUVEC cells(p<0.05);Flow cytometry results show that the apoptotic cell rate of the experimental group stimulated by serum exosomes is reduced compared with the control group(p<0.05);The results of the cycle showed that the G2 phase cells in the experimental group stimulated by serum exosomes were significantly reduced compared with the control group,and the effect of serum exosomes on AML cell lines promoted cell proliferation by shortening G2 phase.4.After interfering with FN in OCI-AML3 cell lines by lentivirus,extracted the exosomes of the cell line down-regulated for FN,and co-cultured the exosomes with OCI-AML3,THP-1,and HL60 cell lines.The CCK8 experiment results showed that Compared with the control group,the cell proliferation of the interference group was inhibited(p<0.05).The flow cytometry results showed that the cell apoptotic cell rate of the interference group was significantly increased compared with the control group.Conclusion(s):1.AML serum exosomes are rich in a large amount of protein,and the protein of exosomes derived from the serum of AML patients and healthy subjects are different.Bioinformatics suggests that differential proteins are mainly involved in immune response regulation,cell proliferation,migration and,apoptosis.2.The differentially expressed exosome proteins Clu,FN,and ANG were highly expressed in serum exosomes from AML patients,while ACTN4 was low expressed in serum exosomes from AML patients.The exosomal proteins FN and ANG were differentially expressed in different types of AML,and their expression levels were closely related to AML types.It suggests that AML serum exosomes have the potential to be biomarkers the diagnosis and typing of AML in vitro.3.Clu,FN and ANG could not be detected in the supernatant after extracted exosome,and almost all of them were packaged in exosomes;Moreover,the expression levels of FN and ANG are closely related to the progression of AML disease and cytogenetics,suggesting that AML serum exosome protein is an ideal biomarker for in vitro biopsy,and its expression level can be used together with cytogenetic stratification to evaluate the prognosis of AML patients.4.AML serum exosomes act on recipient cells,and participate in the progression of AML by promoting the proliferation of AML cells,inhibiting apoptosis,and interfering with the AML cell cycle;By promoting the growth of endothelial cells and endothelial cells forming tubes,they participate in the formation of AML blood vessels.5.The exosomal protein FN participates in the regulation of the occurrence and development of AML by promoting the proliferation of AML cells and inhibiting the apoptosis of AML cells.