Study On The Progression Of Acute Myeloid Leukemia Via Up-regulation Of S100A4 By Bone Marrow Mesenchymal Stem Cell-derived Exosome

Background and PurposeAcute myeloid leukemia(AML)is a malignant disease of myeloid hematopoietic stem/progenitor cells.Mainly characterized by abnormal proliferation of primitive and immature myeloid cells in bone marrow and peripheral blood.In recent years,the application of combined chemotherapy has made great progress in the treatment of AML in adults,but the overall long-term survival rate remains low.More and more studies have shown that the bone marrow hematopoietic microenvironment plays an important role in keep stemness,invasion,and chemotherapy resistance of leukemia.Mesenchymal stem cell(MSC)are members of the stem cell family.They have self-replication and strong differentiation potential,and can differentiate into osteoblasts,chondrocytes,adipocytes and other cell types.As an important part of the bone marrow microenvironment,MSC can regulate hematological malignant cells not only through direct contact but also by secreting a variety of factors.Exosome(EXO)are vesicles secreted by living cells with diameter of 40-160 nm.In recent years,studies have shown that EXO are important media for the interaction between cells.The EXO has a typical lipid bimolecular layer structure containing abundant protein,nucleic acid,and other biologically active substances,which are involved in material transportation and signal exchange among cells.Previous studies have shown that BM-MSC-EXO can affect tumor proliferation,migration,invasion,stemness,and drug resistance.However,there is no study on the relationship between BM-MSC-EXO and AML cell invasion,stemness,and drug resistance.Therefore,we propose the scientific hypothesis that BM-MSC-EXO mediates the "communication" of AML cells and affects the invasion,stemness,and drug resistance of AML cells.Based on this scientific hypothesis,we intend to further demonstrate by carrying out the following studies:first,isolate and identify BM-MSC-EXO,and further verify the enhanced invasion,stem cell-like trait,and drug resistance of AML cells treated with BM-MSC-EXO in vitro.By consulting the literature,it is found that S100A4 plays an important role in the occurrence and development of AML,such as stemness maintenance,invasion,and drug resistance,and related literature shows that S100A4 can be regulated by BM-MSC-EXO.Based on this,we speculate that BM-MSC-EXO can affect the invasion and drug resistance of AML cells through S100A4,and explore the downstream target signal pathway,in order to have a new understanding of the treatment strategy of leukemia.Part Ⅰ:BM-MSC-EXO can maintain the stemness of AML cells,promote the proliferation,migration,invasion and drug resistance of AML cellsPurposeExplore the impact of BM-MSC-EXO to AML cells by treating AML cell lines with EXO or phosphate buffer saline(PBS).Methods1.Specimen collection:bone marrow samples from 10 healthy donors were collected.Mononuclear cells were isolated by density gradient centrifugation,and BM-MSC were cultured by adherent culture method to observe the morphology of MSC with an inverted microscope.The surface antigen of MSC was identified by flow cytometry.2.The cell supernatant of passage 4-6 BM-MSC was collected,and the EXO in the cell supernatant was extracted by EXO kit.The morphology of EXO was observed by a transmission electron microscope,the size and concentration of EXO were observed by nanoparticle tracking analysis,and the specific molecular markers of EXO,CD63,and TSG101,were detected by western blotting.3.Transwell assays were used to detect the migration and invasion ability of AML cells treated with BM-MSC-EXO and control group.4.Flow cytometry,colony formation,real-time quantitative PCR,and CCK-8 assays were used to detect the stemness,proliferation and drug resistance of BM-MSC-EXO to AML cells.Results1.The morphology of MSC was uniform with an inverted microscope,it was fusiform or long fusiform,the local arrangement was swirl or reticulation,and the degree of cell fusion was 80-90%.2.The round vesicular structure of EXO was observed with a transmission electron microscope.The diameter of EXO was 62.5-295.5 nm according to the tracking analysis of nanoparticles.The specific markers of EXO expression,CD63 and TSG101,were detected by western blotting.3.Transwell assays showed that the migration and invasion ability of AML cells was significantly stronger than that of the control group after BM-MSC-EXO uptake by AML cells(p<0.05).4.After AML cells took up BM-MSC-EXO,flow cytometry showed that,except for CD123 in KASUMI-1 cells,the expressions of CD34 and CD123 in the other groups were higher than those in the control group(p<0.05).The results of cell colony formation showed that the number of colonies in the EXO group was higher than that in the control group(p<0.05).The qPCR results showed that the expressions of stem-related genes OCT-4,BMI-1,KLF-4,NANOG and SOX2 in the EXO group were significantly higher than those in the control group(p<0.05),except OCT-4 and BMI-1 in KASUMI-1 cells.The results of CCK-8 showed that when AML cells were treated with different concentrations(4,8,16,32,64,128,256)μM Ara-C,the proliferation ability of AML cells decreased with the increase of drug concentration,and the viability of EXO group was significantly higher than that of the control group(p<0.05).SummaryBM-MSC-EXO can promote the proliferation,migration,and invasion of AML cells,maintain the stemness of AML cells and enhance the resistance of AML cells to Ara-C,suggesting that BM-MSC-EXO may provide a new research method and theoretical basis for the treatment of AML.Part Ⅱ:BM-MSC-EXO promotes the invasion and drug resistance of AML cells by up-regulating S100A4PurposeTo clarify the mechanism of the effect of BM-MSC-EXO on AML cells.Methods1.qPCR and WB were used to detect the expression of S100A4 in AML cells treated with BM-MSC-EXO and PBS.2.Transwell assays were used to detect the migration and invasion ability of AML cells treated with BM-MSC-EXO to AML cells after S100A4 knocking down.3.Cell colony formation and CCK-8 assays were used to detect the proliferation ability of BM-MSC-EXO-treated AML cells after S100A4 knocking down and the effect of combined Ara-C treated AML cells on drug resistance of AML cells after knocking down S100A4.4.Transcriptome sequencing was used to screen the invasion and chemoresistance related genes after S100A4 knocking down.5.Transwell assays were performed to detect the migration and invasion of AML cells after SOX10 knocking down.6.Cell colony formation and CCK-8 assay were used to detect the effect of ABCA8 knocking down on the proliferation and Ara-C resistance of AML cells.7.The expression profile of LncRNA in BM-MSC-EXO was detected by exosomal transcriptome sequencing.Results1.qPCR and WB showed that the expression of S100A4 in the BM-MSC-EXO group was significantly higher than that in the control group(p<0.05).2.The results of cell migration and invasion showed that the ability of migration and invasion of AML cells after S100A4 knocking down in the BM-MSC-EXO group was weaker than that in BM-MSC-EXO group(p<0.05).3.Cell colony formation and CCK-8 assay showed that the ability of cell proliferation after S100A4 knocking down in BM-MSC-EXO group was lower than that in BM-MSC-EXO group(p<0.05).When BM-MSC-EXO and control group were treated with 32 μM Ara-C,the cell survival rate of BM-MSC-EXO group was significantly higher than that of the control group(p<0.05).4.RNA-seq screened a total of 334 differentially expressed genes,of which 113 were up-regulated and 221 were down-regulated.At the same time,it was found that SOX5,SOX10,SOX13,ABCA8,ABCC3,ABCG4 which were invasion and drug resistance related were activated.qPCR results showed that only SOX10 and ABCA8 decreased along with the decrease of S100A4 expression(p<0.05).WB also confirmed that SOX10 and ABCA8 decreased with the down-regulation of S100A4 expression.5.Transwell assays showed that after SOX10 knocking down,the ability of migration and invasion of AML cells was weaker than that of the control group(p<0.05).6.Cell colony formation and CCK-8 analysis showed that after ABCA8 was knocked down,the proliferation ability of AML cells was lower than that of the control group(p<0.05).The proliferation ability of AML cells treated with Ara-C after ABCA8 knockdown was significantly lower than that of the control group(p<0.05).7.The results of sequencing showed that a total of 3001 LncRNA,were detected in BM-MSC-EXO.We screened the top 30 LncRNA,expressed by cluster analysis and found that only VIM-AS1,RN7SL1,RP11-323N12.5,and RP11-334E6.12 LncRNA were reported in the literature,and these four LncRNA was highly expressed in tumors,which were related to prognosis.SummaryBy regulating the S100A4-SOX10/ABCA8 axis,BM-MSC-EXO promotes the migration and invasion of AML cells,and enhances the drug resistance of AML cells to Ara-C,suggesting that the BM-MSC-EXO-S100A4 signal axis may be a new theoretical basis for AML therapy.Part Ⅲ:The expression and clinical significance of S100A4 in AMLPurposeBy using the cancer genome atlas(TCGA)public database,the effects of S100A4,a key gene found in the previous study,on the prognosis of AML patients and the possible pathway and mechanism of S100A4 are explored in depth,which provides a favorable clue and reliable basis for further study on the role of S100A4 in the development of AML.Methods1.Download the relationship between S100A4 mRNA expression and clinical features in patients with AML and normal subjects with complete survival follow-up data and clinical data from the TCGA database.Univariate Cox regression and multiple Cox regression analysis were performed by R software.The Kaplan-Meier survival curve was used to evaluate the correlation between S100A4 and prognosis.2.R software was used to analyze the differentially expressed genes in patients with high and low expression of S100A4 by gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG).3.Single sample gene set enrichment analysis(ssGSEA)was used to analyze the immune infiltration status of AML patients,and the difference of immune infiltration status of S100A4 high/low expression group was compared.Results1.Gene expression profiling interactive analysis-2(GEPIA2)showed high expression of S100A4 in AML patients and low expression in normal controls(p<0.05).Kaplan-Meier showed that the overall survival(OS)of AML patients with high S100A4 expression was worse than that of AML patients with low S100A4 expression(p=0.00078).Univariate Cox analysis showed that age(p<0.01),history of neoadjuvant therapy(p=0.033),bone marrow basophil(p=0.02),and S100A4 expression(p=0.0011)were related to the prognosis of the disease.Multivariate Cox analysis showed that age(p<0.001),bone marrow basophil(p=0.008),and S100A4 expression(p=0.0011)were independent risk factors for poor prognosis.2.Based on the median of S100A4 expression level,patients were divided into S100A4 high/low expression group.A total of 888 up-regulated and 1655 down-regulated genes were identified by differential gene analysis.GO enrichment showed that the differential genes were mainly involved in the process of cell adhesion.KEGG enrichment showed that the differential genes were mainly involved in the pathway related to cytokine regulation.3.ssGSEA results showed that the infiltration levels of activated CD4+T cells,type 17 helper T cells(Th17)and type 2 helper T cells(Th2)in the high expression group of S100A4 were significantly higher than those in the low expression group(p<0.05).SummaryThe increased expression of S100A4 may be a new target to judge the prognosis of patients with AML.The expression of S100A4 was correlated with cell adhesion,cytokine regulation,and immune cell infiltration.Conclusion1.BM-MSC-EXO can promote the proliferation,migration,and invasion of AML cells,maintain the stemness of AML cells and enhance the resistance of AML cells to Ara-C.2.By regulating the S100A4-SOX10/ABCA8 axis,BM-MSC-EXO promotes the migration and invasion of AML cells,and enhances the drug resistance of AML cells to Ara-C.3.The expression of S100A4 was related to the prognosis of patients with AML and the degree of immune cell infiltration.