Autocrine Exosomes Regulate The Proliferation And Apoptosis Of Acute Myeloid Leukemia Cells

Objective: Acute myeloid leukemia is a malignant proliferative disease derived from hematopoietic stem/progenitor cells and one of the most common hematological malignancies worldwide.There is no effective treatment for this disease.Fully understanding the pathogenesis of AML is extremely important for finding new therapeutic targets.Recent studies have confirmed that exosomes are closely related to the occurrence and development of AML.In this study,we mainly discussed the following questions: 1.Whether AML cell-derived exosomes affect parent cell proliferation and apoptosis,and the role of CXCL8-CXCR2 autocrine loop in it.2.Whether the CXCL8-CXCR2 autocrine loop affects glycolytic enzyme activity and cell proliferation through the PI3K/AKT/HIF-1α pathway.The role of CHAF1 A gene in exosome release,glycolysis and its correlation with prognosis.Methods:1.Firstly,exosomes extracted from three cell lines(HL60,THP1 and U937)were identified,and appropriate cell lines were screened by CCK8 proliferation assay for subsequent experiments.This part of the experiment was divided into the following four groups;Control group,5μg/ml,10μg/ml,and 15μg/ml exosome treatment groups.CCK8 assay was used to detect cell proliferation in different groups.Flow cytometry was used to detect cell apoptosis in different groups.Glucose consumption and lactate production were determined separately using kits.2.The TCGA database was used to analyze the expression and prognostic correlation of CXCR2 in AML,after treatment with different concentrations of CXCR2 inhibitor SB225002,it was observed whether it could reverse the proliferation-promoting effect and inhibit the apoptosis effect of AML autocrine exosomes.Then,the western blotting method was used to detect the expression of CXCL8,the ligand CXCL8 of CXCR2,in AML exosomes,and whether the use of neutralizing antibodies to block CXCL8 affected the proliferation-promoting effect and inhibit the apoptosis effect of AML autocrine exosomes.3.Different concentrations of autocrine exosomes were added to the cells,and glucose consumption and lactate production were detected separately using kits.The CXCR2 inhibitor SB225002 was used to block the exosome target,and the expression levels of glycolytic enzyme HK2,LDHA and ENO1 were detected by western blotting.To verify possible signaling pathways affecting glycolysis,the expression of PI3 K,AKT,and HIF-1α in different groups was further detected.In addition,to investigate whether the PI3 K inhibitor LY294002 can reverse the effect of autocrine exosomes on the PI3K/AKT/HIF-1α pathway.Finally,the proliferation and apoptosis of cells after the simultaneous addition of P13 K inhibitors and autocrine exosomes were observed.4.Firstly,24 genes related to exosome pathway in AML were screened out by single gene enrichment analysis,the first 5 genes were screened according to the corrected P value,and the expression levels of these five genes in AML and normal people were compared,and finally CHAF1 A was used as the gene of interest.The expression of CHAF1 A in pan-carcinoma and cell localization in various malignancies were analyzed,and the localization of CHAF1 A in U937 were verified by immunofluorescence and its expression in different stages and age groups of AML patients were verified by TCGA.In order to verify whether the screened gene CHAF1 A affects the secretion of exosomes,a CHAF1 A overexpression plasmid was constructed and transfected into U937 cells,the secretion of exosomes in the two groups was compared by western blotting and nanoflow cytometry.Finally,correlation analysis was used to predict the correlation between CHAF1 A gene and glycolytic enzyme gene,and the relationship between CHAF1 A and clinical features and prognosis was also analyzed.Results: 1.We extracted and identified exosomes from three cell lines(HL60,THP1 and U937),and the results showed that the extracts were gray round vesicles with complete envelope and clear edge.The size of the vesicles was mainly between 100-200 nm,and all of them expressed exosome surface specific membrane proteins TSG101 and CD63,but did not express Calnexin.Among them,exosomes secreted by U937 cells had the strongest ability to promote their proliferation.Therefore,U937 cells were selected as the cells used in the subsequent experiments.At 24 h and 48 h after exosome treatment,the proliferation ability and apoptosis inhibition ability of cells were enhanced with the increase of exosome dose.The cell proliferation ability and apoptosis inhibition ability were the strongest in Exo 15μg/ml group,and the weakest in Exo 5μg/ml group.2.CXCR2 is highly expressed in AML and is negatively correlated with prognosis(P<0.05).Inhibition of CXCR2 receptors can reverse he proliferation ability and apoptosis inhibition ability of AML autocrine exosomes.CXCR2 receptors are highly expressed in U937 cells and CXCL8 ligands are highly expressed in exosomes secreted by U937 cells.After blocking CXCL8 with neutralizing antibodies,the proliferative ability of cells decreased significantly,while the apoptosis level increased,that is,the proliferation ability and apoptosis inhibition ability of AML autocrine exosomes were reversed3.Increases in glucose consumption and lactate production were observed at 24 h and 48 h after exosome treatment of U937 cells,and were positively correlated with exosome dose.Exo 15 μg/ml had the highest glucose consumption and lactic acid content,and Exo 5 μg/ml group had the lowest.The expression level of glycolytic enzyme increased after exosome treatment of cells,while the level of glycolytic enzyme was significantly reduced after blocking the CXCL8-CXCR2 autocrine circuit with CXCR2 inhibitors.Exosome treatment of cells can promote the expression of PI3K/AKT/HIF-1αsignaling pathway-related proteins,while inhibition of CXCR2 receptor blocking exosomes can significantly reduce this promotion.In addition,exosomes can reverse LY294002 inhibition effect on the PI3K/AKT/HIF-1α signaling pathway.After blocking the function of PI3 K with LY294002,the proliferation ability and apoptosis inhibition ability of AML autocrine exosomes were weakened.4.AML samples with high expression of the CHAF1 A gene were enriched with exosome pathway-related gene sets.The CHAF1 A gene is highly expressed in most tumors;Among them,the expression level of CHAF1 A in AML was in the forefront.CHAF1 A is localized to the nucleus of the AML cell line U937 and is consistent with most tumor cell localization.Its expression was different in different types of AML patients,but no difference was seen in age groups.After overexpressing the CHAF1 A gene,U937 cells secrete more exosomes.The expression of CHAF1 A was positively correlated with the expression of glycolytic enzymes,and the survival of the CHAF1 A high expression group was significantly lower than that of the low expression group of CHAF1A(P<0.05).Conclusion:1.Autocrine of AML exosomes affects cell proliferation and apoptosis.2.AML exosomes produce biological effects through CXCL8-CXCR2 receptor ligands.3.Autocrine exosomes are involved in the regulation of glycolysis and affect cell proliferation by influencing the PI3K/AKT/HIF-1α pathway.4.CHAF1 A gene is involved in regulating exosome release and glycolysis,and is negatively correlated with disease prognosis.