AML Exosomes Transfer PKM2 Protein To Reshape Glycolysis Of Human Umbilical Vein Endothelial Cells

Object:To explore how AML-derived exosomes change the glycolytic rate-limiting enzyme pyruvate kinase 2（PKM2）of HUVECs by affecting the glycolysis,and then affect its function,so as to find a new idea for AML treatment from the microenvironment.Methods:1.Extraction and identification of AML-Exo:Exosomes of AML cell line U937/HL-60/THP-1 cells were extracted by ultracentrifugation method,and the levels of ALIX,TSG101 and PKM2 proteins were detected by Western blot.The morphology of exosomes was observed under electron microscopy,and the particle size of exosomes was detected by nanoparticle tracking analysis.2.HUVECs were treated with AML-Exo for 24h,then the exosomes were stained with PKH67 dye,and the nuclei were stained with Hoechst33345 nuclear dye.The uptake of AML-Exo by HUVECs was observed under a confocal laser microscope.3.HUVECs were treated with AML-Exo for 24h,and the biological function of HUVECs was detected by the following indicators:cell viability was detected by CCK8 assay,cell migration was detected by Transwell chamber assay,and angiogenesis ability was detected by matrix gel tube test.4.The proton efflux rate of glycolysis was measured by Seahorse XFe Extracellular Flux Analyzer,and the expression level of PKM2 was detected by RT-q PCR and Western blot.5.The PKM2 level of HUVECs was overexpressed and knocked down by virus infection,and HUVECs were divided into wild type（WT）,negative control group（NC）and knockdown/overexpression group（KD/OE）to detect the activity of cell glycolysis and migratory angiogenesis,respectively.6.The PKM2 gene of U937 was knocked down and overexpressed by virus infection,and AML-Exo^WT,AML-Exo^NC,AML-Exo^shpkm2,AML-Exo^oepkm2 was extracted and acted on HUVECs.PBS was used as the control group to detect the cell glycolytic activity and migratory angiogenesis ability.Results:1.The typical double concave membrane structure of U937/HL-60/THP-1 exosomes was observed under electron microscope.The particle sizes of the three cell lines were（121.4±40.4）nm,（113.8±56.4）nm,（124.7±35.9）nm,respectively.Western blot results confirmed the expression of ALIX and TSG101 exosomal-labeled proteins in the exosomes of the above cell lines,and PKM2 protein was expressed in high abundance.2.Laser confocal analysis showed that AML-Exo was actively uptake by HUVECs in vitro,and exosomes with green fluorescence were accumulated in the cytoplasm of HUVECs.3.Compared with the PBS control group,HUVECs treated with U937-Exo,HL-60-Exo and THP-1-Exo have significantly increased vitality,migration and angiogenesis.4.The effect of AML-Exo on the vitality and biological behavior of HUVECs was related to the increased glycolysis level caused by the up-regulation of PKM2 protein content and activity in HUVECs.5.The overexpression of PKM2 in HUVECs was similar to that of AML-Exo treatment,which enhanced the viability,mobility and angiogenesis of HUVECs at increasing glycolytic level.The PKM2 knockdown in HUVECs was contrary to the effect of AML-Exo treatment,which showed reduced viability migration and angiogenesis of HUVECs at reducing glycolytic level.6.Compared with PBS group,the glycolysis level of HUVECs in U937-Exo^shpkm2group was not significantly changed,while the basic glycolysis and compensatory glycolysis levels in U937-Exo^NC group and U937-Exo^WT group increased significantly.Compared with the U937-Exo^NC group,the vitality,migration and angiogenesis of HUVECs in the U937-Exo^shpkm2 group were significantly decreased.7.Compared with U937-Exo^NC and U937-Exo^WT groups,HUVECs in U937-Exo^oepkm2group showed significantly increased glycolysis level,vitality,migration and angiogenesis.Conclusion:AML-Exo can alter glycolysis level and reshape human umbilical vein endothelial cells by transferring PKM2 protein,promoting microenvironmental angiogenesis.