Molecular Mechanism Of MYO1C-specific Enrichment In Extracellular Vesicles Of Glioma Vascular Endothelial Cell

Glioma is the most common primary intracranial brain tumor,as a highly vascularized tumor,the perivascular microenvironment of glioma is crucial for the occurrence and development of glioma.Vascular endothelial cells in the perivascular microenvironment can support glioma cell survival through intercellular communication,and extracellular vesicles(EV)are highly concerned as an important carrier of intercellular communication,and they can transport many "cargos" such as proteins,lipids,nucleic acids and other components,thereby changing the metabolism and physiological state of recipient cells.Glioma-derived human endothelial cells(GhEC)were isolated in our laboratory,and compared with normal human brain vascular endothelial cells(NhEC),it was found that only extracellular vesicles released by GhEC can promote the migration ability of glioma cells by transmitting MYO1C,while there is no difference in the expression level of MYO1C between GhEC and NhEC.It has been reported that extracellular vesicles of different origin have their own unique biogenetic pathways,which lead to the loading and transport of different bioactive molecules.The extracellular vesicle cargo loading study found that its contents did not directly reflect the matrix composition of the mother cell,and the "cargo" could only be loaded into the extracellular vesicle under certain conditions.Based on the previous work of the laboratory,we pay special attention to the molecular mechanism of specific enrichment of MYO1C in extracellular vesicles by glioma vascular endothelial cells,and the significance of this mechanism for glioma progression.Transmission electron microscope(TEM)and Nanoparticle Tracking Analysis(NTA)found no differences in particle size and appearance between two kinds of EVs,and Western blot verified the expression of typical EV markers.Considering that the specific enrichment of MYO1C in GhEC-EV may be due to the different vesicle protein loading mechanisms of GhEC and NhEC,we performed proteomic mass spectrometry analysis of GhEC and NhEC,and screened 188 differentially expressed proteins(the screening criteria were GhEC/NhEC,FC>1.5).Combined with literature reports,we screened 9 proteins that may be involved in regulating extracellular vesicle cargo loading from 188 proteins,combined with Western Blot verification,screened 4 of them:RAB31,RAB27B,FAS and SDC2.By knocking down these four proteins in GhEC,it was found that after knocking down RAB31,RAB27B or FAS,the enrichment degree of MYO1C in GhEC-EV decreased.Through protein immunoprecipitation,we found that RAB31 and FAS interacted with MYO1C,respectively.Interestingly,only overexpression of RAB31 in 293T cells promoted MYO1C enrichment in their EVs.In addition,we overexpressed MYO1C in glioma cell U87MG and glioma stem cell GSC2,respectively,and found that their invasion ability was enhanced,and the proliferation ability of GSC2 was also promoted.Then,we found that GhEC-EV after knocking down RAB31 weakened the promotion of glioma cell invasion ability.In addition,we overexpressed MYO1C in glioma cells and found that glioma migration and invasion were enhanced.The high expression of RAB31 in glioma vascular endothelial cells mediates the specific enrichment of MYO1C in extracellular vesicles of glioma vascular endothelial cells in the perivascular microenvironment of glioma,thereby supporting the occurrence and progression of glioma.This study can provide a basis for the treatment of the vascular microenvironment targeting glioma.