Inhibition Of SNARE Complex Expression Activates The Rab11a-myosin9 Pathway Causing Diminished Exosome Secretion And Release Of MVB-Like EV Clusters In Head And Neck Cancer

Background:Head and neck cancer is a broad group of cancers that occur in the head and neck.Its complex cancer types and serious complications directly threaten people’s quality of life and health,and its increasing prevalence and mortality have made its pathogenesis and treatment a focus of research.The study of exosomes released by tumor cells has become an important direction of research in the diagnosis and treatment of the disease in recent years,and the role of exosomes in regulating the function of cancer cells in tumor cells is gradually being understood.It has been reported that exosome release is significantly higher in tumor cells than in normal cells,and this is also true in head and neck cancer cells,making the regulation and control of exosomes an effective therapeutic tool for cancer treatment.In the study of exosomes,it has been found that tumor exosomes can activate many intracellular signaling pathways to regulate tumor cells and promote malignant biological behavior,while inhibition of tumor exosomes partially inhibits the development of tumors.In our preliminary studies of head and neck cancer,we found that inhibition of exosome secretion in head and neck cancer cells resulted in enhanced apoptosis and reduced proliferation.To investigate the mechanism by which head and neck cancer cells resist damage due to inhibition of exosome secretion.Methods: The expression of MVB co-localized with autophagy after inhibition of SNARE complex was detected by immunofluorescence;observation of the size and concentration of vesicles encapsulating exosomes by transmission electron microscopy and nanoparticle tracking analysis.;the expression of vesicular CD63/VAMP2 protein,the transfection efficiency of transfected siRNA and the expression level of autophagy-related proteins were detected by cytochemical staining and western blotting;the Annexin V-FITC Apoptosis Detection Kit was used to detect the phosphatidylserine ectoplasm of cell membranes;protein spectrum was used to detect the proteins related to the interaction of SANAP25,VAMP2 and Rab11 a.Cell scratches were used to detect the effects of head and neck cancer cells HN4 and CNE2 cells inhibiting the release of MVB-like EV aggregates on cell migration.Transwell was used to detect the effects of head and neck cancer cells inhibiting the release of MVB-like EV aggregates on cell invasion.CCK-8 was used to detect the effect of HN4 and CNE2 cells inhibiting the release of MVB-like EV aggregates on cell proliferation,and flow cytometry was used to detect the effect of head and neck cancer cells inhibiting the release of MVB-like EV aggregates on apoptosis.Results:(1)The intracellular autophagic pathway was not activated after knockdown of synaptosome associated protein 25(SNAP25),a component of the SNARE complex,and VAMP2 resulted in the inhibition of exosome release from HN4 in head and neck cancer cells.(2)After the release of HN4 exosomes from head and neck cancer cells was inhibited,cellular release of large vesicles was confirmed to belong to MVB-like EV aggregates.(3)Protein profiling identified the protein myoglobulin(Myosin9)associated with Rab11 a interaction;(4)After the release of HN4 exosomes from head and neck cancer cells was inhibited,the Rab11a-Myosin9 complex mediated the exocytosis of phosphatidylserine in the cell membrane to induce the release of MVB-like EV aggregates.(5)Inhibition of HN4 exosome release from head and neck cancer cells knocked down the expression of Rab11 a and Myosin9 protein levels in head and neck cancer cells,preventing the release of MVB-like EV aggregates,which accumulate intracellularly and lead to reduced migration,invasion,and proliferation and enhanced apoptosis,and PLSCR1 protein can play a synergistic role in phosphatidylserine exocytosis.Conclusion: SNARE complex SNAP25 and VAMP2 knockdown in head and neck cancer cells leads to inhibition of exosome release.Instead of fusion and degradation with lysosomes,the accumulated MVB in the cell is secreted into the extracellular cell through MVB-like EV aggregates.The secretion process is through the Rab11a-Myosin9 complex carrying phosphatidylserine to release the MVB-like aggregates encapsulated in the intracavitary vesicles to the outside of the cell,thus alleviating cell damage caused by intracellular MVB aggregation and maintaining cell homeostasis.