VAMP2 Helps MVB Which Has Been Transported By Exocyst Complex To Bind With Membrane Protein SNAP25 To Promote The Secretion Of Exosomes And Then The Progression Of Head And Neck Cancer

Background: Squamous cell carcinoma,whichis the sixth most common cancer in the world,is the most common malignancies in head and neck cancer,with a high probability of recurrence and metastasis.Effective treatment for it remains a major problem in the medical community.A large number of studies have shown that the secretion of exosomes of tumor cells increases,and the exosomes secreted by tumor cells can promote the progression of tumor.Our previous studies have shown that exocyst complex can promote exosome production and promote the malignant biological behavior of head and neck cancer.The analysis basing on STRING database showed that exocyst complex had close interaction with SNAP25 and VAMP2.Objective: The purpose of this study was to explore the effects of SNAP25 and VAMP2 on exosome secretion and malignant biological behavior of head and neck cancer,and to study the related mechanism.Methods: The interaction between different proteins was analyzed by STRING and HNC database and verified by Co-IPassay.After knocking down of SNAP25 or VAMP2 by use of specific si RNA,the exosome concentration of the supernatant in cell culture was detected by nano-size tracking analyzer(NTA).The expression of CD63 in head and neck cancer cells was detected by western blotting.The distribution and quantity of CD63 in head and neck cancer cells were observed by immunofluorescence staining.The density of intracellular multivesicular bodies(MVBs)and intraluminal vesicles(ILVs)was observed by transmissionelectron microscopy.Co-IP and western blotting were used to compare the intensity of interaction between Sec3,Sec10,Exo7,SNAP25,or VAMP2 and CD63 in head and neck cancer cells before and after knockdown.The expressions of SNAP25 and VAMP2 in laryngeal carcinoma and adjacent tissues were compared by immunohistochemistry,and the expressions of SNAP25 and VAMP2 in head and neck cancer cells and normal nasopharyngeal epithelial cells were compared by western blotting.Western blotting,CCK-8 and Annexin V-FITC assays were used to explore the effects of SNAP25 and VAMP2 on the proliferation and apoptosis of tumor cells and their mechanisms.Results:(1)Exocyst complex had close interaction with SNAP25 and VAMP2.(2)Knockdown of SNAP25 and VAMP2 reduced exosome secretion in head and neck cancer cells.(3)After the knockdown of SNAP25 and VAMP2,the number of MVBs and ILVs in HN4 cells increased.(4)The dysfunction of exocystcomple inhibited the interaction of SNAP25 and VAMP2 with CD63,and downregulation of VAMP2 inhibited the interaction of SNAP25 with CD63.However,downregulation of SNAP25 and VAMP2 does not affect the interaction of exocyst complex with CD63,and the downregulation of SNAP25 expression did not affect the interaction between VAMP2 and CD63.(5)SNAP25 and VAMP2 were highly expressed in head and neck cancer tissues and cell lines.(6)After knockdown of SNAP25 and VAMP2,the proliferation ability of head and neck cancer cells was decreased and apoptosis level was increased;At the same time,the ability of the culture supernatant of the head and neck cancer cells to promote the proliferation and to inhibit the apoptosis of other head and neck cancer cells decreased after the knockdown of SNAP25 and VAMP2.Conclusion:(1)VAMP2 binds to MVBs which have been transported to plasma membrane under the help of exocyst complex,and then promotes the release of exosomes by interacting with membrane protein SNAP25 and inducing the fusion between plasma membrane and MVBs.(2)Both SNAP25 and VAMP2 are highly expressed in head and neck cancer,which promote the tumor progression by promoting the secretion of exosomes.This study contributes to the understanding of the mechanism of the occurrence and development of head and neck cancer,and provides a theoretical basis for the development of new treatment methods and therapeutic drugs for head and neck cancer.