The Function And Mechanism Of SYT7 In Exosome Secretion And Promoting Angiogenesis And Metastasis In NSCLC

BackgroundLung cancer is the leading cause of cancer-related mortality,and non-small cell lung cancer(NSCLC)accounts for?85%of all lung cancers.Despite advances in a combination of surgery,radiation,chemotherapy,bio-targeted therapy,and immunotherapy,the 5-year overall survival rate for patients with lung cancer is still only 19%.The key reason is that there is heterogeneity in NSCLC and the exact pathogenesis is unclear.It is of great clinical significance to explore the molecular mechanism of NSCLC and to develop new specific targeted drugs.Angiogenesis is the anatomical basis and prerequisite of tumor blood supply.It is necessary for tumor growth,metastasis and diffusion.In the process of angiogenesis,angiogenic factors and the signal transduction between tumor cells and vascular endothelial cells are crucial.At present,several angiogenesis targeting drugs have been applied in the clinical treatment of NSCLC,and certain progress has been made.Tumor patients benefit from anti-angiogenic therapy targeting VEGF and its receptors.However,angiogenesis targeted therapy brings the risk of drug toxicity and metastasis to patients.The emergence of new ideas,new targets and new methods are urgently needed for anti-angiogenesis in tumor patients.Exosomes are small vesicles secreted through the fusion of multivesicular bodies(MVBs)with cell membranes.Exosomes are approximately 30-150 nm in diameter and are surrounded by a lipid bilayer.Cells secrete exosomes into the extracellular environment through exocytosis,which can mediate intercellular material and information transfer through a variety of pathways.This is a new type of communication pathway between cells.Soluble N-ethyl-maleimide sensitive factor attachment protein receptor(SNARE)complex is the core mechanism of MVBs fusion to cell membrane.SNARE plays an important role in the regulation of exosome release.SNAREs are generally classified into two types:vesicle-SNAREs(vesicle-SNAREs,v-SNAREs)located on the membrane of the vesicle,and target-SNAREs(target-SNAREs,t-SNAREs;Syntaxin-1a/3/4,etc.).The v-SNAREs and t-SNAREs between the membranes pair to form an a helix bundle consisting of four chains(called the SNARE complex).Then the fusion of lipid membrane and the release of material in the vesicle occur.Studies have shown that exosomes derived from tumor cells can promote the proliferation and invasion of tumor cells and promote tumor angiogenesis.Moreover,exosomes can be used as drug delivery vehicles for targeted delivery of chemotherapeutic drugs or gene therapy substances.Therefore,studies of the formation and transport mechanisms of tumor exosomes can provide us with an in-depth understanding of the biological behavior patterns of tumors.This is helpful to establish a new idea of exosome-based tumor anti-angiogenesis and metastasis therapy.Our previous studies confirmed that Synaptotagmin 7(SYT7)can promote metastasis of NSCLC in vivo and in vitro.Recent studies have shown that SYT7 is also highly expressed in gastric cancer,macrophages and osteosarcoma cells,and plays a role in promoting cell proliferation and migration.However,the mechanism of SYT7 in regulation of tumor metastasis remains unclear.SYT7 belongs to the synaptic binding protein(SYT)family.Previous studies of SYT7 mainly focused on the exocytosis process of lysosomes,neurons and pancreatic beta cells,etc.The role of SYT7 in exosome secretion remains unclear.SYT7 expressed on liposomal membranes of various cells and induces Ca2+-triggered lysosomal exocytosis and plasma membrane repair through C2 domain.SYT7 also interacts with Syntaxin-1a(STX1 A)and Syntaxin-3(STX3)of the SNARE complex in a Ca2+-dependent manner through the C2 domain,and induce exocytosis of insulin in human pancreatic beta cells.SYT1 is one of the most studied members of the SYT family.The interaction between its C2 domain,anionic phospholipids and the SNARE complex plays a key role in membrane fusion.It can promote the pairing of SNARE complex between vesicle membrane and target membrane,and regulate membrane fusion and exocytosis.Based on these,we assume that the SYT7 and SYT1 may have similar functions in regulating exocytosis in the membrane fusion mechanism triggered by SYTs/SNARE.Part ? The expression pattern and clinical significance of SYT7 in NSCLCObjectiveTo investigate the expression pattern and clinical significance of SYT7 in NSCLC.Methods1.Explore the expression pattern of SYT7 in tumors,and evaluate the diagnostic ability of SYT7 in tumors using the TCGA pan-cancer data.2.The expression pattern of SYT7 in NSCLC in TCGA database was analyzed.And the prognostic significance of SYT7 in NSCLC was analyzed in Kaplan-Meier(K-M)plotter database.3.The cancer and paracancerous lung tissue samples of clinical NSCLC patients were collected.Immunohistochemical technique(IHC)was used to detect the expression level of SYT7 protein in NSCLC tissues.And the correlation between SYT7 protein and clinicopathological characteristics of NSCLC patients was analyzed.4.Univariate and multivariate Cox regression analyses and K-M survival curves were performed to evaluate the relationship between SYT7 and overall survival(OS)in clinical NSCLC patients.Results1.Analysis of pan-cancer data showed that SYT7 was highly expressed in 18 tumor types.2.In both LUAD and LUSC data sets of TCGA,the expression level of SYT7 was higher than that of the control group.In the K-M plotter database,the high expression of SYT7 was associated with poor OS in patients.3.In NSCLC cancer tissues(N=154)and paracancerous tissues(N=136),the expression level of SYT7 protein in both LUAD and LUSC cancer tissues was higher than that in paracancerous tissues.In LUAD patients(n=86),high expression of SYT7 was significantly associated with gender,degree of differentiation,tumor size,and TNM stage.4.Univariate Cox regression analysis and K-M survival curve showed that high SYT7 expression predicted poor OS in LUAD patients.Multivariate Cox regression analysis showed that high SYT7 expression(HR 1.823,95%CI 1.009-3.292,P=0.046)was an independent prognostic factor for OS in LUAD patients.Advanced TNM stage(HR 3.202,95%CI 1.735-5.909,P<0.001)was also an independent prognostic marker for patient survival.In LUSC patients(n=68),univariate Cox regression analysis showed that high SYT7 expression was a prognostic factor for poor OS.K-M survival curve confirmed that SYT7 protein expression was significantly correlated with OS in LUSC patients,and OS gradually decreased with the increase of SYT7 level.Multivariate Cox regression analysis showed that high SYT7 expression(HR 2.329,95%CI 1.132-4.788,P=0.022)was also an independent prognostic factor for OS in LUSC patients.ConclusionsSYT7 is highly expressed in several tumor types and possess good diagnostic value.SYT7 is highly expressed in NSCLC and can be used as a prognostic biomarker.Part ? SYT7 regulates the secretion of exosomes and promotes the angiogenesis and metastasis through exosomesObjectiveTo clarify the role of SYT7 in regulating exosome secretion,to study the role of SYT7 in NSCLC cell angiogenesis through exosome pathway,and to explore the role of SYT7 in tumor growth,angiogenesis and metastasis through exosomes in vivo.Methods1.The expression of SYT7 protein in extracellular vesicles(EVs)was analyzed by searching the exosome database Vesiclepedia.2.SYT7 overexpression and control lentivirus were constructed.SYT7 overexpression(SYT7-OE)A549 and SYT7-OE H1299 cells and control cells(Ctrl-OE)were established.Exosomes from cell supernatant were extracted by ultracentrifugation method.And then exosomes were identified by transmission electron microscopy(TEM),nanometer particle size analysis(NTA)and protein markers.3.Exosomes isolated from the supernatant of SYT7-OE A549,SYT7-OE H1299 and control cells were co-cultured with human umbilical vein endothelial cells(HUVEC).The exosomes were labeled with PKH26,and the exosomes phagocytosis test was observed under fluorescence microscope after 12 hours of co-culture.4.The migration ability of HUVEC was detected by scratch test and Transwell migration test.The absorbance(OD)at 48 hours of co-culture in each HUVEC group was determined by CCK-8 assay to evaluate the proliferation ability.Tube formation assay was performed to assess the ability of cells to form vascular rings.5.used to The subcutaneous tumor-forming model in nude mice constructed by subcutaneous injection of A549 cells.When the subcutaneous tumor grew to a certain extent,the cell supernatant exosomes of SYT7-OE A549 and Ctrl-OE group were injected into the subcutaneous tumor of the two groups,three times a week,respectively.Subcutaneous volume was measured twice a week.Nude mice were sacrificed at 28 days to measure tumor volume and weight.The subcutaneous tumor tissues were embedded in paraffin,prepared sections and stained with HE.Expression of Ki-67,N-cadherin,Vimentin,E-cadherin and CD31 were assessed by IHC staining.In addition,A549 cells were injected into the tail vein to construct the lung metastasis model in nude mice.The exosomes of the above two groups were injected into the tail vein at about 20 days later,exosomes were injected 3 times a week,and the number of pulmonary nodules was assessed 1 month later.Results1.SYT7 protein was identified in EVS of colorectal cancer cells,renal cancer cells,lung cancer cells and several other cells.2.The exosomes of A549 and H1299 cells in the SYT7-OE group and the control group could be seen with a clear bilayer structure at about 100 nm under the TEM.NTA results showed that the maximum diameter of exosomes secreted by cells in both the SYT7-OE group and the control group was about 100 nm.And the original particle concentration of supernatant in the SYT7-OE group was higher than that in the control group.Moreover,the expression of CD9,CD63 and CD81 in exosomes of SYT7-OE group was higher than that of control group.3.After exosomes of NSCLC co-cultured with HUVEC,the intracellular phagocytosis of exosomes by HUVEC was observed under fluorescence microscope.4.After the phagocytes of exosomes that secreted by SYT7-OE cells,the migration ability,proliferation ability and tube formation ability of HUVEC were enhanced compared with cells in control group.5.The weight and volume of subcutaneous tumors in the SYT7-OE A549 exosomes injection group were higher than those in the control group.IHC staining results also indicated that the expression levels of Ki-67,N-cadherin and Vimentin in the tumor tissues were higher than those in the control group,while the expression of E-cadherin was lower than that in the control group.Moreover,CD31 staining showed that more vascular rings were formed than in the control group.In the model of lung metastasis through tail vein,more metastases were formed in the lungs of nude mice injected with SYT7-OE A549 exosomes through tail vein.ConclusionsSYT7 promotes NSCLC cell secretion of exosomes,and in addition,SYT7 promotes NSCLC angiogenesis and metastasis through exosomes.Part ? The molecular mechanism of SYT7 in regulating exosome secretion and promoting angiogenesisObjectiveTo investigate the mechanism by which SYT7 promotes NSCLC angiogenesis through the exosome pathway,and to explore the molecular mechanism by which SYT7 regulates exosome secretion.Methods1.The NSCLC data of TCGA were used to determine the molecules positively correlated with the expression of SYT7 in NSCLC,and the down-regulated differentially expressed molecules in the shSYT7 chip results were intersected with the above molecules.K-M Plotter database was used for survival analysis to screen differentially expressed molecules with prognostic significance.2.RT-qPCR assay was used to verify the selected target molecules in NSCLC cells.3.The expression of CEP55 protein in extracellular vesicles was explored by searching the Exocarta database.WB was used to detect the expression levels of CEP55 and mTOR signaling pathway proteins in SYT7-OE cells.The co-localization of SYT7 and CEP55 in A549 cells was observed by immunofluorescence assay.The protein levels of SYT7 and CEP55 in the exosomes of SYT7-OE and control cells were detected,and the changes of protein expression in HUVEC after co-culture of exosomes were analyzed.4.CEP55 was knocked down by lentivirus transfection in SYT7-OE cells,and the knockdown efficiency was verified by RT-qPCR and WB.Exosomes were extracted and co-cultured in HUVEC,and the level of CEP55 protein in exosomes and HUVEC was detected.The effects of CEP55 knockdown on HUVEC migration,proliferation and tube formation were evaluated by scratch test,Transwell migration test,CCK-8 test and tube formation test.5.Transwell migration assay and Transwell invasion assay were used to evaluate the changes of invasion and migration abilities after CEP55 knockdown in SYT7-OE cells.The expression level of mTOR signaling pathway protein after CEP55 knockdown was detected by WB.6.STX1A and STX3 were knocked down by lentivirus transfection in A549 SYT7-OE and 1299 SYT7-OE cells,respectively.RT-qPCR and WB were used to verify the cell knockdown efficiency.Exosomes were extracted from cell supernatant by ultracentrifugation method,and the expression of exosome markers CD9,CD63 and CD81 were evaluated.The expression levels of CEP55 and mTOR signaling pathway proteins levels after STX1A or STX3 knockdown in cells was detected by WB.Results1.We screened 33 downstream genes of SYT7.Further survival analysis showed that 29 of these molecules had prognostic significance in NSCLC,and their high expression predicted a worse prognosis in NSCLC patients.2.Twelve of these molecules were selected by literature search,and the results of RT-qPCR showed that the mRNA expression levels of them in SYT7-OE A549 cells were higher than those in the control group.3.The search of target molecules in Exocarta revealed that CEP5 5 was identified in exosomes secreted by various tumor cells.The protein levels of CEP55 and p-Akt and p-mTOR were up-regulated in SYT7-OE cells.SYT7 and CEP55 were co-localized in the cytoplasm of A549 cells.SYT7 and CEP55 proteins were present in NSCLC exosomes and were transferred into HUVEC cells through exosomes.4.After CEP55 knockdown in NSCLC cells,the ability of SYT7 to promote HUVEC migration,proliferation and tube formation through the exosome pathway was inhibited.5.After CEP55 knockdown in SYT7-OE A549 cells and SYT7-OE H1299 cells,the invasion and migration abilities of cells decreased,and the protein levels of p-Akt and p-mTOR in cells were also down-regulated.6.Knockdown of STX1A or STX3 reduced the amount of exosomes secreted by NSCLC cells.The protein expression level of CEP55 in the NSCLC cells increased,and the protein expression levels of p-Akt and p-mTOR were also up-regulated.ConclusionsSYT7 regulates the mTOR signaling pathway through CEP55,and thereby promoting the invasion and metastasis of NSCLC.SYT7 promotes angiogenesis by delivering CEP55 protein to endothelial cells via exosomes.Knockdown of STX1A or STX3 can inhibit the ability of SYT7 in promoting exosome secretion.