| Background and objective Glioblastoma(GBM)is the most malignant tumor in glioma and belongs to WHO IV grade.The pathological appearance of the tumor tissue is characteristic,including proliferation of vascular endothelial cells and large number of immature blood vessels,combined with large hemorrhage and necrosis.Therefore,high vascularization and endothelial cell proliferation are also used to define the characteristics of glioblastoma.In recent years,the incidence of glioblastoma has become higher and higher,and its incidence accounts for about 70% of gliomas.Because of the limited treatment methods,a large number of patients die each year.At present,the pathogenesis of glioblastoma is still unclear,as well as the mechanisms of angiogenesis and cell invasion.So far,the therapy for GBM is still based on surgical resection,supplemented by radiotherapy and chemotherapy.Therefore,it is very urge nt to further study the pathological mechanism of glioblastoma and find more specific treatment methods.recently,a large number of studies have linked the tumorigenesis and its progress with exosomes.Exosomes are nanoscale microvesicles with a function of cell to cell communication.They are produced by the fusion of multivesicle bodys(MVBs),which is produced by the endocytic system,with the plasma membrane and released to the extracellular space.It contains a variety of substances such as proteins,micro RNAs,and m RNAs.By transferring these contents to recipient cells,exosomes play a very key role in long-distance signal transmission between cells.Studies have confirmed that tumor cells can produce great number of exosomes that are significantly higher than normal tissues.In glioblastoma,there are also many studies that suggest exosomes can play a role in promoting tumor cell proliferation,neovascularization,chemoresistance,and immune escape.If the secretion of exosomes can be controlled to achieve the purpose of affecting the development of tumors,this will have very profound significance for the treatment of glioblastoma.Although there are papers showing that hypoxia,immune response and other factors can stimulate the release of tumor exosomes,the molecular biology mechanism in tumor cel s still needs further research.VMP1 is a vesicular membrane protein whose function is related to the formation of various vesicles involved in the transport of substances,especially in the process of autophagy and endocytosis.Besides,VMP1 also plays an important role in many types of tumors such as pancreatic cancer,ovarian cancer,and colon cancer.In pancreatic cancer,the expression of VMP1 enhances cancer cell resistance to chemotherapeutic agents.O ur study found for the first time that the expression of VMP1 was significantly increased in glioblastoma.More importantly,a large amount of VMP1 expression can be detected in the isolated and purified exosomes of tumor tissues.Therefore,the purpose of our study is to explore whether the function of VMP1 is related to the formation of exosomes.Whether VMP1 regulates the exosome secretion of glioblastoma.What is the role of VMP1 regulated exosomes play in the development of GBM? By investigating these problems,we are trying to provide new targets and treatment strategies for the treatment of glioblastoma.Methods: In this subject,we firstly collected the tissue specimens of GBM and other grades of glioma patient,and determined the expression leve l of VMP1 protein by protein immunobloting and immunohistochemistry.Then,immunoelectron microscopy was applied to observe the functional localization of VMP1 in GBM cells,followed with isolation and purification of celluler organelles to verify the subcellular localization of VMP1.Secondly,stably transfected overexpressing and sh VMP1 U251 cell lines were established.The exosomes of the culture medium were purified and the effect of VMP1 on exosome secretion was explored by protein immunoblotting and particle size analysis techniques.Further,we used immunofluorescence double labeling technology in primary cultured GBM cells and U251 cells to study the colocalization of VMP1 with MVBs and exosome marker proteins.In the study of the mechanism by which VMP1 regulates the exosome secretion,we established a model of autophagy activation or repression in the VMP1 overexpressing or knockdown U251 cells.By quantifing the exosomes,we tried to confirm the hypothesis that VMP1 supresses the autophagy induced degradation of MVB.Next,we verified the effect of VMP1-regulated U251 cell-derived exosomes on the proliferation and angiogenesis of vascular endothelial cells by in vitro tube formation and rat arterial endothelial sprouting experiments.Finally,nude mouse model of intracranial tumorigenesis was established using VMP1 overexpressing or knockdown U251 cells to verify the effect of VMP1 on tumor growth and to detect the role of VMP1 in angiogenesis by immunofluorescence histochemistry..Result: Experime nt 1: The expression of VMP1 in glioblastoma tissue was significantly increased.(1)By comparing the expression level of VMP1 in GBM patient specimens and lower grade glioma tissue and normal adjacent tissue,it was found that VMP1 expression increased significantly in GBM tissue,while the difference of VMP1 between grade II and III gliomas and normal tissue is not obvious.(2)VMP1 is only expressed in GFAP-positive tumor cells in GBM tissues,whereas almost no VMP1 expression is found in astrocytes and neurons in normal brain tissues.(3)The expression of VMP1 in various glioblastoma cell lines was higher than that of normal astrocytes.Experime nt 2: VMP1 participates in the formation of exosomes and regulates the exosome secretion of glioma cells.(1)VMP1 forms large vesicles in U251 cells after transfected with GFP-VMP1.The late endosomes in tumor tissues were isolated and purified and we found that VMP1 is present in late endosomes.Immunoelectron microscopic observations further confirmed this structure are MVBs.(2)By immunofluorescence double-labeling technique,we found that VMP1 can colocalize with MVBs and exosome markers C D63,Alix and Rab27 both in primary GBM cells and in U251 cells.(3)Exosomes in culture medium of VMP1 overexpressing or knockdown U251 cells were purified for exosome proteins quantification.We found that VMP1 can promote the secretion of exosomes,and reducing its expression inhibits the biogenesis of exosomes in GBM cel s,while this process does not affect the morpholo gy of the exosomes.(4)Overexpression of VMP1 promoted the punctate formation of exosome markers,CD9 and CD81,and induced the accumulation of CD81 punctate near the plasma membrane.Knockdown of the expression of VMP1 inhibits this process.Experime nt 3: VMP1 promotes the secretion of exosomes by inhibiting the autophagic degradation pathway of MVBs.(1)After autophagy was activated in the U87 and U251 cells by starvation conditions,the change of VMP1 was opposite to that of LC3 II.Autophagy induced by rapamycin caused a decrease in VMP1 level,indicating that VMP1 is associated with autophagy,but it may play the opposite role.(2)By overexpressing VMP1 in U251 cells,the expression of LC3 II decreased significantly,while the expression of LC3 II increased after sh VMP1,which proved that VMP1 inhibits the activity of autophagy.(3)Using LC3 dual-color fluorescence to indicate autophagic flux activity,it was found that the overexpression of VMP1 significantly inhibited the formation of autolysosomes.When autophagy was activated by rapamycin,VMPl overexpressing cells also had lower autophagic flux activity,which further verified the inhibition role of VMP1 in autophagy.(4)Rapamycin-activated autophagy caused a decrease in exosome secretion of U251 ce lls,while cells overexpressing VMP1 inhibited the exosome reduction caused by activation of autophagy.At the same time,sh VMP1 inhibited the increase of exosome secretion caused by blocking autophagy activity.Experime nt 4: Knockdown of VMP1 inhibits the exosome secretion of GBM,which suppresses the angiogenesis of endothelial cells and further inhibits the growth of GBM.(1)U251 cell-derived VMP1 positive exosomes could enter HUVECs under co-culture conditions and promoted the proliferation and tube formation ability of HUVECs.(2)It was found that exosomes secreted by VMP1 over-expressing U251 cells promoted vascularization of endothelial cel s,by the rat artery endothelial sprouting assay.(3)In vivo tumorigenesis experiments in nude mice showed t hat cells over-expressing VMP1 formed tumors with bigger size.But the volume of tumors formed by sh VMP1 cells was significantly smaller than the control group,and the experimental animals also had a higher survival rate.(4)CD31,CD34,and SMa were sta ined in the tumor tissues of the in vivo tumorigenesis experiment.The results showed that the number of blood vessels formed by sh VMP1 cells was significantly less than that of the control group,and tumors formed by overexpressing VMP1 cells showed richer angiogenesis.Conclusion In this study,by studying the role of VMP1 in glioblastoma,we obtained the following conclusions:(1)The expression of VMP1 in GBM cells was significantly increased,and was significantly higher than other grades of glioma.(2)VMP1 is a constituent of exosomes and can regulate the biogenesis of exosomes.(3)VMP1 inhibits the autophagic activity of GBM cells,and promotes the biogenesis of exosomes by inhibiting the degradation of MVBs.(4)VMP1 promote vascular endothelial cell proliferation and angiogenesis by promoting the secretion of exosomes by GBM cells.(5)Decreased VMP1 expression significantly inhibits tumor progression and improves patient survival. |
PDF Full Text Request