Effects Of Up-regulation Of Cx43 Expression In Exosomes On Glioblastoma Microenvironment Angiogenesis Under Hypoxic Conditions And Its Mechanism

Objective:In the human tumor microenvironment,the ability of tumor cells to invade and promote angiogenesis under hypoxic conditions is stronger.In addition to the recognized VEGF,exosomes play an important role in the regulation and promotion of blood vessel formation in remote area.This article will clarify that the up-regulation of Cx43 expression in exosomes plays an important role in promoting angiogenesis in glioblastoma（GBM）under hypoxic microenvironment,which enhances tumor metastasis and invasion ability.Through this study,we will have a more comprehensive understanding of the pathological mechanism of GBM,and lay a new theoretical basis and provide new drug targets for the prevention and treatment of gliomas.Methods:1.Isolation and identification of exosomes secreted by glioblastoma U251 cells under normoxic and hypoxic conditions.First,we placed U251 in a normal oxygen incubator.When the degree of cell fusion reached about 70%,the medium was changed to DMEM without serum,and then divided the cells into two groups:One group continued to cultivate under normoxic conditions,and the other group moved to hypoxic conditions.48 h later,the culture medium was collected,and the exosomes were extracted and separated by differential centrifugation,which were called normoxia-exosomes（Nor-Exo）and hypoxia-exosomes（Hypo-Exo）,respectively.The morphology of Nor-Exo and Hypo-Exo was observed by TEM.The marker proteins CD63,TSG101 of exosomes and the expression of Cx43 in exosomes were detected by Western blot.The size distribution of exosomes was observed by malvern particle size analyzer.2.To determine Hypo-Exo have some influence on the proliferation,wound healing,invasion and angiogenesis of HUVECs and confirm the role of exosomal Cx43 on them.HUVECs were cultured in complete culture medium containing 10%FBS.The cells were divided into four groups:PBS group（Cont）,Nor-Exo group,Hypo-Exo group,Hypo-Exo+^37,43 gap27 group.The exosomes of last three groups were added to HUVECs at a concentration of 100μg/ml for co-incubation.The effects of Hypo-Exo on the proliferation,wound healing,invasion of HUVECs were detected 24 and 48 hours later,and vascularization of HUVECs were detected 10hours later.3.To verify the effect of Cx43 protein in Hypo-Exo on the uptake of exosomes by HUVECs.A U251 stable transfected cell line with silenced Cx43 was constructed,and exosomes were cultured and extracted under hypoxic conditions.This group of exosomes was named Cx43-sh RNA-Hypo-Exo.The cells were divided into four groups:Nor-Exo group,Hypo-Exo group,Hypo-Exo+^37,43gap27 group,Cx43-sh RNA-Hypo-Exo group.The effect of Cx43 protein in exosomes on the uptake of exosomes by HUVECs and their uptake efficiency were counted.Four groups of exosomes were stained with Dio,and then the exosomes were incubated with HUVECs at a concentration of 100μg/ml.The uptake of exosomes by HUVECs were photographed by fluorescence microscopy in each group for 4 h,8 h,and 12hours.Results:1.Electron microscopy,Malvern particle size analysis and Western blot results showed that most of the exosomes were biconcave disc-shaped,with a diameter of about 80～130 nm,and no peaks appeared.Exosomal membrane specific molecular markers CD63 and TSG101 could be detected.The expression of Cx43 in Hypo-Exo was significantly higher than that in Nor-Exo（p<0.05）.2.The exosomes of four different groups were co-incubated with HUVECs for24 h,and there was no significant difference in the proliferation of four groups of cells;when co-incubated for 48 h,the proliferation capacity of the cells in the Hypo-Exo group was significantly enhanced compared to that of Nor-Exo group（p<0.01）.Cx43 inhibitor,^37,43gap27,significantly inhibited the proliferation of HUVECs induced by Hypo-Exo（p<0.01）;Wound healing and invasion experiments showed that Hypo-Exo group could significantly promote wound healing and invasion of HUVECs at 24 h and 48 h,and the ability to promote wound healing was stronger at 48 h（p<0.01）.Similarly,^37,43gap27 could significantly inhibit the wound healing and invasion of HUVECs induced by Hypo-Exo;We found that compared with the Nor-Exo group,the tube forming ability of the HUVECs co-incubated with Hypo-Exo was significantly enhanced（p<0.05）in vitro angiogenesis experiments,while the tube forming ability of the Hypo-Exo+^37,43Gap27 group was significantly decreased（p<0.05）.These results suggested that exosomes extracted from U251 cells cultured under hypoxia conditions could significantly promote HUVECs proliferation,wound healing,invasion and vascularization,which may be related to the increased expression of Cx43 in Hypo-Exo.3.Cell uptake experiments showed that HUVECs could take up exosomes with green fluorescence;HUVECs could take up a large amount of exosomes into cells within 12 h,and the uptake rate of Nor-Exo by HUVECs was about 45%;the uptake rate of Hypo-Exo by HUVECs was about 73%,which was significantly higher than that of Nor-Exo（p<0.01）;The Cx43-sh RNA-Hypo-Exo and Hypo-Exo+^37,43Gap27groups were absorbed by HUVECs with an uptake rate of about 41%and 37%,respectively.Compared with Hypo-Exo,their uptake rate was significantly reduced.The above results suggested that Hypo-Exo could be taken up by HUVECs in large amounts.Inhibiting Cx43 expression in Hypo-Exo or knocking down Cx43expression could significantly inhibit or reverse the higher uptake of Hypo-Exo by HUVECs（p<0.01）.Conclusions:1.The expression of Cx43 is increased in exosomes secreted by glioblastoma Under hypoxic conditions.2.Exosomes secreted by glioblastoma with hypoxia significantly enhance the proliferation,wound healing,invasion and vascularization of glioblastoma.3.The uptake rate of exosomes by human vascular endothelial cells increases significantly,which is related to the up-regulation of Cx43 expression in exosomes secreted by glioblastoma with hypoxia.The detailed mechanism needs further study in the future.