Up-regulation of Cx43 expression in exosomessecreted by glioblastoma under hypoxia promotes proliferation of HUVECs

OBJECTIVE To study the effect of exosomes secreted by glioblastoma under hypoxic conditions on the proliferation of HUVECs and its possible mechanism. METHODS Exosomes secreted by U251 cells under normoxia（21% O;） and hypoxia（3% O;） conditions were extracted by ultracentrifugation. The extracted exosomes were characterized by electron microscopy,NTA and Western blotting. The expression of Cx43 in normoxic and hypoxia was detected by Western blotting.Exosomes were labeled with Dio, and then incubated with HUVECs for 4, 8 and 12 h, then the uptake assay was detected under fluorescence microscope. The exosomes were co-incubated with HUVECs and their effects on proliferation were examined by CCK-8 assay.RESULTS Western blotting confirmed that the expression of the marker proteins TSG101 and CD63 was only expressed in exosomes. The results of NTA experiment showed that the exosomes ranged in size from 30 to 150 nm,and the double-layered membrane structure was observed by electron microscopy. The above results demonstrate that exosomes are successfully extracted. Dio staining results indicate that Nor-Exo, Hypo-Exo can be taken up by HUVECs. The results of CCK-8 showed that the proliferation ability of Nor-Exo and Hypo-Exo cells was higher than that of the control group（P<0.05）, and the proliferation of Hypo-Exo cells was stronger than that of Nor-Exo（P<0.05）, and the proliferation of the gap27 + hypo-exo group was significantly lower than that of the hypo-exo group（P<0.05）. Western blotting results showed that the expression of Cx43 in Hypo-Exo was significantly higher than that of Nor-Exo（P<0.01）. The results of the uptake experiments showed that the increase of Cx43 in the exosomes promote the uptake of HUVECs, while the knockdown of Cx43 in the exosomes was reduced. CONCLUSION Hypo-Exo significantly promotes proliferation of HUVECs. Its mechanism may be related to the up-regulation of Cx43.