Role of Legumain on Tumour Invasion and Human Endothelial Cells Tube Formation

Objective To investigate the role of Legumain, an asparaginyl endopeptidase overexpressed by tumor tissue, in tumor invasive growth and angiogenesis. Method Western-blot was use to study the expression of Legumain in cells under normal or hypoxic condition. The migration of MDA MB231 cells were detected by Transwell invasion assay. The tube formation by HUVEC was detected by tube formation test. The binding of Legumain to integrin was analyzed by immunohistochemical method. Result The expression of Legumain in both MDA MB231 and HUVEC were increased under hypoxia. Using fluorescence double staining method,hypoxic MDA MB231 cells were found to have an increased in size of the cell body. Legumain was found, as shown in green dots, inside of the cells. Some of the Lugumain were found on the cell surfaces and overlapping with integrin (red)on the margin of the cell surfaces ( yellow). The formation of endothelial tube was reduced by Legumain inhibitor (an asparaginly endopepidase inhibitor, AEPI). Compared with the control group (21.3±1.73),the numbers of tube were (13.2±0.85),( 5.33±0.35)and ( 0.57±0.23), (P0.001). Results of cell migration assay showed that legumain stimulated the migration of MDA MB231 cells ( 170.90± 59.27 and 321.22±22.39),when compared with the control group (67.56± 7.58,P0.05). Conclusion Overexpression of Legumain was found to promote the migration of hypoxic MDA MB31 cells and tube formation by HUVECs. The effect might be due to the binding of legumain to integrinβ1.