Effects Of IL-17on VEGF-a Expression And Vasculogenic Mimicry In Mouse Melanoma Cells In Vitro

ObjectiveTo detect the influence of IL-17on the vascular endothelial growth factor (VEGF)-a expression levels of B16cells. To investigate the effect of IL-17on vasculogenic mimicry (VM) of B16cells. Our research was aming at elucidating the effect of IL-17on angiogenesis in melanoma which based on our early research.MethodsMouse B16melanoma cells were cultured in vitro. We treated the cells various concentrations of recombinant mouse IL-17, and the control group without IL-17. We detected the proliferation ability and the VEGF-a expression of B16cells by MTT assay ELISA analysis respectively when treated when treated with various concentrations of IL-17. We examined the migration and invasion ability of the B16cells by transwell migration and invision assays, the MMP-2and MMP-9activities of the cells by gelatin zymography assay, and the ability of VM formation of the cells in three-dimensional cultures when treated with various concentrations of IL-17. At last, westem-blot and immunofluorescence staining was performed to determine the expression of IL-17receptor (IL-17R) of the B16cells.Results1. The proliferation of the B16cells was not changed by MTT assay when treated with various concentrations (5、10、20、50、100ng/mL) of IL-17, compared with the control group (P>0.05). The VEGF-a levels in the supernatants of the B16cells treated with various concentrations (10、50、100ng/mL) of IL-17were higher than that in the control group by ELISA (P<0.05), which was IL-17concentration-dependent.2. The B16cells treated with various concentrations (10、50、100ng/mL) of IL-17. The cells in various concentrations of IL-17groups were found through the membrane more than that in the control group in transwell migration assay (P<0.05) and invision assay (P<0.05), both were IL-17concentration-dependent. The activity of MMP-9of the B16cells in the experimental groups was increased in a concentration-dependent manner (P<0.05) by Gelatin zymography assay.And the activity of MMP-2in lOng/mL IL-17group was found not changed more (P>0.05),but enhanced in50and100ng/mL IL-17groups (P<0.05).The ability of VM formation of the B16cells was not changed in three-dimensional cultures when treated with various concentrations of IL-17, compared with the control group (P>0.05). Western-Blot reveals that high concentration of IL-17R protein was found expressed in the B16cells (P<0.05). Immunofluorescence staining shows that IL-17R was located both on the membrane and in the cytoplasm of B16cells.Conclusion1. IL-17can induce the VEGF-a expression of B16cells. IL-17dose not directly effect the proliferation of the cells.2. In our research IL-17may not stimulate the VM formation of B16cells in vitro. But IL-17can promote the migration and invasion abilities of B16cells by increasing the activities of MMP-2and MMP-9of B16cells, which shows its potential ability to promote tumor metastasis.3. IL-17R is expressed in B16cells. IL-17may play its role in the above effects of B16cells via activating IL-17R signalings. Its exact mechanism needs to be confirmed in further research.