Figure 2.

miR-218 inhibits HUVEC migration, proliferation and tube formation in vitro, and inhibits VEGFA expression. (A) Overexpression of miR-218 suppresses the recruitment of HUVECs in a co-cultured system. Cancer cells cultured in the bottom of a 24-well plate were used to recruit HUVEC seeding on the upper chamber of Transwell insets. ^****P<0.0001 compared with the LV-NC group. (B) Overexpression of miR-218 decreased the recruitment of HUVECs through the conditioned medium (CM), which was collected from the ACHN, 786O and 769P/LV-miR-218 or LV-NC cells. HUVECs were seeded on the upper chamber of the Transwell insets within 16 h. The migrated cells in six random fields per well were counted (magnification, ×200). ^****P<0.0001 compared with the LV-NC group. (C-E) miR-218 overexpression reduced the proliferation of HUVECs. HUVECs were treated with serum-free medium (SFM) or diluted CMs for 24, 48 and 72 h before the CCK-8 assay. **P<0.01 compared with the LV-NC group. (F) miR-218 overexpression suppressed the tube formation of HUVECs. HUVECs diluted in SFM or CMs were added into Matrigel-coated wells and incubated for 4 h. The representative images of tube-like structures were captured (left), and the tube number in the whole field was counted (magnification, ×100) (right). ^****P<0.0001 compared with the LV-NC group. (G) Real-time PCR was used to analyze the expression level of VEGFA in ACHN, 786O and 769P cells transfected with LV-NC or LV-miR-218. ^***P<0.001 and ^****P<0.0001 compared to the LV-NC group. (H) The VEGFA protein level was analyzed by western blot analysis. (I) The concentration of secreted VEGFA protein in the CMs was determined by ELISA. ^****P<0.0001 compared to the LV-NC group. These data are representative of three independent experiments. HUVECs, human umbilical vein endothelial cells; VEGFA, vascular endothelial growth factor A.