Figure 4.

Overexpression of mir-218-1 Promotes Endothelium-like Network Formation

(A) mir-218-1 enhances network formation. Control and mir-218-1 stable cells were seeded on Matrigel-coated wells, and network formation was assessed 18 hr after seeding. mir-218-1 cells displayed an increased ability to align into network structures compared with control cells (n = 3 experiments). A representative image is shown. (B) Anti-miR-218-5p inhibits network formation. HTR8/SVneo cells were transiently transfected with anti-miR-218-5p, and network formation assays were performed. Compared with an NC oligo (anti-NC), cells transfected with anti-miR-218-5p showed a decreased ability to form network structures (n = 3). One representative experiment is shown. (C) mir-218-1 stimulates network formation in a co-culture of trophoblasts and HUVECs. Control (EV) or mir-218-1 stable trophoblasts (green) were seeded on Matrigel at a one-to-one ratio with HUVECs (red), and cells were allowed to co-localize and form networks for 18 hr. mir-218-1-HUVEC co-culture showed a more complex network with a larger total length (n = 3). Examination of networks formed on the left showed that, in co-culture with control (EV) trophoblasts, HUVECs formed intact branches. However, in co-culture with mir-218-1-overexpressing cells, the networks formed by HUVECs were not intact (white arrowheads). Representative images are shown. **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent SEM. Scale bars, 500 μm.