FIGURE 7.

Twist, a transcription factor of miR-10b, promotes angiogenesis and is regulated by thrombin and heparin. A, HMEC-1 cells were transfected without (Blank) or with vector control (Mock) or pcmv-Twist (TWIST) followed by Twist mRNA (top panel) or protein detection (bottom panel). Normalized band densities are shown above the figures. B, HMEC-1 cells were treated as in A. The expression of miR-10b was measured by RT-PCR. Normalized band densities are shown above the figures. C, cell migration was induced by overexpression of Twist in vitro. HMEC-1 cells were transfected without (Blank) or with Mock or Twist as described in A followed by wound healing assay. The widths of the scratches were quantified. D, cell tube formation was induced by overexpression of Twist in vitro. HMEC-1 cells (3 × 10⁴) that were transfected without or with a control vector (Mock) or pcmv-Twist (TWIST) were seeded onto Matrigel-coated plates followed by tube formation assay. The statistical analysis of tube length is shown on the right. E and F, HMEC-1 cells were treated with 100 ng/ml of heparin (H100), no factors (control), 2 units/ml of thrombin (T2U) or heparin and thrombin (H100 + T2U) for 24 h, followed by real-time PCR analysis and Western blot of Twist expression. Heparin treatment inhibited Twist expression, whereas thrombin treatment increased Twist expression at mRNA level (E) and protein level (F). Band intensities normalized to β-actin for each of the duplicate determinations are shown. **, p < 0.01.