Figure 3.

Depletion of miR-224/452 abolishes migration/invasion and lung metastasis

A Stable knockdown of miR-224/452 by specific miRZip anti-microRNA constructs was verified by TaqMan® MicroRNA single assays.

B, C Detection of the invasive and migratory potential of miR-224/452-depleted SK-Mel-147 cells by (B) Boyden chamber and (C) wound closure assay 24 h after seeding/scratching. Fold changes were calculated relative to SK-Mel-147.ZIP-Scr (set as 1). Representative images of three independent scratch assays for each ZIP-miR are shown.

D, E Ablation of miR-224/452 in SK-Mel-147 reverses EMT. Expression of the indicated proteins was measured by Western blot (D). Changes in E-cadherin (Cy3) and vimentin (Cy3) expression as well as cytoskeleton rearrangement (Phalloidin/TRITC) as visualized by confocal laser scanning microscopy (CLSM) (E).

F Knockdown of miR-224/452 leads to complete lack of pulmonary metastases. Representative hematoxylin and eosin sections of lungs (a–d). Visible metastatic tumor infiltrates with atypic large cell shape und nuclei with high mitotic activity in the miRZIP-Scr group (a, b). Normal lung structure without tumor infiltrates in mice (n = 5 per group) injected with miR-224/452 ablated SK-Mel-147 (c, d). Magnification (×20) of the marked area is shown (b, d).

G MiR-224/452 expression is sufficient to induce tumor invasion. The effect of miR-224/452 overexpression in shE2F1-treated SK-Mel-147 was investigated by Boyden chamber assay. Fold changes of invasion were calculated relative to SK-Mel-147.ZIP-Scr (set as 1). E2F1 expression was detected by immunoblotting. Actin served as a loading control.

Data information: Bar graphs are represented as means ± SD, n = 3 (A) or n = 5 (B, G), *P ≤ 0.05, two-sided Student's t-test.