MiR-335 inhibition induces apoptosis and suppresses invasion of human astrocytoma U87-MG cells. (A) Time-dependent effect of antagomir-335 on cell viability. U87-MG cells were transfected with 100 nM antagomir-335 for the indicated times. (B) Effect of antagomir-335 transfection on colony formation. (C) Cell invasiveness was detected by transwell invasion assay. U87-MG cells were transfected with 100 nM antagomir-335 or antagomir-NC. Graph is the representative of three independent experiments. (D) Phase-contrast imaging and (E) Hoechest 33258 staining of U87-MG cells. (F) Apoptotic cells were detected by TUNEL assay. (G) Effect of antagomir-335 on caspase3/7 activity. U87-MG cells were transfected with 100 nM antagomir-335 or antagomir-NC for 72 h. (H) Effect of antagomir-335 on expression of DAAM1 (left-panel) and phosphorylation of MLC (right-panel) in U87-MG cells. Cells were transfected with 100 nM antagomir-335 or antagomir-NC for 48 h. Data represent the means ± SD. Statistical differences compared with the controls are given as *, P < 0.05; **, p < 0.01; ***, P < 0.001. Original magnification in (C, D, F), 200 ×; Original magnification in (E), 400 ×.