miR-130b overexpression promoted invasion and MMP-2 activities in A549 cells. A549 cells stably overexpressing miR-130b or mock control (mock) (A), A549 cells transfected with the miR-130b mimic (130b) or negative control miRNA mimic (NC) (B), and NCI-H1755 cells transfected with the miR-130b inhibitor (130b) or negative control miRNA inhibitor (NC) (C) were subjected to invasion assays. The transfected cells were added to the upper chambers of Matrigel-coated transwell membrane inserts, and the lower chambers were filled with medium. Cells were cultured for 20 h (A549 cells), or 48 h (NCI-H1755). Fluorescence derived from invasive cells was measured. Data are means ± standard deviations of three independent experiments. *p < 0.05 and **p < 0.01 for t-tests. (D) miR-130b expression level was examined by real-time qPCR. Primary cultured NSCLC cells were seeded into Nunclon Sphera 96U-well plates and incubated for 48 h. The medium was replaced with that containing Matrigel and incubated for 72 h. The expression of miR-130b was compared in NSCLC specimens with or without vascular invasion (E), lymphatic invasion (F), and pleura cancer cell invasion (G). Relative expression normalized to U6 snRNA is shown (means ± standard deviations). *p < 0.05 for t-tests. (H) MMP-2 levels in A549 cells stably overexpressing miR-130b or control vector (mock) were analysed by real-time qPCR. The relative expression of MMP-2 normalized to GAPDH is shown (means ± standard deviations) from three independent experiments. Conditioned medium from A549 cells stably overexpressing miR-130b or control vector (mock) were subjected to gelatine-zymography (I) and western blotting analysis with anti-MMP-2 antibodies (J). Uncropped zymography and Western blot data is shown in Supplementary Fig. S9. Representative results from three independent experiments are shown. Data are means ± standard deviations from three independent experiments. *p < 0.05 and **p < 0.01 for t-tests.