Fig. 5
From: miR-301a promotes lung tumorigenesis by suppressing Runx3
The effects of miR-301a and Runx3 on cell proliferation and migration in A549 and H1299 cells. qPCR analyses of miR-301a (a) and its target gene RUNX3 (b) expression in NSCLC cell lines (PC9, H1975, A549, and H1299) compared to a noncancerous bronchial cell line (16HBE). Total RNAs were extracted from the cell lines and used for reverse transcription and real-time PCR. (c) Western blotting analyses of RUNX3 expression in A549 and H1299 cells transfected with anti-negative-control (anti-NC) or LNA-anti-miR-301a (anti-miR-301a). (d) Quantification of RUNX3 expression (c) using Image J with β-actin as a reference. (e) Cell viability of A549 cells transfected with only the transfection reagent, RNAiMAX (None), anti-negative control (anti-NC, 10 pmol), or LNA-anti-miR-301a (anti-miR-301a, 10 pmol). A549 cells were incubated for 48 h and 72 h after transfection, and cell viability was determined by counting cells labeled with CCK-8. (f) Cell viability of H1299 cells. H1299 cells were transfected and measured as in (e). (g) Western blotting shows the efficacy of siRNA knockdown in A549 cells co-transfected with LNA-anti-miR-301a and siRNA-RUNX3 or siRNA-control. (h) A549 cells were transfected with anti-NC & siRNA control (siRNA-Pool), anti-miR-301a & siRNA-Pool, or anti-miR-301a & siRNA-RUNX3. At 48 h after transfection, A549 viability was determined as in (e). (i) Migration of A549 cells was determined with a transwell migration assay. (j) The percentage of migratory cells in (i). (k) Western blotting analyses of β-catenin expression in A549 and H1299 cells transfected with Anti-NC or Anti-miR-301a. (l) Western blotting analyses of fibronectin, vimentin, N-cadherin, E-cadherin, MMP9 and MMP2 expression in A549 and H1299 cells transfected with Anti-NC or Anti-miR-301a. Values represented the mean ± s.d. of three independent experiments. **P < 0.01 or *P < 0.05 indicates a significant difference between the indicated groups (two-tailed, unpaired Student’s t-test in d and one-way analysis of variance (ANOVA) in others). NS, not significant