Fig. 6

Inhibition of NSCLC cell proliferation and motility by overexpression of miR-335-5P. a-b CCK-8 assay of cell viability in NSCLC cell lines transfected with miR-335-5p mimics at 24, 48, and 72 h. c Representative images of the results of clonogenic analysis of cell proliferation in NSCLC cells. Bar charts showing the clonogenic growth of cells. d Flow cytometric analysis of the NSCLC cell lines (miR-335-5p vs. miR-NC cells). Cells were harvested at 72 h after transfection and stained with propidium iodide. The percentage of cells in each cell cycle phase is shown in the inset of each panel, in which the values represent the mean ± SD of three measurements. e A wound healing assay was performed to observe the role of miR-335-5p transfection in cells. The data showed that the speed with which the cells migrated towards the scratch was lower in cells transfected with the miR-335-5p mimics than in the control cells. f Flow cytometry assay of A549 and H1299 cells (cells transfected with miR-335-5p mimics vs. miR-NC). Cells were harvested at 72 h after transfection and stained with Annexin V/FITC and propidium iodide (PI). g Wound healing assay was performed to observe the role of cells transfected with the miR-335-5p mimics; the speed with which cells migrated towards the scratch was lower in the cells transfected with the miR-335-5p mimics than in the control cells. h Overexpression of miR-335-5p inhibits invasion and migration of NSCLC cells. The A549 and H1299 cell lines were transfected with miR-335-5p mimics and were allowed to migrate through 8-μM pore Transwells. The cells that migrated were stained and counted in at least three microscopic fields (magnification, × 100). Then, cells were treated as described before and allowed to invade through the Matrigel-coated membrane in the Transwells. The invasive cells were stained and counted under a light microscope. i The A549 and H1299 cells were treated with or without miR-30a-5p mimics for 72 h, respectively. The expression levels of p-EGFR, EGFR, p-Src, Src, p-FAK, FAK, p-AKT, AKT, p-ERK, ERK, EMT marker, snail and cyclin D1 were analyzed by western blotting. j Ectopic miR-335-5p led to lower p-EGFR expression in CPNE1-overexpressing cells than in the control cells. *P < 0.05; **P < 0.01; ***P < 0.001