Overexpression of miR-138 inhibits cell proliferation and migration in vitro. (A) GBM cell proliferation with miR-138 overexpression. Patient-derived primary GBM cells (GBM12, 28 and 43) were transfected with a series of concentration of miR-138 mimics or negative control (miR-Ctrl). After 4 days of transfection, viable cells were measured by CellTiter-Glo Luminescent Cell Viability Assay. (B) Cell proliferation analysis by fluorescence live cell imaging every four hours on GBM12-RFP cells after transfection of 25 nM miR-138 or miR-Ctrl. (C) Representative fluorescence images of GBM12-RFP cells at 96 h after transfection of 25 nM miR-138 or miR-Ctrl. (D) Apoptosis analysis on GBM cells (GBM12, 28 and 43) after transfection of 25 nM miR-138 or miR-Ctrl followed by Annexin V-PI double staining for flow cytometry. Percentage of apoptotic cell populations were indicated in red-dotted box on each representative cytograms (n = 3). (E) Cell migration assay in Trans-well chamber plates. GBM cells transfected with 25 nM miR-138 or miR-Ctrl were cultured in 8 µm pore size Boyden chamber inserted into 24-well plates. The top chamber was removed 4 days later, and the migrated cells in the bottom chamber were visualized by staining with 0.5% crystal violet. Each images represent stained cells from each well obtained by bright field microscope. (F) Wound healing assay to evaluate the change of physical gap-closure by miR-138 overexpression. At Day 0, monolayer of GBM cells were scratched vertically followed by transfection of 25 nM miR-138 or miR-Ctrl. Representative cell images were taken at 4 days later by bright field microscope. All error bars indicates standard deviations (n = 3), and the p-values were determined by two-tailed student t-test. ***p < 0.001.