Dual‐luciferase reporter assay

The U87 cells were cotransfected with pcDNA3/miR‐4295 or anti‐miR‐4295 and the wild‐type or RUNX3‐3′UTR‐mut with the control in 48‐well plates. The luciferase fluorescent intensity was measured with an F‐4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) 48 hrs after transfection, and firefly luciferase activity was normalized to that of Renilla luciferase. U87 and U251 cells were cotransfected with pGL3‐pmiR‐4295 and N‐myc inhibitors or control oligonucleotides. Firefly and Renilla luciferase activities were measured using the Dual‐Luciferase Reporter Assay System (Promega cat. no. E1910) 48 hrs after transfection, and Renilla luciferase activity was normalized to firefly luciferase activity.