miRNA Regulation of COX-2 and BCL9 in Cell Lines

Approximately 8,000 HEK293T cells per well or 12,000 MKN45 cells per well were plated into 96-well plates and were cotransfected with 50 nmol/L of miRNA mimic (or NC), 50 ng luciferase reporter (pmirGLO-COX-2-3′UTR, or pmirGLO-BCL9-3′UTR), or 50 ng luciferase reporter (pmirGLO-COX-2-3′UTR-mut, or pmirGLO-BCL9-3′UTR-mut) using HilyMax kit (DOJINDO, Japan). Previously constructed plasmid pGL3-basic-LEF/TCF promoter and plasmid pRL-SV40 were used as the irrelevant and positive controls, respectively^{45 (link)}. After 48-hour incubation, according to the instructions of dual luciferase activity detection kit (Promega, USA), the fluorescence intensity of COX-2 3′UTR or BCL9-3′UTR was measured.