Investigating miR-155 Regulation of BAG5

Bioinformatics analysis tools including miRanda (21 (link)), miRWalk (22 (link)) and TargetScan (23 (link)) were used to predict the possible target mRNA of miR-155. A luciferase reporter gene assay was conducted using the Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer's protocol. The 3′UTR segments of the BAG5 including the wild type (WT) or mutant (Mut) miR-155 binding sites were inserted into pGL3 luciferase vector (Promega Corporation). The transfected cells were seeded onto 24-well plates at a density of 1×10⁴/well. 293 cells were then co-transfected with WT-BAG5/Mut-BAG5 plasmids and miR-155 mimic/NC by Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cultured for 48 h, transfected cells were harvested and the luciferase activity was detected using a Dual Luciferase™ reporting system (Promega Corporation). The luciferase activity was normalized to that of Renilla luciferase activity.