Luciferase reporter assay

The PPARG 3′-UTR containing the miR-128 target sequence (position 82–88) and mutant sequences (a single-base mutant in the 3′UTR) were chemically synthesized (GenScript, Nanjing, China) and cloned into the PGL3 control vector (Promega Corporation, Madison, WI, USA) downstream of the luciferase gene using the XbaI site. The resulting plasmids were designated PPARG 3′-UTR-luci-wild type (WT) and PPARG 3′-UTR-luci-mutant (MUT). HEK293 cells were transfected with either PPARG 3′-UTR-luci-WT or PPARG 3′-UTR-luci-MUT in 24-well plates using Lipofectamine 2000 transfection reagent according to the manufacturer's instructions, and co-transfected with miR-128 mimic, antagomir-128 or the same concentration of negative control. Each well was also co-transfected with the pRL-TK plasmid (Promega Corporation) to determine the transfection efficiency. Subsequently, firefly and Renilla luciferase activity levels were measured in cells that were harvested 24 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega Corporation). Each transfection was performed in triplicate.