Luciferase Reporter Assay for miR-339-5p Binding Sites

The human MDM2 3′-UTR sequences (1580 bp, 38-1617 nt from the start of 3′-UTR) containing three putative miR-339-5p binding sites were amplified by PCR using following two primers: Forward primer 5′-ACT AGT TAT AAC CCT AGG AAT TTA GAC AAC C -3′ and Reverse primer 5′-AAG CTT ACA TCA TTA CTC CCA TCC CTT AC-3′. These two primers contain HindIII and SpeI recognization sites at the 5′ end of the primers, respectively. The PCR products were subcloned into the 3′ end of the pMIR-luciferase reporter vector (Ambion) at HindIII and SpeI sites. The mutations of three putative miR-339-5p binding sites were introduced using a Quikchange II XL Site-Directed Mutagenesis Kit (Stratagene/Agilent Technologies). Luciferase reporter assays were performed as previously described [18 (link)]. In brief, firefly pMIR-luciferase reporter vectors (100 ng) were transfected into cells in 6-well plates together with miR-339-5p mimic (100 nM) or scrambled miRNA mimic as a negative control by using Lipofectamine 2000. pRL-SV40 vectors (5 ng) that express Renilla luciferase (Promega) were co-transfected to normalize the transfection efficiency. Luciferase activities were measured at 24 h after transfection by using the Dual Luciferase Assay Kit (Promega).