Luciferase miRNA target reporter assay

For the MMP-19 and P21 3′-UTR luciferase assays, we constructed dual-luciferase vectors (pmiR-RB-REPORT dual-luciferase vector) containing either wild-type or mutant miR-4270 or miR-423-3p binding sites in the 3′-UTRs of MMP-19 or P21, respectively. Mutant binding sites were constructed by substituting four nucleotides in the seed region. NCI-H157 cells were co-transfected with the luciferase reporter gene constructs and miR-4270, and NCI-H1299 cells were co-transfected with the luciferase reporter gene constructs and miR-423-3p. Both cells lines were also co-transected with the luciferase reporter gene constructs and negative control oligonucleotides (miR-NC). After 48 h, cells were lysed and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). A plasmid containing an expression cassette for Renilla luciferase, pRenilla, was co-transfected and used to normalize the firefly luciferase values expressed by the luciferase reporter gene constructs.