Dual glo luciferase assay system

Oligos specific to the wildtype (WT) PART1 miRNA 937-5p binding region and the mutated version of the sequence (MUT) are listed in Table S2. To make double stranded sequences for cloning, the oligos were admixed into oligo annealing buffer and heated to 90 °C for 3 min, followed by cooling to 37 °C for 15 min. The WT and MUT annealed oligos (ThermoFisher Scientific) were cloned into the multiple cloning site of the pmirGLO Dual-Luciferase miRNA Target Expression Vector (ThermoFisher Scientific, using SacI and XhoI restriction enzymes (New England Biolabs Ltd.). The confirmed vectors were co-transfected into HCC1806 cells with the pRLTK vector (Promega ThermoFisher Scientific), using TransIT-BRCA transfection reagent. 24 h later the mirVana miRNA negative control mimic or mimic-hsa-miR-937-5p (ThermoFisher Scientific) was transfected into the cells using TransIT-BRCA. The resulting firefly and renilla luciferase activity in the cells were measured 24 h later using the Dual-Glo^® Luciferase Assay System (ThermoFisher Scientifc) with a SpectraMax^® M3 Multi-Mode Microplate Reader (ThermoFisher Scientific). Binding of the mimic sequence to the luciferase reporter vector would inhibit production of luminescence.