Luciferase Assay for miR-99a Regulation

The luciferase experiments were performed in HEK293T cells. HEK293T cells were seeded at 1 × 10⁴ cells per well in a 96-well plate and were transfected the following day with Lipofectamine with the following molecules: the synthetic miRNA precursor miR-99a (ID: AM17100; Thermo Fisher Scientific), and negative control miR-C (ID: AM17110; Ambion), Cy3 (ID: AM17020; Thermo Fisher Scientific) and the pmirGLO plasmid (Promega Corporation, Madison, WI, USA) containing the luciferase reporter and also the Renilla gene (control) versus pmirGLO3′-UTR-E2F2 or pmirGLO3′-UTR-EMR2. The transfection efficiency was ∼95%, and luciferase activity was measured 48 h after transfection with the dual luciferase reporter assay as previously described.^{39 (link)} In each case, the miR-99a concentrations were measured by titrating the miR-99a with each pmirGLO3′-UTR mRNA construct to establish a dose–response relationship between 10 and 80 nM (data not shown). Results were conducted in triplicate in at least three independent experiments.