2.4 Luciferase reporter assay
The Segment of 3’-UTR encompassing the miR-370-3p binding sites was amplified from the Sirt3 gene as the wild type (WT), correspondingly, a mutant 3’-UTR sequence was obtained using a mature technology, site-specific in vitro mutation. WT or mutant 3’-UTR was subsequently inserted downstream of the luciferase gene in the pmirGLO-control vector (Promega, USA). The NCI-H295R cells were seeded onto 96 well plates. After culturing for 24 h, co-transfection of miR-370-3p vector and negative control (NC) along with a pmirGLO-control vector carrying WT or mutant 3’-UTR of Sirt3 into cells were performed using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. Then luminescence signal was recorded using the Dual-Luciferase Assay kit (Promega, USA) as indicated in the instructions. All assays were performed in triplicate in a single experiment and repeated for six times.