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.