miRNA-target Interaction Validation

The procedure used has been described in detail in our earlier study22 (link). The human FOXO4 wild type or mutated 3′-UTR sequence containing the miR-150 binding site was cloned into the psiCHECK-2 vector. The primer sequences are listed in Supplementary Table 1. Cells were seeded into 24-well plates and co-transfected with miR-150 or control vector and wild-type or mutated foxo4 3′-UTR using Lipofectamine 2000. Both firefly and Renilla luciferase activities were measured after the 48 h transfection using the Dual-Luciferase Reporter 1000 Assay System (Promega, USA) and were detected by the GloMax TM 20/20 detection system (E5331, Promega, USA) according to the manufacturer’s instructions. Luciferase activities were normalized to Renilla luciferase.