Luciferase reporter assay

The fragment of wild type or mutant SOCS3 3’untranslated region (3’UTR) containing predicted binding site of miR-455-5p was synthesized and subcloned into pmirGLO vectors (Promega, Madison, WI, USA) to establish SOCS3-Wt/Mut. Phusion Site-Directed Mutagenesis Kits (Thermo Fisher Scientific) were used to mutate the predicted binding site. The above vectors were then co-transfected with miR-455-5p mimics or miR-NC into ESCs using Lipofectamine 2000 (Invitrogen). The luciferase activity was measured with a dual luciferase® reporter assay system (Promega) after 48 h of co-transfection [³⁷].