Pmirglo based luciferase reporter plasmids

The pmirGLO-based luciferase reporter plasmids (Promega, Madison, WI, USA) containing wild-type HIPK2 (termed HIPK2-Wt) or HIPK2 mutated at the putative miR-221-3p binding sites (termed HIPK2-Mut) were designed. The desired pmirGLO-HIPK2-Wt and pmirGLO-HIPK2-Mut plasmids with either miR-221-3p-specific miRNA mimic or miRNA mimic negative control (NC) were delivered into HEK293T cells by liposome-mediated transfection methods. Forty-eight hours later, a portion of the HEK293T cell lysate (100 μL) was added to an equal amount of Renilla luciferase, and the luminescence was determined by a microplate reader (SpectraMax M5, Molecular Devices, USA). Another part of the HEK293T cell lysate (100 μL) was added to an equal amount of firefly luciferase, and the luminescence was determined by a SpectraMax M5 microplate reader. The luminescence of firefly luciferase was analyzed relative to that of Renilla luciferase.

The putative binding sites of C/EBPβ and TFF3 were obtained by the JASPAR (http://jaspar.genereg.net/). The pmirGLO‐based luciferase reporter plasmids (Promega, Madison, WI, USA) containing wild‐type TFF3, wild‐type TFF3 truncated the putative C/EBPβ binding sites or TFF3 mutated at the putative C/EBPβ binding sites were designed. HEK293T cells (Shanghai Beinuo Biotech Ltd., Shanghai, China) were seeded into 6‐well plates in triplicate and cotransfected with well‐designed pmirGLO‐based reporter plasmids and plasmids carrying C/EBPβ gene using the dual‐luciferase reporter assay system (D0010, Beijing Solarbio Science & Technology Co., Ltd, China). Relative luciferase activity was normalized to that of Firefly luciferase.