Reporter vector construction and luciferase reporter assay

pmirGLO dual-luciferase carrier (Promega, Madison, WI) was cloned into the downstream region of the pmirGLO dual-luciferase carrier (MIR17HG-piR-Wt), which was determined by the amplified MIR17HG gene. Similarly, pmirGLO dual-luciferase vectors were constructed to contain the putative miR-377 binding sequence in the MIR17HG gene (MIR17HG-miR-Wt) or its respective mutant sequence (MIR17HG-miR-Mut), the putative miR-153 binding sequence in the MIR17HG gene (MIR17HG-miR-Mut1 and MIR17HG-miR-Mut2) or its respective mutant sequence, and the FOXR2 3’UTR (FOXR2-miR-Wt) or its respective mutant sequences (FOXR2-miR-Mut). Lipofectamine 3000 was used to transfect HEK293T cells with pmirGLO dual-luciferase vector (wild-type or mutant fragment) and pre-piR-{"type":"entrez-nucleotide","attrs":{"text":"DQ590027","term_id":"108081281","term_text":"DQ590027"}}DQ590027 or pre-piR-NC. The transfection method and luciferase activity were determined by the Promega Fluorescein Enzyme Activity System after 48 h of transfection.