Pmir report luciferase system

The luciferase reporter vectors for the wild-type or mutant ACOX1 3' UTR containing the miR-31-5p binding site were constructed into pMIR-REPORT luciferase system (Thermo Fisher Scientific). The primers used here are shown in Table S1. Mutated ACOX1 3' UTR was generated by replacement of the target sequence UCUUGC to AAGCUU using two-step mutagenesis PCR. The vectors expressing firefly luciferase reporter fused with wild-type or mutant ACOX1 3' UTR were co-transfected with miR-31-5p antisense oligomers or scramble control into OSCC cells. After 24 h, cells were harvested and the two distinct luciferase activities were measured using Dual-luciferase reporter assay according to the manufacturer's instructions (Promega; Madison, WI, USA). Renilla luciferase activity was used to normalize the extent of firefly luciferase activity.

HEK-293T cells (1×10⁵) were seeded in a six-well plate at 70%–80% confluence. The luciferase reporter vectors for the wild-type (wt) or mutant (mut) p53 3′-UTR containing miR-330-5p binding site were constructed into pMIR-REPORT luciferase system (Thermo Fisher Scientific). The vectors expressing firefly luciferase reporter fused with wt or mut p53 3′-UTR were co-transfected with miR-330-5p or miR-NC into cell. After 24 hours, cells were harvested, and the two distinct luciferase activities were measured using Dual-luciferase reporter assay according to the manufacturer’s instructions (Promega Corporation, Fitchburg, WI, USA). WT1-AS containing the predicted miR-330-5p-binding site was amplified using PCR and subcloned into a pmirGLO luciferase Target Expression Vector (Promega Corporation) to form the WT1-AS wt (pmirGLO-WT1-AS-wt) vector. The mutated miR-330-5p-binding sequence was constructed that was named as pmirGLO-WT1-AS-mut vector. The HEK-293T cells were co-transfected with PrirGLO, pmirGLO-WT1-AS-wt, prirGLO-WT1-AS-mut, and miR-330-5p mimics or negative control using Lipofectamine 2000, and the relative luciferase activity was measured using the Dual-luciferase reporter assay Kit (Promega Corporation) after 24 hours.