Apsicheck 2 vector

Double-stranded oligonucleotides corresponding to the wild-type (WT-3′-UTR) or mutant (MUT-3′-UTR) miR-501-3p binding sites in the 3′-UTRs of the potential target genes were synthesized (GenePharma, Shanghai, China) and ligated into apsiCHECK™-2 Vector (Promega, Madison, WI, USA). The detailed procedure has been described previously [19 (link)].

To construct a luciferase reporter for wild-type (WT) BMP-2 3'UTR, we synthesised this UTR sequence and sub-cloned it into the NotI and XhoI sites in the apsiCHECK-2 vector (Promega). A mutant BMP-2 3'UTR was generated by site-directed mutagenesis using Phusion^™ High-Fidelity DNA Polymerase (Thermo Scientific) according to the manufacturer’s protocol. All constructs were confirmed by sequence analysis. For transfection, 5 × 10⁴ 293T cells were plated in complete medium on a 24-well plate. The next day, the cells were transfected with 50 nM of miR-93-5p mimics or NC mimics and inhibitor using Lipo3000 (Thermo Scientific) according to the manufacturer’s protocol. Three groups of cells (miR-93-5p NC group, miR-93-5p mimic group and miR-93-5p inhibitor group) were harvested 72 h after transfection in cell lysis buffer and then assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Beyotime, Shanghai) and a luminometer according to the manufacturer’s protocol. Transfection of each construct was performed in triplicate for each assay, and the ratios of Renilla luciferase activity to firefly luciferase activity were averaged for each experiment. For protocol see: dx.doi.org/10.17504/protocols.io.irbcd2n.