Construction of the vector and luciferase reporter assay

The expression vector for miR-21-5p (pcDNA-miR-21-5p) was generated by cloning the genomic fragments encompassing the corresponding miR-21-5p precursor and its 5′- and 3′-flanking sequences into the pcDNA6.2 plasmid (cat. no. K246020; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fragments of the 3′-untranslated region (UTR) of potential target mRNAs containing putative binding sites for miR-21-5p were inserted downstream of the stop codon of Firefly luciferase in the psi-Check2 vector (cat. no. C8021; Promega Corp., Madison, WI, USA) as previously described (33). To yield mutant constructs, mutations were introduced in the potential miR-21-5p binding sites using the QuikChange Site-directed Mutagenesis kit (cat. no. 210515; Stratagene, San Diego, CA, USA). The luciferase reporter constructs and pcDNA-miR-21-5p (or empty plasmid) were co-transfected into HEK-293T cells in a 48-well plate for reporter assays using Lipofectamine™ 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), respectively. Renilla and Firefly luciferase activities were assayed using the Dual-Luciferase^® Reporter 1000 assay system (cat. no. E1960; Promega Corp.). The effect of miR-21-5p on target gene expression was detected by calculating the relative value of Firefly luciferase activity normalized to Renilla luciferase activity.