Pmirglo luciferase target expression vector

To verify the prediction, dual luciferase reporter assay was conducted as reported elsewhere (18 (link)). The predicted binding mode was obtained using bioinformatic prediction by software targetscan 5.1 (http://www.targetscan.org; Whitehead Institute for Biomedical Research, Cambridge, MA, USA). PCR was performed to amplify the 3’-end fragment of lncRNA XIST containing the predicted miR-185-5p-binding site and to subclone into a pmirGLOluciferase target expression vector (Promega, USA) as the XIST wild-type vector. And the mutated miR-185-5p-binding site was constructed as XIST Mutant vector. 293T cells were co-transfected with 200 ng of either pmirGLO-XIST-wide type (WT) or pmirGLO-XIST-mutant (MUT) vector and 80 ng of miR-185-5p mimics or inhibitor and their negative controls with Lipofectamine 3000 (Invitrogen). The verification of combination between miR-185-5p and CCND2 was realized via co-transfection with pmirGLO-CCND2-WT or pmirGLO-CCND2-MUT and miR-185-5p mimics or negative control. And the relative luciferase activity was measured using Dual-luciferase Reporter Assay Kit (Progema, USA) after 48 h of transfection.

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.