Pmirglo luciferase reporter gene vector

The 3′-UTR fragments of ADAMTS-7 containing the wild-type (WT) miR-423-binding site or a mutant (MUT) miR-423-binding site were amplified by Shanghai GenePharma Co., Ltd. and cloned into the pmirGLO luciferase reporter gene vector (Promega Corporation). The generated luciferase reporter plasmids were designated as WT-ADAMTS-7-3′UTR and MUT-ADAMTS-7-3′UTR. WT-ADAMTS-7-3′UTR or MUT-ADAMTS-7-3′UTR were co-transfected with miR-423 mimics (50 nM) or mimics control (50 nM) into 293T cells using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h of incubation at 37°C, the activity of luciferase was determined using a dual-luciferase reporter assay system (cat. no. PR-E1960; Promega Corporation). The firefly lucif-erase activity was normalized to Renilla luciferase activity.

The combination between SNHG14 and miR-181b-5p were predicted using StarBase 3.0 (https://starbase.sysu.edu.cn/index.php). Cells were seeded into 24-well plates at a density of 5x10⁴ cells/well and cultured at 37˚C for 24 h. The fragment of SNHG14 containing the predicted wild-type (WT) or mutant (MUT) miR-181b-5p-binding sequences were amplified by Shanghai GenePharma Co., Ltd., and inserted into the pmirGLO luciferase reporter gene vector (Promega Corporation) to produce the reporter plasmids SNHG14-WT and SNHG14-MUT, respectively. Subsequently, the cells were co-transfected with SNHG14-WT/SNHG14-MUT and miR-181b-5p mimic/mimic-NC using Lipofectamine 2000 reagent for 24 h. The culture medium was then removed, and the cells were rinsed twice with PBS. The cells were added to the cell lysates, which were swirled for 10 min and centrifuged at 12,000 x g for 10 min at 4˚C, and the supernatant was subsequently transferred to a new Eppendorf tube. A dual luciferase kit (cat. no. D0010; Beijing Solarbio Science & Technology Co., Ltd.) was used to measure luciferase activity according to the manufacturer's protocol. Firefly luminescence and Renilla luminescence were detected by enzyme markers, and Renilla luminescence was used as internal reference.