Luciferase assay
The 3’untranslated region (UTR) of wild type (WT) HINT1 and its mutant (MUT) was amplified before being cloned into pGL3 luciferase vectors (Promega Corporation) using the Q5 Site‑Directed Mutagenesis kit (New England BioLabs, Inc., cat. no. E0554S), as per the protocol provided by the manufacturer. Cells were subjected to co-transfection with miR‑205‑3p inhibitor (or miR‑NC) at 10 nM concentration and 200 ng/well of luciferase reporter vectors for the HINT1 gene with either the WT or MUT type 3’UTR. This was carried out utilizing Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) as per the guidelines stipulated by the manufacturer. Post-transfection, the relative luciferase activities were measured for 48 hrs with the Dual‑Luciferase Reporter Assay kit (Promega Corporation), per package guidelines. Renilla luciferase activity was utilized for normalization against firefly luciferase activity.