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.