Dual glotm luciferase assay system

A fragment of the 3′-UTR of HCP5 or HMGB1, and muted HCP5 or HMGB1 that contains the binding site for miR-29b-3p was cloned into pMiR-report vector (Ambion, USA). Cells were co-transfected with HCP5-wt/HCP5-mut or HMGB1-wt/HMGB1-mut and miR-29b-3p mimic/mimic NC using Lipofectamine 3000. A Dual Glo^TM Luciferase Assay System (Promega) was used to measure the luciferase activity.
PCR was performed to amplify the partial sequences of HCP5 which contained the putative binding sites of miR-29b-3p. The sequences were then cloned into the pmirGLO Dual‑Luciferase miRNA Target Expression Vector (Promega Corp., Fitchburg, WI, USA). Gene Art^TM Site‑Directed Mutagenesis system (Thermo Fisher Scientific, Inc.) was used to induce site‑directed mutagenesis of miR-29b-3p complementary bases in the sequences of HMGB1. Then, cells were transfected with the constructed wild‑type (WT) and mutant (MUT) reporter vectors, respectively, in the presence of miR-29b-3p mimics or miR‑NC. The activity of luciferase was measured using the Dual‑Luciferase Assay system (Promega) and then normalized to Renilla luciferase activity based on the manufacturer’s instructions. The assay was performed in triplicate experiments.