Pgl3 dual luciferase vector

SNHG6-wild type and SNHG6-mutant (The potential binding site of miR-325-3p was muted) was designed and cloned into PGL3 dual-luciferase vector (Promega, Madison, WI, USA) by Genechem (Shanghai, China). And GITR-wild type and GITR-mutant was designed and cloned into PGL3 dual-luciferase vector as well by Genechem. miR-325-3p plasmids or negative control plasmids were co-transfected with plasmids, stated before respectively, for two days. Then the dual-luciferase reporter assay system was used to assess the relative luciferase activities.

A 3′ untranslated region (3′UTR) fragment (1078-bp) of the HMGA2 containing the let-7d binding region was synthesized and purified by Generay biotechnology (Shanghai, China). Then the fragment was ligated into pGL3 dual luciferase vector (Promega, Madison, USA). IK cells at a density 5 × 10⁵ cells per well were co-transfected with 1 μg of pGL3-HMGA2 vector and 40 pmol of let-7d mimic or antagomir (Ribobio, Guangzhou, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Transfection of the negative control of mimic and antagomir was also performed as a control. After 24 h, the luciferase activities of firefly and Renilla were continuously measured by a Dual Luciferase Reporter Assay kit (Vazyme). The firefly luciferase activities were normalized to the Renilla luciferase activities to calculate the relative luciferase activity of HMGA2. The sequence of let-7d mimic, antagomir and their negative control we used are as following:

miR-NC: 5′-UCACAACCUCCUAGAAAGAGUAGA-3′;

let-7d mimic: 5′-AGAGGUAGUAGGUUGCAUAGUU-3′;

antagomir-NC: 5′-UUUGUACUACACAAAAGUACUG-3′;

let-7d antagomir: 5′-AACUAUGCAACCUACUACCUCU-3′.