STAT3 3'UTR Luciferase Assay

The putative binding sites of the 3′-untranslated region (UTR) of the human genes for miR-340-5p targeting were predicted using the TargetScan Human computational methods (http://www.targetscan.org/). The partial 3′-UTR fragment of STAT3, the wild STAT3 including predicted binding sites, and the mutant STAT3 inserts with an opposite mutation in the miRNA seed sequence binding sites, were cloned into the pMIR-REPORT™ miRNA Expression Reporter Vector (Applied Biosystems). All primers were designed as shown in Table 1. HEK293T cells were seeded in triplicate at a density of 2 × 10⁵ cells per 24-well plate. Luciferase activity was monitored using the Luciferase Assay Kit (Promega) and normalized to Renilla luciferase activity, as previously described [21 (link)].