Prl renilla luciferase control reporter vector

The wild-type 3′UTR of TRAF5 mRNA containing predicted miR-135a binding sites and mutant 3′UTR of TRAF5 were inserted into pmiRGLO vectors (Promega Corporation). BGC-823 or SGC-7901 cells were seeded in 24-well plates and cotransfected with wild-type or mutant 3′UTR of TRAF5 luciferase reporters and miRNA mimics or inhibitor using Lipofectamine 2000. After 48 hours, luciferase activity was detected using dual-luciferase reporter system (Promega Corporation) and normalized to Renilla activity. The coding sequence of TRAF5 mRNA was amplified by PCR using KOD-plus-Ver.2 kit (KOD-211; Toyobo) and cloned into pCMV-Tag2B expression vector. The NF-κB reporter plasmid was purchased from Beyotime Company (D2206 1 µg; Beijing, China). pRL Renilla Luciferase control reporter vectors were purchased from Promega Corporation.

The 3′-UTR sequence of human ZEB2 gene was amplified using PCR and cloned into a psiCHECK-1-based luciferase plasmid (Addgene, Inc., Cambridge, MA, USA) in which the Renilla luciferase sequence was replaced with a firefly luciferase sequence (restriction enzyme sites: NheI/SgfI) from pGL-4.22 vector (Addgene, Inc.). pRL Renilla luciferase control reporter vectors (Promega Corporation) was co-transfected as an internal reference. The construction of the psiCHECK plasmid containing the mutated 3′-UTR of ZEB2 was performed as previously described (26 (link)–28 (link)). In brief, cell transfection was performed using Lipofectamine 3000 transfection reagent according to the manufacturer's protocol. Cells (3×10⁵) were co-transfected with 1 µg plasmids, and miR-30a mimics, inhibitor or Ctrl miRNA for 24 h, then the dual luciferase assay was performed using a Dual Luciferase Assay Kit according to the manufacturer's instructions (Promega Corporation). In the present study, firefly and Renilla luciferase values were detected; for the evaluation of relative luciferase activity, the firefly luciferase activity was normalized to the Renilla luciferase value.