Dual luciferase reporter assay system kit

For wild‐type luciferase reporter vector construction, the 3′ UTR of CCND1 (from 1900 to 2635 nt) was amplified by PCR and ligated into the pMir‐GLO luciferase reporter vector (Promega, Madison, WI, USA). For mutant luciferase reporter vector construction, the mutant CCND1 3′UTR (replaced TGTTACA with AAAAACA) was cloned into the MCS region of pMir‐GLO luciferase reporter vector with Sac I and Xba I. The primers for wild‐type and mutant CCND1 3′UTR were as follows: F: 5′‐gggagctcCTGTCCCACTC CTACGATAC‐3′, R1 (wild‐type): 5′‐tctctagaTGTAACATCAAAGGCAGAAGG‐3′, and R2 (mutant): 5′‐tctctagaTGTTTTTTCAAAGGCAGAAGGTTTGTGT‐3′. The luciferase reporter assay was conducted as we previously described before [15]. Briefly, the BGC823 cells were seeded into 12‐well tissue plates 24 h before transfection, and then co‐transfected with 5 nm siRNA and 1 mg plasmid using the Lipofectamine 2000 Reagent (Invitrogen), according to the manufacturer's instructions. After another 48 h, cells were assayed using the Dual‐Luciferase Reporter Assay System Kit (GeneCopoeia, Rockville, MD, USA). All experiments were performed in triplicate, and data were pooled from three independent experiments.

The wildtype and mutant NR2F1-AS1 fragment were amplified by PCR and ligated into the pEZX-FR01-dual luciferase reporter vector (GeneCopoeia, USA). GC cells were seeded into 12-well-tissue plates 24 h before transfection, and then co-transfected with 5 ng siRNA and 1 mg plasmid using the Lipofectamine 2000 Reagent (Invitrogen), according to the manufacturer’s instructions. After another 48 h, cells were assayed using the Dual-Luciferase reporter assay system kit (GeneCopoeia, USA). All experiments were performed in triplicate and data were pooled from three independent experiments.