Dual luciferase reporting analysis system

Luciferase reporter assays were performed as previously described. Briefly, the cells were seeded in a 24-well plate. AGS GC cells stably overexpressing BCL6 and control lentivirus were transfected with 1 μg truncated FZD7 promoter-luciferase reporter, 0.5 μg empty vector, and 0.1 μg β-galactosidase reporter. After 48 h of transfection, cells were harvested in lysis buffer. Luciferase activity was measured using a dual-luciferase reporting analysis system (Promega, Madison, WI, USA) according to the manufacturer’s instructions on a Varioscan flash instrument (Thermo Fisher Scientific, Carlsbad, California, USA).

The 3′-untranslated regions (3′-UTR) of LINC01094 or SLC2A3 mRNA containing wild type (WT) or mutant (MUT) binding site was artificially synthesized and inserted into the pmiR-RB-REPORT^TM plasmid (RiboBio, Co., Ltd., Guangzhou, China) using restriction endonuclease. Meanwhile, the empty plasmid was taken as a negative control. The correctly sequenced luciferase reporter plasmids WT and MUT were co-transformed with NC mimic or miR-184 mimic into HEK293T cells, respectively. After 48 h of transfection, the cells were collected and lysed and centrifuged for 3–5 min followed by the collection of supernatant. Then, the relative luciferase unit (RLU) was evaluated on a dual-luciferase reporting analysis system (Promega Corporation, Madison, WI, United States) using a Renilla luciferase detection kit (YDJ2714, Shanghai Yuduo Biotechnology, Co., Ltd., Shanghai, China). The relative fluorescence activity was expressed as the ratio of the Renilla RLU value to the firefly RLU value.

Established protocols were used to perform a dual-luciferase reporter assay to investigate the binding activity between LINC00680 and miR-320a in 293T cells. The binding site for LINC00680 and miR-320a was predicted by the bioinformatics website (http://starbase.sysu.edu.cn/). To construct a LINC00680-WT luciferase reporter vector, we cloned a fragment of LINC00680 that contains the predicted binding site of miR-320a into the PHY-811 vector (Hanyi Biotechnology, Shanghai, China). Next, we mutated the assumed binding site for miR-320a in LINC00680, creating a vector that we named LINC00680-MUT. The LINC00680-WT or LINC00680-MUT vector was then transfected into 293T cells in combination with negative control or miR-320a mimics using Lipofectamine^® 2000. After 48 h of transfection, luciferase activity was determined using a dual-luciferase reporting analysis system (Promega, WI, USA) referring to the manual. Relative luciferase activity was measured and normalized to renin luciferase activity. These experiments were replicated three times in total.