Dual luciferase reporter gene detection system

The promoter region of miR-19a was fragmented from the distal side to near side and fused with the cDNA of the Firefly and Renilla luciferases in the GV354 vector (GeneChem). All the constructed vectors were identified by sequencing. HEK-293T cells were sorted in 24-well plates in a 37°C incubator with 5% CO₂ at 5 × 10⁴ cells per well. The vectors above were delivered into cells and maintained at 37°C for 12 h. The activity of Firefly and Renilla luciferase was examined on a dual-luciferase reporter gene detection system (Promega, Madison, WI, USA). The PTEN 3′UTR containing the wild-type (WT) binding sequence with miR-19a and the corresponding mutant-type (MT) sequence were constructed and inserted into pGL3 promoter vectors to construct pGL3-PTEN-WT and pGL3-PTEN-MT luciferase reporter vectors. After that, the miR-19a mimic or control was cotransfected with 500 ng pGL3-PTEN-WT or pGL3-PTEN-MT vectors into 2 × 10⁴ cells, and 50 ng pRL-SV40 Renilla luciferase vector was cotransfected as well to examine the transfection efficacy. After 48 h, the relative activity of Firefly and Renilla luciferase was examined using the dual-luciferase reporter gene detection system again.

TargetScan (http://www.targetscan.org/vert_72/) was utilized to predict the binding sites of SP1 and miR-124-3p, while dual luciferase reporter gene assay to verify the targeting relationship between them. Wild type (Wt)-SP1 and mutant (Mut)-SP1 were formed by pMIR-Report luciferase vector (Ambion, TX, USA). Next, H9C2 cardiomyocytes were co-transfected with Wt-SP1 or Mut-SP1 and miR-124-3p mimic or mimic NC via Lipofectamine 2000 (Invitrogen). H9C2 cardiomyocytes were collected to determine luciferase activity in a dual luciferase reporter gene detection system (Promega, Madison, WI, USA) [52 (link)].

Pre-designed and synthesized the wild-type and mutant fragments of circ-PTPDC1 and ELK1, and inserted them into the pGL3 promoter vector (Realgene, Nanjing, China). MKN45 and AGS cells were seeded on a 96-well plate and cultured in a medium containing 10% FBS, and incubated in a 37 °C, 5% CO2 incubator. They were co-transfected with luciferase reporters and miR-139-3p mimics. After 48 hours of incubation, the fluorescein in the cells was collected according to the instructions Enzyme Mars and detected by the dual luciferase reporter gene detection system (Promega, Madison, Wisconsin, USA).

The bioinformatics website RNAhybrid was used to predict the potential binding sites between MONC and miR-636. MONC wild-type and mutant dual-luciferase reporter vectors were purchased from Liaoning Baihaobio Biotech Co., Ltd. (Liaoning, China), and co-transfected with miR-636 agomir or NC (GenePharma, Shanghai). The bioinformatics website, miRDB, was used to predict the potential binding sites between miR-636 and GLCE. GLCE wild-type and mutant dual-luciferase reporter vectors were purchased from Liaoning Baihaobio Biotech Co., Ltd., and co-transfected with miR-636 agomir or NC. A dual-luciferase reporter gene detection system (Promega, Madison, WI, USA) was used to detect luciferase activity.

The wild-type (WT) or mutant-type (MT) fragments of SATB1-AS1 or OAS2 3ʹuntranslated region (3ʹUTR) containing miR-580 target sequences were synthesized and inserted into the pGL3 promoter vector (Promega). These sequences were named SATB1-AS1-wt, SATB1-AS1-mut, OAS2 3ʹ-UTR-wt and OAS2 3ʹ-UTR-mut, respectively. The above vectors, miR-580 mimic or mimic control were co-transfected into 293T cells by using Lipofectamine 2000 (Invitrogen). The cells were collected 48 h after induction. The luminescence was assessed with a dual luciferase reporter gene detection system (Promega). The results were then normalized to Renilla luciferase activity [25 (link)].