Gv354 vector

The recombinant luciferase reporter plasmids with different fragments of SAV1 promoter were constructed using the GV354 vector, a dual-luciferase plasmid containing both firefly luciferase gene and Renilla luciferase gene, was obtained from Shanghai Genechem. First, the different DNA fragments containing the TEAD binding site locating in SAV1 promoter were cloned using PCR. Then, the target fragment was cloned into GV354 using T4 DNA ligase. The recombinant luciferase reporter plasmids were verified by the DNA sequencing of Huada Genomics Institute (BGI, Guangzhou, China).

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.

The wild type (WT) and mutated (MUT) sequence of SIRT1 promoter region was fragmented from distal end to proximal end and constructed with firefly and Renilla luciferase cDNA into GV354 vector (GeneChem, Shanghai, China), respectively. HEK-293T cells were seeded into 24-well plates at the density of 5 × 10⁴ cells/per well 24 h before transfection, and extracted constructs were then co-transduced with oe-NC/oe-FOXQ1 plasmids into cells, followed by12-h incubation at 37°C. Dual-luciferase reporter analysis was performed according to the manufacturer’s instructions (Promega, Madison, Wisconsin) and the luciferase activity of firefly and Renilla luciferases was evaluated using the dual luciferase reporter assay system (Promega, Madison, Wisconsin). The ratio of firefly luciferase activity to Renilla luciferase activity indicated the relative activity of luciferase.