Pglo basic

The luciferase reporter vector pGLO-basic (Promega, Beijing, China) containing the wild-type (WT) or mutant AFAP1-AS1 sequences 1/2 (Mut 1/2) were transfected into A549 cells. The pGLO plasmids containing the full-length RRM2 3′ UTR or its corresponding mutant were co-transfected with an miR-139-5p mimic or inhibitor into A549 cells. After 48 h of incubation, the cells were harvested and luciferase activity was determined using a dual luciferase assay kit (Promega).

Using the TargetScan 8.0 database (http://www.targetscan.org/, accessed on 1 January 2020), the binding sites of miRNAs matched to the target genes were predicted. Figure 5 depicts the algorithm-predicted binding site of miRNA to the target genes. Luciferase reporter assay was employed to confirm the combination of miRNA and target genes. Following the manufacturer’s protocol, the position of the sequence region, containing the putative binding sequence of each miRNA, was inserted into a luciferase reporter vector pGLO-basic (Promega, Madison, WI, USA). The mutated sequence of the target gene was constructed into a pGLO-basic vector. The sequences constructed in the reporter vector and mutant vector were confirmed by sequence analysis. pRL-TK Renilla Luciferase Reporter vector (Promega, Madison, WI, USA) was used as an internal control vector. HEK 293 cells were seeded into 96-well plates with the confluent rate about 80% and co-transfected with either reported vector (0.01 mg/well each), miRNA mimic/mutated vector (10 nM) and internal control vector. After 48 h co-transfection, luciferase activities were measured by a microplate reader (Infinite M1000, TECAN, Männedorf, Swiss). Renin luciferase activity was defined as the standardization of firefly luciferase activity.