Reporter assay system kit

Oligonucleotide pairs containing miR-454-3p target region and miR-374b-5p target region or their mutant target regions were designed and ordered from Sangon (Shanghai, China). After annealing, all double-stranded segments were inserted into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, U.S.A.). Dual luciferase assays were carried out in HEK-293T cells which were seeded in a 96-well plate (Corning/Costar, Acton, MA, U.S.A.) at a density of 1   × 10⁴ cells per well. Eight hours later, relative miRNA mimics and control mimics were co-transfected into cells. Two days later, the luciferase activities were assessed with a Reporter Assay System Kit (Promega, Beijing, China) and were normalized to Renilla.

The UQCRC2 3′-UTR containing the miR-370 binding site was amplified using PCR and inserted upstream of the promoter in the psiCHECK-2 vector (Promega, Madison, WI, USA). The 3′-UTR of mutant or wild-type UQCRC2 was co-transfected into cells along with miR-NC or miR370 mimics using Lipofectamine 2000 (Invitrogen). After 48 h, luciferase activity was measured using a Reporter Assay System Kit (E1910; Promega, Beijing, China). The activity of firefly luciferase was determined relative to that of Renilla luciferase. Each experiment was performed on three independent occasions.

Through TargetScan database, miR-9-3p was predicted one binding site with 3′ -UTRs of FOXC1, BCL11A or FAM171A1, and miR-135b-3p was predicted one binding site with 3′ -UTRs of RGMA (Fig. S3A). To construct wide type (wt) reporter plasmids, around 200 bp of sequences upstream and downstream of the binding site were amplified and inserted between NotI and XhoI of psiCHECK-2 (Promega, USA) vector. Then the binding sites were mutant to construct mutant type (mut) vectors. For dual-luciferase reporter assay, MDA-MB-231 cells were evenly distributed into 96-well plate with 8 × 10³ cells per well. After 12 h, miRNA mimics and wt/mut-reporter vectors were co-transfected for another 24 h incubation. Luciferase activity was measured by the Reporter Assay System Kit (Promega, 017319). Firefly luciferase activity was normalized to Renilla luciferase. The sequences of primers used to construct wt/mut reporter plasmids are listed in Table S2.

Oligonucleotide pairs that contained the desired miR-22 target region or mutant target region were designed and ordered from Sangon, Shanghai, China. After annealing, these double-stranded segments were inserted into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA), between the SacI and SalI sites. The insertions were verified by sequencing. Dual-luciferase assays were performed using 1 × 10⁴ UM-UC-3 cells per well in a 96-well plate (Corning/Costar, Acton, MA, USA). After the cells attached for 8 h, they were cotransfected with 50 ng of respective reporter constructs with either 50 nM of miRNA mimics or control miRNA. After 48 h, a Reporter Assay System Kit (Promega, Beijing, China) was used to measure the luciferase activity. There were three replicates for each transfectant. Firefly luciferase activity was normalized to constitutive Renilla luciferase activity.

HeLa cells were seeded in 24-well plates 1 day prior to transfection using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Cells were transfected with 0.25 μg of either pGL3-basic vector or pGL3-basic-SLC15A3 promotor reporter constructs. In all cases, 0.025 μg of pRL-TK plasmid (Promega, Germany) was cotransfected for normalization of transfection efficiency. Overexpression of NF-κB was accomplished by co-transfection with 0.25 μg pcDNA3.1( + )-mNF-κB plasmid. Cells were harvested 24 h after transfection, and luciferase activity was measured by a Reporter Assay System Kit (Promega, Germany) and then expressed as x-fold activation relative to the activity of non-mNF-κB transfected cells.