Prl tk renilla luciferase reporter vector

The pGL3 vector (Promega, Madison, SUA) was used to construct the reporter vectors, and the fragment of wild‐type 3′UTR of FOXO3 or PERP containing the putative miR‐629 binding site was amplified from human genomic DNA and cloned into the downstream of the pGL3 vector, For the mutant, point mutations were introduced into the seed region of miR‐629 bindings sties. For the luciferase reporter assay, cells were co‐transfected with wild‐type or mutant reporter vectors, respective miRNAs (mimics NC or miR‐629 mimics) and pRL‐TK renilla luciferase reporter vector (Promega) by Lipofectamine 200 reagent (Invitrogen) according to the manufacturer's protocol. Relative luciferase activities were determined by Dual‐Luciferase Reporter Assay system (Promega) at 48 hours after co‐transfections.

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.

Cells with 70% confluence were co-transfected with 2.5 μg of each pMIR-REPORT^™ construction and 0.05 μg of the pRL-TK Renilla Luciferase reporter vector (Promega, Charbonnières-les-Bains, France) by using the Lipofectamin 2000 reagent (Invitrogen, ThermoFisher Scientific). After 48 h of transfection, the reporter assays were carried out with the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Aliquots of cell lysates were transferred to a 96-well microplate and successively incubated with the Firefly Luciferase and Renilla Luciferase substrates. The luminescence signal was recorded with the FluoStar Optima Plate reader (BMG Labtech). Firefly luciferase values were first normalized to those of Renilla luciferase values, and then to luciferase expression of the empty pMIR-REPORT^™ (assigned a value of 1) for each of the 3’UTR constructions (S1 Table). Assays were run in duplicates from at least three independent experiments.

By searching the Target Scan 6.2 database (http://www.targetscan.org/) to find algorithm-based binding sites of miR-145, we found a predicted binding site to be at positions 278–284 in the 3′-UTR of KLF4 mRNA. The sequence region 2161–2520, containing the putative binding sequence of miR-145, was inserted into a pMIR-REPORT^TM Luciferase miRNA Expression Reporter Vector (Applied Biosystems) according the manufacturer's protocol. Moreover, we made another pMIR construct encompassing a mutated seed sequence for miR-145 (Wild type, GACTGGAA; mutant, GACGTCAA) by using a PrimeSTAR^® Mutagenesis Basal Kit (TaKaRa). The mutation of the vector was confirmed by sequence analysis. The pRL-TK Renilla Luciferase Reporter vector (Promega, Madison, WI, USA) was used as an internal control vector. 253J B-V cells were seeded into 96-well plates at a concentration of 0.1 × 10⁴ per well on the day before the transfection. The cells were co-transfected with either reporter vector (0.01 μg/well each) and 20 nM miR-145 or nonspecific non-coding siRNA (Dharmacon, Tokyo, Japan). Luciferase activities were measured at 24 h after co-transfection by using a Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol. Luciferase activities were reported as the firefly luciferase/Renilla luciferase ratio.

The promoter sequence of YB-1 target gene was cloned into pGL3 firefly luciferase reporter vector (Promega, USA) with sequence-specific primers (Table 1). Then, the recombinant pGL3 was co-transfected into melanoma stem cells or breast cancer stem cells with pRL-TK renilla luciferase reporter vector (Promega, USA). The activities of firefly luciferase of pGL3 and renilla luciferase of pRL-TK were determined following the dual-luciferase reporter assay protocol recommended by Promega. The cells were washed with PBS and then lysed with passive lysis buffer (Promega, USA). Subsequently, the cell lysate was subjected to the detection of firefly luciferase activity (M1), followed by the examination of renilla luciferase activity (M2). The promoter activity was calculated according to the following formula: Promoter Activity = M1/M2.