Dual luciferase reporter gene assay system

StarBase database was utilized to predict the binding sites between miR-335-5p and circ_0064288 / ROCK1 3’UTR, respectively, and the binding fragments of circ_0064288 and ROCK1 3’UTR were amplified by PCR. The amplification products were inserted into the PGL3-promoter plasmid vector (Promega, Madison, WI, USA) to construct the circ_0064288 and ROCK1 wild-type (WT) plasmids; the binding fragment was mutated using gene mutation technology to construct the circ_0064288 and ROCK1 mutant type (MUT) plasmid. The recombinant plasmids were co-transfected with miR-335-5p mimic (or mimic NC) respectively in HEK-293 T cells. After 48 h, the cells were collected. Fluorescence values were measured using a dual-luciferase reporter gene assay system (Promega, Madison, WI, USA).

The sequence of circSCAP was cloned downstream of p-GLO Dual-Luciferase vector (Jidan, Guangzhou, China). Mutations were performed in the binding sites. The cells were cultured in a 24-well plate until showing 60–70% confluence, and then the constructed report vectors containing wild-type fragment or mutant type fragment together with renilla vector and miRNA mimics or miR-NC were co-transfected into 293-T cells using LipofectamineTM 3000 reagent. The cells were collected after 48 h for luciferase detection with the dual-luciferase reporter gene assay system (Promega, Madison, WI, USA). The firefly luciferase activity was normalized based on Renilla luciferase activity.

To detect the Wnt/β-catenin activation in EEECs, TOP/FLASH and FOP/FLASH reporter gene system (GenePharma Company, Shanghai) were selected to test the Wnt signaling pathway and the Promega dual-luciferase reporter gene assay system was used to measure the reporter activity. TOP/FOP values were used to represent the result. A higher value of TOP /FOP indicates a stronger Wnt pathway activity.

We used 24-well plates for transcriptional activity assays. HEK293T cells were transfected with the empty vector pcDNA3.1-3xflag, the WT or mutant FOXL2 expression vector, and the above-mentioned reporter gene constructs using Lipofectamine 2000 reagent (Invitrogen). Four groups were co-transfected into HEK293 cells, each containing 500 ng of either pcDNA3.1-3xflag, pcDNA-WT, pcDNA-MT1, or pcDNA-MT2, in addition to 500 ng of luciferase reporter plasmid (pGL3-StAR) and 40 ng of pRL-TK plasmid (Promega). The total DNA content of each well was maintained at 1,040 ng/well. Cells were incubated with plasmid in DMEM for 8 h and then in complete medium for 48 h. Luciferase intensity was measured using an EnSpire Multiplex Plate Reader (PerkinElmer, Waltham, MA, United States) with a dual-luciferase reporter gene assay system (Promega). All experiments were performed in triplicate.

The pmirGLO Dual-Luciferase miRNATarget expression vector was purchased from GenePharma (Shanghai, China), and the luciferase reporter plasmid was inserted into the wild-type WT-MFI2-AS1 and Mut-MFI2-AS1 3'UTR sequences. Firefly luciferase was used as the primary reporter gene to regulate mRNA expression, and Renilla luciferase was used as a normalization control. Dual-luciferase reporter gene assay system (Promega) was used 48 h after transfection. The mean luciferase intensity was normalized to Renilla luciferase. Data are shown as mean ± SD and each experiment was performed three times.

The FUNDC1 3′-UTR or Lnc049808 sequence (including the miR-101 binding site) was inserted into the pGL3 luciferase vector (Promega, USA) to construct the luciferase reporter vector. A rapid site-directed mutagenesis kit (TIANGEN, China) was used to generate mutations in the seed region of miR-101 as a mutation control. A dual-luciferase reporter gene assay system (Promega) was used to measure luciferase activity.