Dual luciferase reporter assay system

To generate luciferase reporter expression constructs, full-length circCCDC66 or the 3′-UTR of PDK4 containing the expected binding sites was inserted into luciferase reporter DNA pGL3 vector (Promega Corporation). circCCDC66 mutant DNA or PDK4 3′-UTR mutant DNA was cloned into luciferase reporter DNA pGL3 vector. Plasmids were transfected with miRNAs in 293T cells (ATCC) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h. The measurement of luciferase activity was carried out with a Dual-Luciferase Reporter Assay System. The relative activity was normalized to Renilla luciferase activity.

cDNA oligonucleotides (Sangon Biotech Shanghai, Co., Ltd., Shanghai, China) containing the putative miR-98 target site according to miRBase sequence database (http://www.mirbase.org) within the 3′-UTR of the target gene were synthesized with Hind III and Sac I restriction enzyme digestion (Takara Biotechnology Co., Ltd., Dalian, China) sites and cloned into the multiple cloning site of the pMIR-REPORT Luciferase vector (Ambion; Thermo Fisher Scientific, Inc.). An additional pMIR-REPORT Luciferase construct containing mutant 3′-UTR was generated as a control. Subsequently, HEK293 cells (Cell bank of Chinese Academy of Sciences, Shanghai, China) were cultured in 24-well plates with DMEM and 10% FBS for 24 h and then were transfected with each reporter construct, alongside miR-98 mimic or miR-98 inhibitor, using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Firefly luciferase activity was quantified at 48 h post-transfection using the dual-luciferase reporter assay system (Thermo Fisher Scientific, Inc.).

The RAP1 gene promoter fragment (−1,500/+150 bp) or (−500/+150 bp) was cloned into the polylinker site upstream of the luciferase gene (luc) in the pGL-Basic vector. One recombinant clone for each of the constructs was chosen, and the plasmid DNA was extracted and purified using Qiagen Miniprep kit (27106, Qiagen, Hilden, Germany) following the manufacturer’s instructions.
HCT116 and HEK293 cells were seeded in 6-well plates (5×10⁵ cell/well) with 2 mL of DMEM and 10% FBS and grown until 80% confluence (24–36 hour). Cells were cotransfected with the appropriate rap1 promoter-reporter constructs and the pcDNA3-myc-PRL-3 expression plasmid by using 1 µg/well of luciferase reporter construct, 0.25 µg/well of renilla reporter, and 1 µg/well of pcDNA3-myc-PRL-3 (or empty pcDNA3 vector as a negative control). After 48 hours, cells were lysated and assayed for luciferase activity. Luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System (Thermo Fisher Scientific). All transfections were performed in duplicate, and experiments were repeated at least three times. The firefly luciferase activity was normalized according to the renilla luciferase activity.

The miRNA upstream of CDH3 was predicted using bioinformatics softwares, including GSE93415 (26 (link)) from Gene Expression Omnibus DataSets (https://www.ncbi.nlm.nih.gov/gds/?term=), TargetScan (http://www.targetscan.org/), starBase (http://starbase.sysu.edu.cn/panCancer.php) and Venny 2.1.0 (http://bioinfogp.cnb.csic.es/tools/venny/index.html). The pmiRGLO plasmid vector (Promega Corporation) used in the present study contained both the firefly luciferase reporter gene and the internal control gene, Renilla luciferase. The pmiRGLO CDH3 3′-untranslated regions (3′-UTRs) wild-type (WT) vectors and pmiRGLO CDH3 3′-UTR mutated (MUT) vectors with the miR-665 binding sites replaced by a random nucleotide sequence were constructed. A 24-well plate was then used to seed AGS and HGC-27 cells (2×10⁵ cells/well). The next step was the co-transfection of the cells with pmiRGLO CDH3-MUT or pmiRGLO CDH3-WT and with either miR-665 mimic or mimic-NC using Lipofectamine 3000 Transfection Reagent. After collecting the cells transfected for 48 h, Renilla luciferase and firefly luciferase activities were detected using the Dual Luciferase Reporter Assay System (Thermo Fisher Scientific, Inc.). Firefly luciferase activity was normalized to that of Renilla luciferase activity.

To determine the transcriptional activity of the Wnt pathway, 5×10⁶ A549 and SPC-A1 cells treated as indicated were co-transfected with either the Wnt signaling reporter TOP Flash or the negative control FOP Flash (EMD Millipore, Billerica, MA, USA) according to the manufacturer's protocol. A549 and SPC-A1 cells were transiently transfected with either 2 µg pTOP Flash or pFOP Flash plasmids and 0.5 µg pSV40-Renilla plasmid as an internal control (Promega Corporation) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 48 h. The dual-luciferase reporter assay system (Dual-Luciferase^® Reporter Assay system; cat. no. E1910; Promega Corporation) was used to assay the firefly and Renilla luciferase activity ratio.