Pmir report luciferase mirna expression reporter vector

The whole 3′-UTR of CDK4 and TCF-1 were amplified from genomic DNA of the EL4 cells by PCR. Mutational 3′-UTRs were constructed by replacing 4 or 5 nucleotides in the miR-491 binding sites with PCR repair. The primers were listed in Table. S2. Then, the wild-type 3′-UTRs and mutational 3′-UTRs were individually cloned downstream of the firefly luciferase gene in the pMIR-REPORT luciferase miRNA expression reporter vector (Ambion). HEK293 cells were seeded in a 96-well plate (1 × 10⁴ per well), and each well was co-transfected with 0.2 μg of wild-type or mutant firefly luciferase reporter vectors, 0.01 μg of pRL-TK (Promega) and miRNA-491 mimics (50 nM) or negative-control scrambled miRNA mimics (50 nM) (RiboBio, China) using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, the cells were lysed, and fluorescence activity was detected via dual luciferase reporter assay system (Promega). The firefly luciferase activity was normalized to Renilla luciferase activity.

To generate a reporter vector with a miRNA binding site, three untranslated regions (3′-UTR) of TRPV3 were synthesized by Sangon (China). The construct was inserted into multiple cloning sites (SacI and HindIII sites) downstream of the luciferase gene in the pMIR-REPORT luciferase miRNA expression reporter vector (Ambion, USA). For the luciferase assay, using Lipofectamine 2000 (Invitrogen), 0.1 μg of a luciferase reporter containing 3′-UTR was co-mingled with miR-369-3p mimics or miR-369-3p inhibitors or NC transfection into HEK-293 cells (Shanghai Institute of Cell Biology, China). As an internal control, 10 ng of Renilla luciferase reporter was also included. Forty-eight hours after transfection, cells were collected and double luciferase activity was measured by a luminometer according to the manufacturer's instructions (Promega Corporation, USA).

To generate a reporter vector bearing miRNA-binding site, the 3-untranslated region (3′-UTR) of FoxO4 was synthesized by Sangon (Shanghai, China). The construct was inserted into multiple cloning sites downstream of the luciferase gene (SacI and HindIII sites) in the pMIR-REPORT luciferase miRNA expression reporter vector (Ambion, USA). To test the binding specifcity, the sequences that interacted with the seed sequence of miR-216b-5p were mutated (from AGAGAUU to UAUAUAA for the miR-216b-5p binding site), and the mutant FoxO4 3'-UTR was inserted into an equivalent luciferase reporter plasmid. For the luciferase assay, 0.1 μg of luciferase reporters containing 3′-UTR were co-mingled with miR-216b-5p mimic or miR-216b-5p inhibitor or miR-NC into HEK-293 cells using lipofectamine 2000 (Invitrogen, USA). As an internal control, a 10 ng of renilla luciferase reporters was also included. After 48 h transfection, the cells were collected and double luciferase activity was measured by luminometer according to the manufacturer's instructions (Promega Corporation).

To generate reporter vectors bearing miRNA-binding sites, the 3-untranslated region (3′-UTR) of FoxO4 and its mutation type were synthesized by Sangon (Shanghai, China). The construct was inserted into multiple cloning sites downstream of the luciferase gene (SacI and HindIII sites) in the pMIR-REPORT luciferase miRNA expression reporter vector (Ambion, USA). For the luciferase assay, 0.1 μg of luciferase reporters containing 3′-UTR were cotransfected with miR-499-5p mimic or miR-499-5p inhibitor or NC into HEK-293 cells using lipofectamine 2000 (Invitrogen, USA). As an internal control, 10 ng of renilla luciferase reporters was also included. Forty-eight hours after transfection, the cells were collected, and dual luciferase activities were measured by a luminometer according to the manufacturer’s instructions.

NPC cell lines (CNE2, HONE1, HNE2, HK1, CNE1, 5-8F, HNE1, 6-10B, and C666-1) and immortalized nasopharyngeal epithelial cell line NP69 were derived from the Cell Center of Central South University. All these cells were cultured on RPMI 1640 (Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37 °C and 5% CO₂ and free of mycoplasma contamination.
The sequences of circCAMSAP1 exons 2 and 3 were inserted into the pcDNA3.1(+) circRNA Mini Vector (kindly provided to us by professor Yong Li at Baylor College of Medicine, USA) to construct an overexpression vector. The SERPINH1-Flag, c-Myc-Flag, and SRSF10-Flag overexpression plasmids were purchased from Sino Biological Company (Beijing, China). The SERPINH1 3’UTR wild-type and mutant sequences were inserted into the pMIR-REPORT Luciferase miRNA Expression Reporter Vector (Ambion, TX, USA). The CAMSAP1 promoter sequence was inserted into the PGL3 basic vector (E1751, Promega, USA).
The siRNAs that specifically targeted circCAMSAP1, SERPINH1, c-Myc, and SRSF10 were purchased from Gemma Gene Corporation (Shanghai, China), and the sequence is shown in Table S3.
Lipofectamine 3000 (Life Technologies, NY, USA) was used to transfect plasmids and Hiperfect (Qiagen, Hilden, Germany) was used to transfect siRNAs.

A fragment sequence from the 3′-UTR of EBF1 containing a putative miRNA binding site was amplified by PCR from human B cell genomic DNA. The same procedure was used to generate reporter constructs with mutations in the 3′-UTR of the target gene. 3′-UTR sequences were inserted into pMIR-REPORT luciferase miRNA Expression Reporter Vector (Ambion) using Spe I and Hind III. The primers used for PCR are listed in Additional file 1: Table S2. Jurkat cells were cultured in RPMI 1640 with 10% FBS. Luciferase activity assays were performed as previously described [21 (link)] with 5 μg of firefly luciferase reporter vector containing either the wild-type or mutant oligonucleotides, 0.5 μl of mimic control, and has-mir-1246 mimic. Relative luciferase activity was normalized to renilla luciferase activity for each transfected well. Experiments were performed in triplicate in three independent trials.