Pmir reporter luciferase vector

The LDH-A 3′UTR sequence was generated and added to the pMIR-reporter luciferase vector (Ambion), with a mutant construct also being created in which the normal miR-7 binding site (UCUUCC) was changed to UGAUGA. Then wild type and mutant constructs were validated by DNA sequencing, and cells were seeded in 24-well plates and co-transfected with 0.3 μg of luciferase reporter plasmids, 0.1 μg of Renilla luciferase reporter (internal control) and equal amounts (50 nM) of miR-7 or miR-NC using Lipofectamine 3000 (Invitrogen). Firefly and Renilla luciferase activities were measured 24 h after transfection using a dual luciferase assay kit (Promega).

A 3′UTR fraction from the HIF-1 gene was amplified from genomic DNA by PCR and contained a predicted binding site for miR-194-5p. The fraction after amplification was replicated into a UTR downstream of the luciferase gene in a pMIR-reporter luciferase vector (Ambion, USA). Suitable mutation constructs were applied to exert control. NSCLC cells were cotransfected with the test luciferase reporter plasmid and the Renilla luciferase plasmid. Subsequently, the cells were obtained, the dual luciferase activity was evaluated by the Dual-Glo luciferase test regimen, and Renilla was used to exercise intrinsic regulation.

Target genes were predicted using miRDB (http://mirdb.org/), TargetScan (http://www.targetscan.org/vert_72/), and EVmiRNA (http://bioinfo.life.hust.edu.cn/EVmiRNA#!/). Transcription factors and lncRNA binding sites were predicted using JASPAR (http://jaspar.genereg.net/). For RNA-RNA pull-down assays, biotinylated wild-type miR-6734-5p (miR-Wt Bio), biotinylated mutant miR-6734-5p (miR-Mut Bio), biotinylated NC (NC Bio), and lncRNA OTUD6B-AS1 were synthesized by Sangon Biotech. After 12 h of incubation, miRNAs and lncRNA or miRNAs and mRNA were cotransfected in T24 cells into 6-well plates. Forty-eight hours after transfection, T24 cells were lysed and incubated with Pierce Nucleic Acid-Compatible Streptavidin Magnetic Beads (Thermo Fisher) to bind to biotinylated probes. Bound lncRNAs or mRNA were purified and analyzed by qRT-PCR. For dual-luciferase reporter assay, the 3′-untranslated region (3′UTR) of IDH2 (WT) was amplified by Sangon, and the sequences were inserted into a pMIR-reporter luciferase vector (Ambion, Rockville, MD, USA). A mutated 3′UTR (MUT) was also synthesized and inserted into the luciferase vector. MUT or WT vectors were cotransfected into HEK-293 cells with NC miRNA or miRNA mimics and luciferase intensity measured 2 days later.

mTOR and p70S6K1 3′-UTR regions containing predicted miR-497 binding sites and corresponding mutant sites were amplified by PCR from genomic DNA, and the PCR fragments were inserted into untranslated region (UTR) downstream of the luciferase gene in the pMIR-reporter luciferase vector (Ambion). Luciferase reporter plasmid, β-galactosidase (β-gal) plasmid, and pre-miR-497 and negative control precursors were cotransfected into cells using Lipofectamine 2000 (Invitrogen). Luciferase activities were measured 48 hours after transfection using β-gal for normalization. Primers used for Luciferase reporter constructs as follows:
mTOR Wild-type F: 5′-GCCGAGCTCTTTTCTGAGGCTTTTGTA-3′
mTOR Wild-type R: 5′-GCGAAGCTTCTAGGTCATTCTTCCATC-3′
mTOR Mutant F: 5′-GCCGAGCTCGGTTTGAACCAACTTTCTAGCTGCTGTTGAAGAATATATTGTCAGAAGCTTCGC-3′
mTOR Mutant R: 5′-GCGAAGCTTCTGACAATATATTCTTCAACAGCAGCTAGAAAGTTGGTTCAAACCGAGCTCGGC-3′
p70S6K1 Wild-type F: 5′-GCCGAGCTCTAGCCCTTGAGCCCTGTCC-3′
p70S6K1 Wild-type R: 5′-GCGAAGCTTATTCAGCCCTTTAATCTTCCAC-3′
p70S6K1 Mutant F: 5′-GCCGAGCTCGGAGATAGGGATATCCAGGGGAAGAGGGTGTAGCTGTGGCCCACAAGCTTCGC-3′
p70S6K1 Mutant R: 5′-GCGAAGCTTGTGGGCCACAGCTACACCCTCTTCCCCTGGATATCCCTATCTCCGAGCTCGGC-3′

The 3′-UTR of Wnt5a was synthesised and annealed, then inserted into the SacI and HindIII sites of the pMIR-reporter luciferase vector (Ambion) downstream of the stop codon of the gene for luciferase. The sequences complementary to the binding site of miR-129-5p in the 3′-UTR (Wnt5a: GCAAAAA) were replaced by TACCCCC for mutagenesis of the binding site. These constructs were validated by sequencing. U251 and N3 cells were seeded in triplicate in 24-well plates and cultured for 24 h. The cells were co-transfected with the wild-type or mutated plasmid and indicated amounts of miR-129-5p or miR-NC mimics. A Dual Luciferase Reporter Assay kit (Promega) was used to conduct luciferase assays 24 h after transfection according to the manufacturer’s instructions.

PD-L1 and CD80 3′-UTR regions that contained predicted miR-424-binding sites were amplified by PCR from genomic DNA. The PCR fragments were inserted into the UTR downstream of the luciferase gene in the pMIR-reporter luciferase vector (Ambion). Mutation of the putative miR-424(322) target sequence within the PD-L1 and CD80 3′-UTR were generated using QuickChange Site-Directed Mutagenesis Kit (Stratagene). Luciferase reporter plasmid, β-galactosidase (β-gal) plasmid, and pre-miR-424 and negative control precursors were co-transfected into cells using Lipofectamine 2000. Luciferase activities were measured 48 h after transfection using β-gal for normalization36 (link).
Human: PD-L1-3′-UTR-F: 5′- GCGCTCGAGGGAGACGTAATCCAGCATT -3′; PD-L1-3′-UTR-R: 5′- AATGCGGCCGCCACCTTACAAATACTCCAT -3′
Mouse: PD-L1-3′-UTR-F: 5′- GCGCTCGAGCTATGATCACTCTCCAGAT -3′; PD-L1-3′-UTR-R: 5′- AATGCGGCCGCAAAAAAAGAAAAACAAATG -3′
Human: CD80-3′-UTR-F: 5′- GCGCTCGAGGCCAGAACCCAGATTTCCT -3′; PD-L1-3′-UTR-R: 5′- AATGCGGCCGCCCTCTCTGCCCTACACTGAGA -3′