Pezx fr02 vector

Fragments of miR-200a-3p, including the binding site of TGF-β2, were amplified and inserted into pEZX-FR02 vectors (GeneCopoeia, Amaranth Drive Germantown, Maryland, USA) at the 3′ end of the Firefly Luciferase gene using restriction enzymes BsiWI and XhoI (TaKaRa) and T4 DNA ligase (pEZX-TGF-β2-WT). Mutant pEZX-TGF-β2-MT was generated by mutating complementary to the seed region of miR-200a-3p using mutagenic primers. All constructs were verified by sequence analysis.

The wild-type (Wt) or mutant type (Mut) 3′-UTR of GOLPH3 was cloned into the pEZX-FR02 vectors (ZX002, GeneCopoeia, Guangzhou, China). In the next step, miR-142-5p mimics or miR-NC and the synthesized plasmids were co-transfected into A549 cells using Lipofectamine 2000. After 48 h of transfection, the luciferase activities were examined using the Luc-Pair™ Duo-Luciferase Assay Kit 2.0 (GeneCopoeia, Guangzhou, China).

SIRT1 wild-type and mutant 3′ untranslated regions (3′UTRs) were constructed after the corresponding sequences containing the forecasted or mutant binding sites were inserted into pEZX-FR02 vector (GeneCopoeia Co., Ltd.). HEK293T cells were seeded into 24-well plates at a confluence of approximately 70%, then co-transfected with luciferase reporter and mmu-miR-181a-5p mimics or NC mimics (RiboBio, China) using Lipofectamine 3,000. After incubation for 48 h, luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Vazyme Biotech, China) and a microplate reader.

For luciferase assays, the potential miR-876-5p binding site in the vimentin 3′-UTR was predicted by TargetScan (http://www.targetscan.org) to be at position 47–56 from the vimentin stop codon site. All vimentin 3′-UTR sequences with the wild-type or mutant seed region were synthesized and cloned into the Pezx-FR02 vector (Genecopoeia, USA) downstream from the luciferase stop codon; the new vectors were designated vimentin-WT or vimentin-MUT, respectively. 293T cells were cotransfected with 50 nM miR-876-5p mimics or negative control and 1 µg of vimentin-WT or vimentin-MUT using Lipofectamine 2000 (Invitrogen, USA). Cells were harvested at 48 h after transfection, and luciferase activities were analyzed by the Luc-Pair Duo-Luciferase Assay Kit 2.0 (Genecopoeia, USA).

Targetscan was used to identify a putative miR-34a-5p binding site in FLOT-2, after which wild-type (WT) and mutant versions of this FLOT-2-3'UTR sequence were generated and separately cloned into the pEZX-FR02 vector (Genecopoeia, USA). Cells were then transfected with these reporter plasmids and miR-34a-5p mimics using Lipofectamine 2000. At 48 h post-transfection, a Dual Luciferase Assay kit (Promega, WI, USA) was used based on provided directions.