Pmir report system

To construct p^{MIR-LUC-3′UTR-CD86} and p^{MIR-LUC-3′UTR-ICAM-1}, CD86 3′UTRs containing miR-134 binding sites and ICAM-1 3′UTR containing miR-222 binding site were generated by Chemical synthesis, and then cloned into downstream of luciferase of pMIR-REPORT™ System (Applied Biosystems/Ambion, Austin TX), respectively. Plasmids containing the 3′UTR mutants (mutation of two nucleotides within the miRNAs binding sites) of CD86 and ICAM-1 were also constructed as control.

Double-stranded oligonucleotides corresponding to the wild-type (wt) or mutant (mut) miR-497 binding site in the 3′-UTR of VEGFA and AEG-1 were synthesized and subcloned into the pMIR-REPORT system (Applied Biosystems). Synthesized sequences are listed in Supplementary Table S3. In 96-well plates, Hela cells were cotransfected with 0.1 mg of pLUC-wt-gene or pLUC-mut-gene, 0.01 mg of pMIR-REPORT β-galactosidase plasmid served as an internal transfection efficiency control, and miR-NC or miR-497 mimic with a 50 nM final concentration. At 48 h after transfection, luciferase and β-galactosidase activities were measured using the Dual-Light System (Applied Biosystems).

The recombined lentiviruses LV-Twist and LV-shTwist were constructed in Capan-1 and Bxpc-3 cells respectively. The empty lentiviral vector LV-control was used as a negative control (NC). The production, purification and titration of recombinant lentivirus were prepared as previously descried [25 (link)]. MiR-497 mimic, inhibitor (anti-miR-497) and their matched negative controls (mimic control or anti-control) were synthesized by GenePharma (Shanghai, China). VEGFA over-expressing vector, VEGFA gene-specific short interfering (siRNA) and non-specific control siRNA were purchased from Clontech Laboratories, Inc. The transfection was conducted using lipofectamine 2000 (Invitrogen, CA, USA) according to manufacturer's construction. Complete medium was changed 5 h after transfection. Double-stranded oligonucleotides corresponding to the wild-type (wt) or mutant (mu) miR-497 binding site in the 3′UTR of Twist (Twist-3′UTR-wt and Twist-3′UTR-mu) were synthesized into the pMIR-REPORT system (Applied Biosystems).

Luciferase assays were performed on 293T cells using the pMIR-REPORT System (Applied Biosystems, USA) following the manufacturer's protocol. Briefly, either the pMIR-REPORT-ZFX-WT-3′UTR plasmid or a mutant form of the plasmid (3 μg) was co-transfected with a pMIR-REPORT β-gal control plasmid (1 μg) into 293T cells (10⁶) with or without miR-101 overexpression using 15 mL of Lipofectamine 2000 (Invitrogen). Twenty-four hours later, luciferase activity was measured using a Luciferase Assay Kit (Applied Biosystems). To accomplish this, lysis solution (250 mL) was added to the cells, and the cells were detached from the plate with a cell scraper. The cell lysate was then transferred into a microfuge tube and centrifuged at 4°C for 5 min. The resulting supernatant was transferred into a fresh tube, and 50 mL of cell extract was transferred into a luminometer tube. Following this, 100 mL of Substrate A (ATP solution) and 100 mL of Substrate B (luciferin solution) were added. Following a 2-s pre-measurement delay, a luminometer was used to measure light emission from the tube for 10 s. β-galactosidase activity was tested using a β-Gal Assay Kit (Invitrogen) following the manufacturer's instructions. Relative luciferase activity was obtained by normalizing luciferase expression against β-gal expression.