Psicheck2 construct

CD47 3’-UTR was subcloned into the psiCHECK-2™construct (Promega), and the resulting reporter constructs were used to transfect 293T cells along with either miR-196b-3p mimic or control. Lipofectamine 3000 reagent was used for transfection. SIRT1 3’-UTR psiCHECK-2™ vector and miR-450b-3p mimic were used to co-transfect 293T cells. At 48 h post-transfection, the Dual Luciferase Reporter Assay System was used to analyze the luciferase activity as described before [15 (link)].

The psiCheck2 construct containing dual Renilla and Firefly luciferase reporters was purchased from Promega (Promega). The DDX5-bound sequence on Fabp1 was cloned into a multiple cloning site located downstream of the Renilla translational stop codon. SW480 cells were cultured in 1× DMEM/F12 media (Gibco, Life Technologies). 1× 10% FBS (Gibco, Life Technologies), 1 mM sodium pyruvate (Gibco, Life Technologies) and 1% penicillin streptomycin (Gibco, Life Technologies) were added. The cells were plated on a 96-well plate at 0.5 × 10⁵ cells/ml 1 d before transfection. 100 nM of siCTL or the human DDX5 siRNA pool were introduced to SW480 cells using Lipofectamine 3000 (L3000001; Invitrogen) in Opti-MEM medium according to the manufacturer’s protocol. The transfection mixture was incubated at room temperature for 5 min. 10 μl of the transfection mixture was added to each well and incubated at 37°C for 24 h. 1 μg of psiCheck2 luciferase reporter plasmids were transfected with Lipofectamine 3000 and 1 μl P3000 enhancer reagent in the Opti-MEM medium. After 24 h, the cell lysates were used to measure both Renilla and firefly activities using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.

Fragments (540 bp) of VE‐cadherin and Notch4 3′UTR containing mutant or wild‐type miRNA‐responsive elements were cloned into the psiCheck2 construct (Promega) downstream of the Renilla luciferase ORF. Fragments (2600 bp) of circRNA7 promoter containing mutant or wild‐type miRNA‐responsive elements were cloned into the pGL3 construct (Promega) downstream of the Renilla luciferase ORF. SK cells were plated in 24‐well plates for 24 hours; then, the DNA was transfected into SK cells with Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Luciferase activity was assayed by dual‐luciferase assay (Promega), according to the manufacturer's protocol.