Mir 133b mimic

The wild-type or mutant 3′ UTR of NFATC4 mRNA, containing the miR-133b and miR-532-3p seed sequence targeting sites, was biosynthesized (Genewiz, Suzhou, China) and modified to include Xho I and Sal I restriction enzyme sites. Subsequently, all the obtained sequences were treated with restriction enzymes, Xho I and Sal I, and ligated to the pmirGLO dual-luciferase reporter vector (Promega, Madison, AL, USA) using T4 DNA ligase (TaKaRa, Dalian, China). The plasmids were sequenced to verify the correct insertion (Genewiz, Suzhou, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to co-transfect HEK293T cells with the wild-type or mutant 3′ UTR luciferase reporter plasmids, and the mimics NC, miR-133b mimic, and miR-532-3p mimic (RiboBio Co., Guangzhou, China). The cells were harvested 48 h after transfection, and the luciferase activities were measured using a Dual-Glo Luciferase Assay System (Promega; Madison, WI, USA). Firefly luciferase was used as the normalization control.

Before the transfection, 1×10⁶ 293T cells were inoculated with DMEM medium supplemented with 10% fetal bovine serum for 24 h in six-well plates. When the density of cells reached 50%–80%, cells were injected with 50 nM miR-133b mimic and miR-133b mimic negative control (Ribobio, Guangzhou, China) using 1×tiboFECT CP buffer and reagent (Ribobio), as well as 100 nM miR-133b inhibitor and miR-133b inhibitor negative control (Ribobio), according to the instructions of the manufacturer. Total RNAs and proteins were extracted 72 h after transfection and used for RT-PCR and Western blotting, respectively.
The 293T cells were injected with psiCHECK-2-NP, psiCHECK-3′ UTR and psiCHECK-3′ UTR-m, together with the different doses of miR-133b mimic using Lipofectamine2000 (Invitrogen Carlsbad, CA, USA), according to the manufacturer's instructions. Cells were collected 36 h after injection, and the ratios between Renilla and firefly luciferase were measured using Dual-Luciferase Reporter Assay System (E1910) (Promega, Madison, WI, USA).