miR-32 directly targets the 3′-UTR of tumor necrosis factor receptor-associated factor 3 (TRAF3).
(a) Seed sequence in miR-32 and complementary sequence in the 3′ UTR of TRAF3 mRNA showing seven-mer binding in wild-type (WT) TRAF3 3′ UTR. A deletion mutation of 4 base pairs in the 3′ UTR of TRAF3 was generated by site-directed mutagenesis. This alteration in the 3′ UTR sequence of TRAF3 abrogated the interaction of miR-32 and the 3′ UTR of TRAF3, resulting in translational derepression. (b) Luciferase assays were performed by transfecting HeLa cells with pCMV-β-gal (normalization control), WT TRAF3 3′ UTR and mutated (MUT) TRAF3 3′ UTR plasmids, along with pCMV-miR-32 plasmids. Normalized luciferase light units of control cells are presented as 100 units, and relative light units (RLU) of other treatments are shown accordingly. All experiments were performed three times and data are presented as mean ± SE (error bars). ***P ≤0.0005.