Figure 2.

DRAM2 is a target of MIR144*. (A) Three MIR144* binding sequences located in the DRAM2 3′ UTR. (B) Human primary monocytes were transfected with vehicle control; mimic negative control or MIR144* mimic (10, 20, and 50 nM); inhibitor negative control or MIR144* inhibitor (50, 100, and 150 nM) for 24 h. Expression levels of DRAM2 were determined by real-time PCR. (C) Human primary monocytes were transfected with vehicle control; mimic negative control (50 nM) or MIR144* mimic (10, 20, and 50 nM); inhibitor negative control (150 nM) or MIR144* inhibitor (50, 100, and 150 nM) for 24 h. DRAM2 protein levels were determined by immunoblotting. (D) Human primary monocytes were transfected with vehicle control; mimic negative control or MIR144* mimic (10, 20, and 50 nM); inhibitor negative control or MIR144* inhibitor (50, 100, and 150 nM) for 24 h. Expression levels of DRAM1 were determined by real-time PCR. (E) Schematic representation of the 3 MIR144*-binding sites in the DRAM2 3′ UTR construct (full length; WT, wild type) and generation of deletion constructs (Δ3, deletion of site 3; Δ2,3, deletion of sites 2 and 3). (F) THP-1 cells were cotransfected with vehicle control, mimic negative control, or MIR144* mimic (50 nM) and a series of luciferase reporter constructs (pmirGLO vector (mock), wild type, Δ3 and Δ2,3), which were incubated for 24 h. Luciferase assays were conducted to assess MIR144* targeting of the DRAM2 3′ UTR. (G) Nucleotide alignments among the MIR144* seed sequence, site 3, and mutated sequences 3′ UTR of DRAM2 cloned into pmirGLO vectors. (H) THP-1 cells were cotransfected with pmirGLO vector (mock) carrying site 3 or mutant constructs in addition to the vehicle control, mimic negative control, or MIR144* mimic (50 nM). Following cotransfection, luciferase assays were performed. Experiments were performed 3 times, and data were presented as means ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with the vehicle control (B and D). ns, not significant.