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. 2018 Mar 8;19(3):773. doi: 10.3390/ijms19030773

Figure 4.

Figure 4

Transcription factor AP-2α binds to the miR-25-3p promoter region. (A) Schematic diagram of the AP-2α binding site (arrow, red dot) in the miR-25-3p promoter. The first nucleotide of pre-miR-25-3p was assigned as +1, and the other nucleotides were numbered relative to it. (B) A series of progressive deletion mutants were transfected into growing BHK and C2C12 cells, and the promoter activities were analyzed by dual luciferase activity assay. (C) miR-25-3p-P9 reporter constructs were cotransfected with pc-AP-2α into growing C2C12 cells. Dual luciferase activity was measured 24 h after transfection. Overexpression of AP-2α upregulated miR-25-3p promoter luciferase activity. pcDNA-3.1(+) was used as a control. (D) Site-directed mutagenesis of the AP-2α binding site (CAGG into TGTA) in the miR-25-3p promoter region resulted in the miR-25-3p-P9 luciferase activity being reduced. Data were expressed as the ratio of relative activity, normalized to pRL-TK, and presented as means ± SD (n ≥ 3). (E) Binding of AP-2α to the miR-25-3p promoter region was analyzed by chromatin immunoprecipitation (ChIP). DNA isolated from immunoprecipitated materials was amplified using qRT-PCR. Normal mouse IgG was used as the negative control. Data were normalized by total chromatin (input) and presented as means ± SD (n = 3); ** p < 0.01.