miR-627 targets JMJD1A. (A) Predicted duplex formation between human JMJD1A 3′UTR and miR-627. (B) HCT-116 cells were transfected with GFP-JMJD1A 3′UTR (left) or GFP-JMJD1A 3′UTR-mut (right) plasmids together with plasmids expressing miR-627 or a negative control miRNA. Western blotting was performed with anti-GFP and anti-tubulin antibodies. Relative protein levels were quantified and shown under the gel. The experiment was repeated three times. The mean protein levels ± S.D. are: left panel, NC (1.0 ± 0), miR-627 (0.375 ± 0.007); right panel, NC (1.0 ± 0), miR-627 (0.83 ± 0.09). (C) HCT-116 cells were transfected with pcDNA6.2-GW/EmGFP-miR-negative control or pcDNA6.2-GW/EmGFP-miR627 plasmids. 48 hours after transfection, cell lysates were analyzed by western blots with the indicated antibodies. The experiment was repeated three times. The mean protein levels ± S.D. are: NC (1.0 ± 0), miR-627 (0.26 ± 0.13). (D) HT-29 and HCT-116 cells were treated with various doses of calcitriol for 48h, and western blots were done using the indicated antibodies. Relative protein levels were quantified and shown under the gel. The experiment was repeated three times. (E) HCT-116 cells were transfected with 100 nM LNA-modified miRNA inhibitor specific to miR-627 or the negative control inhibitor, and treated with 500 nM calcitriol for 48h. Total RNA and cell lysates were analyzed by real-time PCR for miR-627 and western blots with the indicated antibodies. The experiment was repeated three times. The mean protein levels ± S.D. are: left panel, Ctr (1.0 ± 0), Calcitriol (0.33 ± 0.08); right panel, Ctr (1.0 ± 0), Calcitriol (1.07 ± 0.17). (F) HCT-116 cells were treated with 500 nM calcitriol for 48 hours. ChIP assay was performed as described in Materials and Methods, using primers specific for the GDF15 promoter and the indicated antibodies. Total RNA was isolated and analyzed for GDF15 expression by real-time PCR. The experiment was repeated three times. Representative images of three independent experiments were shown.