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Figure 1 | BMC Molecular Biology

Figure 1

From: Clock-controlled mir-142-3p can target its activator, Bmal1

Figure 1

mir-142-3p can target Bmal1 3’UTR. (A) Prediction results using three bioinformatic algorithms (TargetScan, PicTar and MicroCosm). The schematic representation of the binding sites for mir-142-3p and mir-448 in the 3’ UTR of Bmal1 is shown on the bottom. (B) A luciferase reporter assay was employed to screen the miRNAs that could target the mouse Bmal1 and human BMAL1 3’ UTR. 293ET cells were co-transfected with luciferase reporter plasmids and mir-142 or mir-448 expression plasmids or control vector, and the normalized firefly luciferase activity was measured (mean ± SD, n = 3). (C) A luciferase reporter assay was employed to confirm that mir-142-3p but not mir-142-5p can target Bmal1 in 293ET cells. Synthetic mir-142-3p mimics or mir-142-5p mimics or control microRNA mimics (mir-NC) were co-transfected with luciferase reporter plasmids into 293ET cells and the normalized firefly luciferase activity was measured (mean ± SD, n = 4). The top sketch modified from the miRBase shows that the pre-mir-142 can produce two mature miRNAs, mir-142-3p and -5p. (D) Schematic representation of the mir-142-3p binding sites in the Bmal1 3’ UTR and mutation of the mir-142-3p binding sites. In addition to the conserved binding site at position 350–358 of the Bmal1 3’ UTR, there is a poorly conserved binding site at position 1–7. (E) 293ET cells were transfected with the empty luciferase reporter vector (LUC) or luciferase reporter plasmids containing the Bmal1 3’ UTR with no mutations (WT) or with mutations (M1,M2 and M1 + M2) in the mir-142-3p binding sites and normalized luciferase activity was measured (mean ± SD, n = 4). (F) GFP reporter constructs containing the wild-type Bmal1 3’ UTR or mutated sequences were co-transfected into 293ET cells with mir-142-pcDNA3.1 or empty vector (pcDNA3.1), and the expression of GFP was determined by western blotting. The western blotting results were quantified from the pixel values in grayscales (mean ± SD, n = 3). Representative western blotting results are shown (bottom). *P < 0.05, **P < 0.01 and ***P < 0.001.

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