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. 2015 Dec 24;7(8):8756–8770. doi: 10.18632/oncotarget.6752

Figure 5. Contribution of potential miR-199a-5p binding sites in MAP3K11 mRNA.

Figure 5

A. and B. Schematic representations of MAP3K11 luciferase reporter constructs containing 3′UTR fragments with individual predicted miR-199a-5p binding sites, (A) F1 and (B) F2. In addition, the sequence of the miR-199a-5p potential binding sites in MAP3K11 3′UTR fragments F1 (A) and F2 (B) were mutated by substituting 3 bases (underlined). Adjacent bar diagrams depict luciferase activity in the specified wild-type (WT) and mutant (Mut) MAP3K11 reporter constructs (10ng) following co-transfection with pre-miR-199a-5p (10 nM) or control miR in TE7 cells for 36 hours. Firefly luciferase activities were normalized to Renilla luciferase activities and expressed as the mean of three independent experiments. All experiments were carried out in triplicate. Error bars represent mean ± S.D. and * represents statistically significant p < 0.05, p values based on two-tailed Student's t test. C. Schematic representation of constructs of different MAP3K11 luciferase reporter constructs containing the full length 3′UTR (FL-3′UTR). (a) The binding sequences of both of the miR-199a-5p potential binding sites are intact in the full-length MAP3K11 3′UTR wild-type (WT) fragment. (b) The binding sequence of miR-199a-5p potential binding site 1 was mutated by substituting 3 bases (underlined) while potential binding site 2 remained intact. (c) The binding sequence of miR-199a-5p potential binding site 2 mutated by substituting 3 bases (underlined) while potential binding site 1 remained intact. (d) Binding sequences of both the predicted miR-199a-5p binding sites in the MAP3K11 full-length 3′UTR were mutated. D. Luciferase activities of the mutated constructs (10 ng) were compared to wild type following co-transfection with pre-miR-199a-5p (10 nM) or control miR in TE7 cells for 36 hours. Firefly luciferase activities were normalized to Renilla luciferase activities and expressed as the mean of three independent experiments. All experiments were carried out in triplicate. Error bars represent mean ± S.D. and * represents statistically significant p < 0.05, p values based on two-tailed Student's t test.