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. 2017 Oct 25;11:316. doi: 10.3389/fncel.2017.00316

Figure 4.

Figure 4

miRNA-324-5p acts onto Ttbk1 and downregulates Tau-tubulin kinase-1. (A) Luciferase reporter assay is done by the co-transfection of luciferase reporter containing wild or mutant 3′-untranslated region (UTR) of Ttbk1 with miRNA-324-5p mimic or its negative control (NC) into HEK293T cells. Luciferase activity was read 48 h after co-transfection. Top panel shows the predicted interaction between wild-type 3′-UTR of Ttbk1 and miRNA-324-5p. The relative activities of luciferase reporter for Ttbk1 3′-UTR are 1.0 ± 0.05 in the group of adding NC (dark-gray bar) and 0.66 ± 0.07 in the group of adding miRNA-324-5p mimic (p = 0.006, n = 24; light-gray bar). The relative activities of luciferase reporter for 3′-UTR of Ttbk1 with mutant miRNA-324-5p binding site are 1.0 ± 0.06 in the group of NC (dark-gray bar) and 0.92 ± 0.05 in the group of miRNA-324-5p (light-gray). The ratio for NC is normalized as 1. (B) The expression of Tau-tubulin kinase-1 was studied by western-blot. The ratios of Ttbk1 to β-actin are 1.0 ± 0.1 in antagomir control mice (light-gray bar, n = 6) and 1.37 ± 0.07 in miRNA-324-5p antagomir mice (dark-gray, p = 0.02, n = 6). The ratios for control mice are normalized as 1. *p < 0.05; **p < 0.01.