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. 2015 Jan 20;23(4):656–666. doi: 10.1038/mt.2014.247

Figure 3.

Figure 3

HDAC5 is the direct target for miR-125a-5p. (a) RNAhybrid predicted miR-125a-5p binding sites and free energy values (−32.9 kcal/mol) in the 3′-UTR of HDAC5. The wild-type (WT, black letters) and mutated (MT, red letters) 3′-UTR of the HDAC5 sequence were cloned into a luciferase reporter plasmid. (b) HEK-293T cells were cotransfected with different ratios of miR-125a-5p and the plasmids containing the wild-type (WT) or mutant (MT) 3′-UTR of HDAC5. Luciferase activity was analyzed with the dual luciferase system. Activity of the WT and MT constructs was normalized to firefly/Renilla luciferase activity. (c) Cells were transfected as in Figure 2 and HDAC5, 7, and 10 protein expression was examined by western blotting 72 hours later. β-actin served as a loading control. (d) Cells were transfected with control-siRNA or HDAC5-siRNA-1/2 (5 μg/ml) and HDAC5, Ki-67, MMP2, caspase 3 and Bcl-xL levels were examined by western blotting 72 hours later. R2N1d cells were transfected as in (d) and 48 hours after transfection, the cell growth (e), apoptosis rates (f), wound healing (HDAC5-siRNA-1) (g), and invasion (HDAC5-siRNA-1) (h) were evaluated. Values are the mean ± SD of three experiments. *P < 0.05 versus untreated control; two-tailed Student's t-test.