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

Figure 4.

Figure 4

Histone deacetylase inhibitors (HDACi) induce synthesis of miR-125a-5p via the RUNX3/p300/HDAC5 complex. (a) Schematic diagram of the miR-125a-5p promoter showing the one predicted p300 binding site (red). (b) R2N1d cells were treated with Trichostatin A (TSA) or transfected with HDAC5 siRNA-1 at the indicated doses, and HDAC5 expression was analyzed by immunoblotting (IB) with β-actin as a loading control. Protein–protein interactions with RUNX3 and acetyl-lysine were analyzed with immunoprecipitation. (c) R2N1d cells were transfected with RUNX3 siRNA-1 (5 μg/ml), p300 siRNA (5 μg/ml), or HDAC5 siRNA-1 (5 μg/ml) then treated with TSA, and miR-125a-5p expression was analyzed with qRT-PCR 48 hours after treatment. (d) Binding of the transcription factor p300 to miR-125a-5p was analyzed with anti-p300 ChIP. (e–h) R2N1d and MDA-MB-231 cells were transfected with control siRNA (5 μg/ml) or RUNX3 siRNA-1/2 (5 μg/ml). At 48 hours post-transfection, RUNX3, Ki-67, MMP2, caspase 3, and Bcl-xL proteins expression was analyzed by western blotting (e) with β-actin as a loading control, and the cell growth (f), invasion (RUNX3-siRNA-1) (g), and wound healing (RUNX3-siRNA-1) (h) rates were evaluated. Values are the mean ± SD of three experiments. *P < 0.05 versus untreated control; two-tailed Student's t-test.