Figure 6
p53 transcriptionally downregulates miR-181a upon to anoxia. (a) Schematic representation of rat miR-181a promoter region containing a potential p53-binding site. (b) ChIP analysis of p53 binding to the miR-181a promoter in NRVCs treated or not with anoxia. The anti-IgG antibody was used as a negative control. (c) Luciferase activity measured from NRVCs infected with adenovirus harboring p53 or β-galactosidase (β-gal) and transfected with empty vector (pGL3) or with constructs containing the WT miR-181a promoter or the miR-181a promoter mutated the putative p53 binding sites (m-BS). Firefly luciferase activities were normalized to Renilla luciferase activities. *P < 0.05 compared with WT construct alone; #P < 0.05 compared with m-BS construct in combination with p53. (d) miR-181a levels by RT-PCR and p53 determined by in NRVCs transfected with adenoviral p53 siRNA or its scrambled form (p53-sc) as control. *P < 0.05 compared with control. (e) NRVCs upon anoxia or not were infected with adenoviral p53 siRNA or its control p53-sc and the percentage of cells with mitochondrial fission or cell apoptotic was determined. *P < 0.05 compared with control. (f) miR-181a levels by RT-PCR and myocardial infarct sizes in the hearts of mouse subjected to MI or not and infected with adenoviral p53 siRNA or its control p53-sc. n = 6 mice per group. Left graph, *P < 0.05 compared with control. Right graph, *P < 0.05 compared with control subjected to MI. Scale bar, 2 mm. (g) Schematic model of miR-181a’s role in regulating mitochondrial fission and cardiomyocytes apoptosis.
