Figure 5.

miR-146a regulates cyclophilin D expression

(A) qRT-PCR analysis of mRNA expression of the Vdac1, Slc25a4, and Ppif genes encoding voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and cyclophilin D (cyclophilin D), respectively, in cardiomyocyte mitochondria after transfection with miR-146a mimics for 48 h (n = 5). (B) miR-146a upregulation decreased cyclophilin D protein expression in mitochondria; however, it had no distinct effect on expression levels of VDAC and ANT. ∗∗p < 0.01 versus control, n = 6. (C) Cardiomyocytes were pretreated with miR-146a mimics before A/R stimulation. Western blot analysis of cyclophilin D protein expression in the mitochondrial fraction is shown. ∗∗p < 0.01 versus control; ^##p < 0.01 versus A/R, n = 6. (D and E) Cyclophilin D mRNA (D) and protein (E) expression levels in cardiomyocyte mitochondrial fraction treated with miR-146a inhibitor for 48 h. ∗∗p < 0.01 versus control, n = 6. (F) Analysis of Ppif (cyclophilin D) gene coding sequence (CDS) region for the potential binding site of miR-146a. (G) miR-146a inhibits Ppif (cyclophilin D) translation by binding to its CDS. Cardiomyocytes were co-transfected with the luciferase construct carrying wild-type cyclophilin D-CDS (cyclophilin D-CDS-WT) or mutated cyclophilin D-CDS (cyclophilin D-CDS-Mut) and miR-146a mimics or mimics negative control, whereupon the cells were harvested for the measurement of luciferase activity. ∗∗p < 0.01 versus control, n = 6. (H) qRT-PCR analysis of miR-146a levels in the cyclophilin D immunoprecipitate obtained from the cardiomyocyte mitochondrial fraction (n = 4).