Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-146a inhibits mitochondrial dysfunction and myocardial infarction by targeting cyclophilin D

doi: 10.1016/j.omtn.2021.01.034

Figure Lengend Snippet: 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).

Article Snippet: TRIzol, fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin solution, Lipofectamine 2000, JC-1 dye, IgG-Alexa Fluor 594 secondary antibody, 4′,6-diamidino-2-phenylindole (DAPI), calcein-AM, and protease inhibitor cocktail were obtained from Invitrogen (Carlsbad, CA, USA). miR-146a mimics, mimics negative control, miR-146a inhibitor, inhibitor negative control, as well as adenovirus vectors carrying genes encoding cyclophilin D (Ad-CypD) and Lacz (Ad-Lacz) were purchased from Sunbio Medical Biotechnology (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Sequencing, Binding Assay, Luciferase, Construct, Negative Control, Activity Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-146a inhibits mitochondrial dysfunction and myocardial infarction by targeting cyclophilin D

doi: 10.1016/j.omtn.2021.01.034

Figure Lengend Snippet: Cyclophilin D is involved in the regulation of cardiomyocyte apoptosis by miR-146a (A) Cardiomyocytes were transfected with miR-146 mimics and simultaneously transduced with adenoviruses carrying either Lacz (Ad-Lacz) or cyclophilin D (Ad-CypD) encoding constructs for 24 h before A/R stimulation. Cardiomyocyte apoptosis was determined by annexin V-FITC/PI staining using flow cytometry. (B) TUNEL staining of cardiomyocytes treated as described above. (C and D) Western blot analysis of Bcl-2, Bax (C), cleaved caspase-9, cleaved caspase-3, and cleaved PARP protein expression levels (D). Representative western blot images are shown in the leftmost column. Bar charts illustrate results of the densitometric analysis. ∗∗p < 0.01 versus NC-m A/R; ## p < 0.01 versus 146a-m A/R, n = 4–7.

Article Snippet: TRIzol, fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin solution, Lipofectamine 2000, JC-1 dye, IgG-Alexa Fluor 594 secondary antibody, 4′,6-diamidino-2-phenylindole (DAPI), calcein-AM, and protease inhibitor cocktail were obtained from Invitrogen (Carlsbad, CA, USA). miR-146a mimics, mimics negative control, miR-146a inhibitor, inhibitor negative control, as well as adenovirus vectors carrying genes encoding cyclophilin D (Ad-CypD) and Lacz (Ad-Lacz) were purchased from Sunbio Medical Biotechnology (Shanghai, China).

Techniques: Transfection, Transduction, Construct, Staining, Flow Cytometry, TUNEL Assay, Western Blot, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-146a inhibits mitochondrial dysfunction and myocardial infarction by targeting cyclophilin D

doi: 10.1016/j.omtn.2021.01.034

Figure Lengend Snippet: Cardiomyocyte miR-146a regulates cardiac I/R injury in a cyclophilin D-dependent manner (A and B) miR-146a f/f /MHC-Cre mice (miR-146a CKO ) were crossed with Ppif f/f /MHC-Cre mice ( Ppif CKO ) to obtain miR-146a CKO Ppif CKO mice and underwent I/R surgery. Myocardial infarct was visualized by Trypan blue (A) and 2,3,5-triphenyltetrazolium chloride staining and assessed as the ratio of infarcted area (IF) to area at risk (AAR) (IF/AAR) (B). ∗∗p < 0.01 versus I/R 146a f/f ; ## p < 0.01 versus I/R 146a CKO , n = 6 per group. (C) Measurements of LDH serum levels. ∗∗p < 0.01 versus I/R 146a f/f ; ## p < 0.01 versus I/R 146a CKO , n = 10 per group. (D and E) Analysis of echocardiographic left ventricular ejection fraction (EF, D) and percentage fractional shortening (FS, E) data at the end of 24 h reperfusion. ∗∗p < 0.01 versus I/R 146a f/f ; ## p < 0.01 versus I/R 146a CKO , n = 8 per group. (F) Representative images of myocardial tissue sections stained with TUNEL and anti-α-actinin antibody. (G) QuantiGication of apoptotic cardiomyocytes in each group. ∗∗p < 0.01 versus I/R 146a f/f ; ## p < 0.01 versus I/R 146a CKO , n = 6 per group.

Article Snippet: TRIzol, fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin solution, Lipofectamine 2000, JC-1 dye, IgG-Alexa Fluor 594 secondary antibody, 4′,6-diamidino-2-phenylindole (DAPI), calcein-AM, and protease inhibitor cocktail were obtained from Invitrogen (Carlsbad, CA, USA). miR-146a mimics, mimics negative control, miR-146a inhibitor, inhibitor negative control, as well as adenovirus vectors carrying genes encoding cyclophilin D (Ad-CypD) and Lacz (Ad-Lacz) were purchased from Sunbio Medical Biotechnology (Shanghai, China).

Techniques: Staining, TUNEL Assay