Fig. 6: MiR-206 and miR-216b jointly target to Atg13.

a miR-206 and miR-216b harbored conserved targeting sites in Atg13 3′-UTR. b Luciferase constructs of wildtype Atg13 3′-UTR (Atg13-WT-3′-UTR), mutated Atg13 3′-UTR in the miR-206-binding site (Atg13-MUT1-3′-UTR), mutated Atg13 3′-UTR in the miR-216b-binding site (Atg13-MUT2-3′-UTR), and mutated Atg13 3′-UTR in the miR-206/216b binding sites (Atg13-MUT1/2–3′UTR) are shown. c HEK293 cells were infected with miR-206/216b mimics or mimics negative control (mimics-NC) along with the luciferase constructs of Atg13-WT-3′-UTR, Atg13-MUT1/2–3′UTR, or the empty vector pGL3. Luciferase activity was measured after 24 h. d H9c2 cells were infected with luciferase constructs of Atg13-WT-3′-UTR, Atg13-MUT1/2–3′UTR, or the empty vector pGL3 along with miR-206/216b antagomirs (miR-206/216b Anta) or antagomir negative control (Anta-NC) for 8 h. Cells were then treated with 10 μM histamine for 12 h. Luciferase activity was measured. e H9c2 cells were infected with adenoviral constructs expressing Atg13 (Ad-Atg13) or β-gal (Ad-β-gal) along with miR-206/216b mimics or negative control (mimics-NC). The level of Atg13 was analyzed by immunoblot. f H9c2 cells were treated with 10 μM histamine with 1 μM pyrilamine or 50 μM cimetidine under hypoxia for 12 h. The level of Atg13 was analyzed by immunoblot. g MiR-206/216b antagomirs (miR-206/216b Anta) or their negative controls (Anta-NC) were transfected into H9c2 cells for 8 h. Then cells were treated with 10 μM histamine under hypoxia for 12 h. For overexpression of miR-206/216b, H9c2 cells were transfected with miR-206/216b mimics or mimics-NC for 8 h. The level of Atg13 was analyzed by immunoblot. h H9c2 cells were infected with adenoviral constructs expressing Atg13 (Ad-Atg13) or β-gal (Ad-β-gal) along with miR-206/216b mimics or mimics-NC for 8 h. Then cells were treated with 10 μM histamine under hypoxia for 12 h. Representative photos of LC3 and p62 immunoblot and the densitometric analysis of the bands are shown. i Cells were treated as h. Quantitative analysis of apoptosis detected by TUNEL assay is shown. For detecting autophagy flux assay, GFP-LC3 plasmids were transfected into cells for 8 h prior to other treatment. The percentage of cells with GFP-LC3 puncta was quantified as the middle panel. j HDC^−/− mice were intravenously injected for 3 consecutive days before MI with miR-206/216b mimics, their antagomirs (miR-206/216b Anta), or their negative controls as described in methods along with 4 mg/kg/d histamine until euthanasia. Atg13 and LC3II levels were detected by immublot. k HDC^−/− mice were treated as j. Myocardial cell apoptosis was analyzed by TUNEL assay. TUNEL-positive nuclei (apoptotic cells) are green. Nuclei stained by DAPI show blue. Cardiomyocytes were labeled with a-actinin. scale bar, 50 μm. n = 3 for the sham group and n = 3 for each of MI groups. l HDC^−/− mice were treated as j. Cardiac function 1 week after MI surgery was analyzed by echocardiographic analysis. LVEF, left ventricular eject fraction. LVFS: left ventricular fraction shortening. n = 4 for the sham group and n = 4 for each of MI groups. *P < 0.05; **P < 0.01