Figure 4

miR-874 regulates necrosis through targeting caspase-8. (a) Enforced expression of caspase-8 reduces necrotic cell death induced by H₂O₂. Cardiomyocytes were infected with adenoviral caspase-8, caspase-8 ^C360S(cysteine to serine mutation at the catalytically active site) or β-gal. Twenty-four hours after infection, cells were treated with H₂O₂. Necrotic cell death was assessed by flow cytometry using a PI exclusion assay, *P<0.05. (b) Enforced expression of caspase-8 attenuates myocardial necrosis upon I/R. Mice were treated as described in methods. Percentage of cells with myosin antibody infiltration was shown, n=6, *P<0.05 versus I/R alone. (c and d) Knockdown of caspase-8 can attenuate the inhibitory effect of miR-874 antagomir on H₂O₂-induced caspase-8 expression and necrosis. Cardiomyocytes were infected with adenoviral caspase-8-siRNA (casp8-siRNA) or its scramble form (casp8-sc), transfected with miR-874 antagomir (anta-874) and exposed to H₂O₂. Cells were harvested at 48 h after treatment for the analysis of caspase-8 levels (c) and PI exclusion (d), *P<0.05 versus H₂O₂+anta-874. (e and f) Caspase-8 target protector can inhibit caspase-8 reduction and necrosis-induced by H₂O₂. Cardiomyocytes were transfected with the target protector (caspase-8-TP^miR-874) or the control (caspase-8-TP^control), and then exposed to H₂O₂. Caspase-8 levels (e) and PI exclusion (f) were analyzed, *P<0.05 versus H₂O₂ alone