The Effect Of MicroRNA-424-CRISPLD2 Pathway On Cardiomyocyte Pyroptosis

Part Ⅰ.Establishment of Cardiomyocyte Pyroptosis ModelObjective: To establish the pyroptosis model of myocardium by myocardial ischemia-reperfusion and cardiomyocyte hypoxia-reoxygenation in vivo and in vitro,evaluating modeling effect,and to detect changes in miRNA expression.Method: Male Balb/c mice of SPF level with 6-8 weeks and H9c2 cardiomyocyte were treated by ischemia-reperfusion and hypoxia-reoxygenation,respectively.After treatment,the concentration and transcription of IL-1β and IL-18 in serum and the supernate of cell culture were detected.Meanwhile,we tested the expression of CRISPLD2 and pyroptosis-related protein,caspase-1 and caspase-11,in cardiomyocyte.Results:(1)The concentration of IL-1β and IL-18 in serum of mice in group I/R and in supernate of H9c2 culture in group H/R were significantly higher than those in group sham and group Ctrl,respectively.The high expression of IL1β and IL-18 in both I/R and H/R group was detected at transcriptional level.(2)Compared with the group sham,the expression of pyroptosis-related protein was remarkably increased.The same results were found in isolated cell experiments.(3)The expression of CRISPLD2 was obviously decreased in cardiomyocyte with pyroptosis.Conversely,the expression of miRNA-424 was significantly upregulated.Conclusions: Either myocardial ischemia-reperfusion or cardiomyocyte hypoxia-reoxygenation can induce pyroptosis.The expression of CRISPLD2 and miRNA-424 are both changed in cardiomyocyte with pyroptosis.Part Ⅱ.The Effect of mi RNA-424 on cardiomyocyte pyroptosis via regulating CRISPLD2 expressionObjective: To establish the pyroptosis model via myocardial ischemia-reperfusion in vivo and cardiomyocyte hypoxia-reoxygenation in vitro and to demonstrate the effect of mi RNA-424-CRISPLD2 pathway on cardiomyocyte pyroptosis.Method: Cardiomyocyte pyroptosis was induced by ischemia-reperfusion and hypoxia-reoxygenation in vitro and in isolated cell experiments.Over-expressed and low-expressed mi RNA-424,respectively.Detected the expression of IL-1β and IL-18 at protein and transcription,and measured the expression of CRISPLD2 and pyroptosis-related protein in cardiomyocyte.Results:(1)Bioinformatics analysis predicted a conserved binding site for mi RNA-424 in the 3’ untranslated region(3’-UTR)of the CRISPLD2 gene.Compared with the control group,mi RNA-424 inhibitor remarkably increased the expression of CRISPLD2,which was obviously decreased when mi RNA-424 was transfected,conversely.(2)Compared with the control group,the expression of IL-1β、IL-18 and pyroptosis-related protease,caspase-1 and caspase-11,was significantly decreasd when mi RNA-424 mimic downregulated the expression of CRISPLD2.Moreover,with the upregulating of the CRISPLD2 expression by mi RNA-424 inhibitor,the expression of IL-1β、IL-18 and pyroptosis-related protease,including caspase-1 and caspase-11,was notably increased.Conclusions: Mi RNA-424 promotes cardiomyocyte inflammatory response and pyroptosis via reducing the expression of CRISPLD2 by binding to the 3’-UTR of the CRISPLD2 gene.