| Cardiovascular disease is one of the major causes of mortality all over the world,however,among a number of cardiovascular disease,ischemic heart disease is the leading cause of mortality.Nowadays,Current reperfusion therapies remain the most effective strategy to rescue the ischemic myocardium.However,reperfusion would induce myocardium reperfusion injury.Researches indicate that myocardial reperfusion also contributes to mitochondrial oxidative stress through generation of reactive oxygen species(ROS)in mitochondrias,then leading to mitochondrial dysfunction and myocardial injury at last.However,the underlying mechanism has not been fully elucidated.MicroRNAs(miRs)as one class of new regulators,regulate target gene expression in transcriptional or translational level by base pairing with specific binding sites located in the 3' untranslated region(UTR)of target mRNA.An ever increasing number of studies have concurred to highlight a fundamental role of microRNAs(mi Rs)in the pathological process of heart diseases.Our previous study has demonstrated that down-regulation of miR-22 involved in estrogen protects cardiomyocytes against oxidative stress through positively regulating CSE and H2 S expression.Further bioinformatics analysis found that miR-22 is potentially target sirtuin-1(SIRT-1)and peroxisome proliferator-activated receptor-? coactivator-1?(PGC1?),both of which are known to provide protection against mitochondrial oxidative injury.However,whether mi R-22 is involved in the regulation of cardiac I/R injury by regulation of mitochondrial function remains to be investigated.Therefore,the present study,using ischemia-reperfusion(I/R)model in rats and hypoxia-reoxygenation(H/R)model in vitro is to clarify the miR-22 expression in cardiomyocytes in oxidative stress injury firstly,then elucidate the role and molecular mechanism in cardiomyocytes in oxidative stress injury.Main results: 1.Up-regulation of miR-22 contributes to myocardial injury upon to I/R insult 1.1.The effect of I/R on the expression of miR-22 in myocardium1.1.1.Rat hearts are subjected to ischemia for 30 minutes,followed by 2 hours and the LDH concentration,CK concentration,cardiomyocytic apoptosis number and infract size are significantly increased,which suggests that the model is constructed successfully.1.1.2.miR-22 expresson in ischemic myocardium is significantly increased in I/R group,as compared with the sham group.1.2.The effect of miR-22 inhibitor on myocardial injury induced by I/RMiR-22 inhibitor was delivered via intra-myocardial injection into the left ventricle myocardium two days before carrying on myocardial I/R surgery.Compared with control group,mi R-22 inhibitor could reduce the LDH concentration,CK concentration,cardiomyocytic apoptosis number and infract size.miR-22 inhibitor per se had no significant effect on serum LDH and CK levels,as well as cardiomyocyte apoptosis.2.Up-regulation of miR-22 contributes to cardiomyocytic injury upon to H/R insult 2.1.The effect of H/R on the expression of miR-22 in cardiomyocytes2.1.1.The apoptotic cardiomyocyte number,serum LDH level are significantly increased and cell vitality are markedly repressed when cardiomyocytes are subjected to hypoxia with 1% oxygen content for 24 hours then followed by reoxygenation for another 2 hours,which also indicates that we build the H/R model successfully.2.1.2.miR-22 expression in H/R group is significantly increased,as compared with control group.2.1.2.p53 and p21 expression in protein were significantly increased,as compared with control group,and NAC could reverse this effect.And the increased effect of mi R-22 exposed to H/R was reversed by NAC and p53 inhibitor.2.2.The effect of miR-22 inhibitor on cardiomyocytic injury induced by H/RCardiomyocytes are transfected with 200 nM miR-22 mimic or mi R-22 inhibitor for 24 hours,followed by H/R or normal treatment,then we measure relative indexs and find that miR-22 could increase supernatant LDH concentration,the number of apoptotic cell and repress the cell vitality to induce cell injury.What's more,it could further aggravate H/R-induced injury in cultured cardiomyocyte.However,this effect could be abolished by miR-22 inhibitor and show certain protective effect.3.Up-regulation of miR-22 contributes to myocardial mitochondrial dysfunction induced by I/R insultCompared with the group which is delivered mi R control via intra-myocardial injection,the group which is delivered miR-22 inhibitor via intra-myocardial injection shows the increased mitochondrial ATP production,membrane potential in mitochondria and reduced the production of superoxide.Mi R-22 inhibitor per se had no significant effect on cardiac mitochondrial function nction.4.Up-regulation of miR-22 contributes to cardiomyocytic mitochondrial dysfunction induced by H/R insultMiR-22 not only could increase the production of superoxide and decrease ATP production,membrane potential in mitochondrias to induce mitochondrial injury,then further aggravating H/R-induced mitochondrial dysfunction.However,mi R-22 inhibitor could reverse this effect and show certain protective effect on mitochondrias in cardiomyocytes,as evidenced by an decrease in mitochondrial superoxide production,and significant increases in mitochondrial membrane potential and ATP production in cardiomyocytes 5.Sirt1 and PGC1? are the targets of mi R-22 in cardiomyocytes.Bioinformatics analysis found that miR-22 is potentially target Sirt-1 and PGC1? and further studys indicate that mi R-22 could directly negatively regulate the transcriptional activity and the expression in mRNA and protein with dual-luciferase reporter,qPCR and western blot,respectively.6.Silencing of Sirt1 with Sirt1 siRNA abolishes the protective effect of miR-22 6.1.The effect of Sirt1 siRNA and miR-22 inhibitor on the expression of Sirt1 and PGC1? in protein levelH/R could repress the protein expression of Sirt1 and PGC1 a in cardiomyocytes.MiR-22 inhibitor not only could increase the protein expression of Sirt1 and PGC1 a,but also reverse the effect of H/R on the protein expression of Sirt1 and PGC1 a.However,the effect of miR-22 inhibitor could be abolished by Sirt1 siRNA and Sirt1 siRNA could aggravate the effect of H/R on the protein expression of Sirt1 and PGC1 a.6.2.The effect of Sirt1 siRNA and miR-22 inhibitor on mitochondrial functions in cardiomyocytesSirt1 siRNA could abolish the protective effect of miR-22 inhibitor on mitochondrias as evidenced by an increase in mitochondrial superoxide production,and significant decreases in mitochondrial membrane potential and ATP production in cardiomyocytes.6.3.The effect of Sirt1 si RNA and miR-22 inhibitor on cardiomyocytic functionSirt1 siRNA could abolish the protective effect of miR-22 inhibitor as evidenced by decreased cell survival as well as increased LDH release and apoptotic cells.Conclusion: The miR-22 expression was increased in the myocardium subjected to ischemia-reperfusion.And miR-22 activates ROS-induced p53/p21 signaling pathway and induce oxidative stesss in cardiomyocytes and myocardium injury by targeting related genes: Sirt1 and PGC1? in mitochondrial function. |
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