| With the extensive development and immediate intervention of drug thrombolysis,percutaneous coronary intervention(PCI)and other reperfusion treatment technologies,the morbidity and mortality of acute myocardial infarction(AMI)has been significantly decreased.However,how to improve the clinical prognosis of those AMI patients has become an another disturbing problem.In clinic,the occurrence of myocardial ischemia-reperfusion injury(MI/RI)often leads to the adverse cardiovascular events such as malignant arrhythmia,no reflow,heart failure or even cardiogenic shock,which badly limites the reperfusion’ beneficial effect on AMI patients.It has been proved that mitochondria mediated apoptosis signaling pathway is over activated in the whole MI/RI process,resulting in a large number of important cardiomyocytes death.As a key protein molecule on the outer membrane of mitochondria,B lymphoma-2/Bcl-2 related X protein(Bcl-2/Bax)play an important role in regulating the activation of mitochondrial apoptosis signaling pathway.Micro RNAs(miRNAs)are a group of highly conserved single stranded noncoding RNA molecules consisted of about 22 nucleotides,which regulate the expression of message RNA(m RNA)by inhibition or degradation in the post transcriptional period.Studies have showed the precursor miR-143 and its mature miR-143-3p participate in MI/RI via apoptosis pathway.However,its molecular mechanism has not been fully elucidated.In the previous study,we have found that Bcl-2 is a potential target site of mir-143-3p by using the Target Scan.Therefore,we speculate that miR-143-3p might be involved in MI/RI via targeting Bcl-2/Bax mediated mitochondrial apoptosis signal pathway.To test this hypothesis,this study will be carried out in the following two parts to reveal the molecular mechanism of miR-143-3p-mediated apoptosis involved in MI/RI.Part Ⅰ: Study on the relationship between miR-143-3p,Bcl-2 and Bax on myocardial ischemia/reperfusion injury in mice.Objective: To explore the expression of miR-143-3p,Bcl-2 and Bax in the myocardium of MI/RI mice.Methods: Twenty C57BL/6 male mice were randomly divided into two groups(10 mice per group): sham group and I/R group.The MI/RI model of mice was established by ligating the left anterior descending artery(LAD)to make the myocardium followed ischemia for 30 min and reperfusion for 24 h.At the end of reperfusion,2,3,5-triphenyltetrazolium chloride(TTC),hematoxylin and eosin(HE)staining were used to detect the situation of myocardial injury in mice.Serum cardiac troponin I(c Tn I)and creatine kinase MB(CK-MB)were detected by enzyme-linked immunosorbent assay(ELISA).And TDT mediated d UTP nick end labeling(TUNEL)was performed to detect the level of cardiomyocyte apoptosis.Finally,the expression of miR-143-3p,Bcl-2 and Bax were quantified by real time fluorescence quantitative polymerase chain reaction(q PCR).Results: The myocardial infarction area of mice in I/R group relative to the Sham group was significantly increased(P<0.05).The sham group showed that the structure of myocardial cells was complete,and cells were arranged orderly with only a small amount of inflammatory cells infiltrated in the intercellular stroma;however,the I/R group exhibited that the myocardial cells was arranged disorderly with loose intercellular stroma,and was infiltrated by a large number of inflammatory cells,and of which some myocardial cells were necrotic.At the same time,compared with sham group,c Tn I,CK-MB and cardiomyocyte apoptosis level in I/R group were also significantly increased(P<0.05).q PCR showed that the expression of miR-143-3p and Bax in I/R group were significantly elevated(P<0.05),while Bcl-2 was significantly decreased(P<0.05).Finally,the spearman correlation test showed that miR-143-3p was negatively correlated with Bcl-2(r=-0.726,P<0.05),and miR-143-3p was positively correlated with Bax(r=0.937,P<0.05).Conclusion: 1.I/R process induces cardiomyocyte apoptosis and promotes myocardial infarction.2.The expression of Bcl-2 was decreased in I/R-induced myocardium,while the expression of Bax and miR-143-3 were increased.Meanwhile,miR-143-3 was negatively correlated with Bcl-2,and positively correlated with Bax.Part Ⅱ: Study the role of miR-143-3p targeting mitochondrial apoptosis signaling pathway in myocardial ischemia/reperfusion injury.Objective: To explore the the role of miR-143-3p targeting mitochondrial apoptosis signaling pathway in mice induced by myocardial ischemia/reperfusion injury.Methods: Sixty male mice were randomly divided into 4 groups(15mice/group): Sham(NC)group,NC + I/R group,AAV-miR-143-3p inh + I/R group,AAV-control + I/R group.Three weeks before the operation,mice in Sham(NC)group and NC + I/R group were injected with the same amount of normal saline(NC)via tail vein.Mice in AAV-mir-143-3p inh + I/R group were injected with AAV-miR-143-3p inhibitor in the same way,and mice in AAV-control + I/R group were injected with the same amount of AAV-control as the control.After 3 weeks,the mice model of MI/RI were established by ligating LAD.At the end of reperfusion,myocardial infarction was detected by TTC staining,serum c Tn I and CK-MB were detected by ELISA,myocardial apoptosis was detected by TUNEL staining,and the expression of miR-143-3p,Bcl-2,Bax,Caspase-3,Caspase-9 and Cytochrome C were detected by q PCR and WB,respectively.Results: Dual luciferase gene report showed that the luciferase activity of bcl-2-3’UTR-wt + miR-143-3p group decreased significantly when compared to the bcl-2-3’UTR-wt + NC mimics group(P<0.05);there was no significant difference between bcl-2-3’UTR-mu + miR-143-3p group and bcl-2-3’UTR-mu+ NC mimics group(P>0.05).q PCR showed that the expression of miR-143-3p was significantly higher in NC + I/R group and AAV-control + I/R group in contrast to the sham(NC)group(P<0.05);Meanwhile,no significant difference in the expression of miR-143-3p between NC + I/R group and AAV control +I/R group was showed(P>0.05).However,compared with NC + I/R group,the expression of miR-143-3p in AAV-mir-143-3p + I/R group was significantly decreased(P<0.05).The results of TTC and ELISA showed that the MI area,c Tn I and CK-MB among NC + I/R group,AAVmir-143-3p inh + I/R group and AAV control + I/R group were increased significantly(P<0.05),when compared with sham(NC)group.And no significant difference in MI area,c Tn I and CK-MB between NC + I/R group and AAV control + I/R group was showed as well(P>0.05).However,compared with NC + I/R group,AAV-mir-143-3pinh +I/R group exhibited significant decline of MI area,c Tn I and CK-MB(P<0.05).At the same time,TUNEL staining results showed basically consistent with the above results.Compared with NC + I/R group,the positive apoptotic cells of myocardial tissue in AAV-mir-143-3p inh + I/R group were decreased significantly(P<0.05).q PCR and WB showed that when compared with sham(NC)group,the m RNA and protein expression of bcl-2 were decreased significantly in NC + I/R group,AAV-mir-143-3p inh + I/R group and AAV-control + I/R group(P<0.05),while m RNA and protein expression of Bax were increased significantly(P<0.05).Compared with NC + I/R group,there was no significant difference in the expression of Bcl-2 and Bax between AAV control + I/R group and NC + I/R group(P>0.05).However,compared with NC+ I/R group,the expression of Bcl-2 in the myocardium of AAV-mir-143-3p inh+ I/R group was significantly increased(P<0.05),and the expression of Bax was also significantly lower(P < 0.05).Interestingly,compared with NC + I/R group,AAV-mir-143-3p inh + I/R group significantly reduced the cytochrome C protein released from mitochondria to cytoplasm(P<0.05),and also obviously decreased the expression of Caspase-3 and Caspase-9(P<0.05).Conclusion: 1.miR-143-3p binds to Bcl-2;2.Low expression of miR-143-3p reduces myocardial infarction area and myocardial cell apoptosis in mice induced by MI/RI;3.Low expression of miR-143-3p down-regulates the expression of related protein of mitochondrial mediated apoptosis signal pathway. |
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