| Part Ⅰ The mechanism of miR-425-5p in acute myocardial infarctionBackgroundAcute myocardial infarction(AM1)refers to myocardial tissue damage caused by coronary artery obstruction,which belongs to the category of acute coronary syndrome.Plaque rupture after coronary atherosclerosis is the most common cause of AMI.Endothelial dysfunction is regarded as the initial pathology of the development of atherosclerosis and occurs throughout the entire process of AMI.Therefore,improving endothelial dysfunction is the key to improve the prognosis of patients with AMI.microRNAs(miRNAs)play an important regulatory role in cardiovascular diseases including AMI,and participate in acute myocardial infarction by regulating endothelial function,which may provide new ideas for the treatment of cardiovascular and other diseases.It was found that miR-425-5p is abnormally expressed during the process of myocardial fibrosis in patients with acute heart failure,and plays an important role in the proliferation,migration and apoptosis of vascular endothelial cells.However,the effect and mechanism of miR-425-5p in AMI are unclear.The first part of this study took AMI rat model and hypoxia reoxygenated cell model(H/R)as the research object,aiming to explore the role and possible mechanism of miR-425-5p in AMI through in vivo and in vitro studies,so as to provide a certain theoretical basis for the mechanism research of AMI.Objective1.To detect the expression changes of miR-425-5p in AMI rat and H/R cell models.2.To study the intervention mechanism of miR-425-5p overexpression on cardiac function injury and endothelial dysfunction in AMI rats.3.To predict and verify the targeted gene PAI-1 of miR-425-5p,and explore the binding relationship between them and its mechanism through co-regulation in vitro.Methods1.Construction of H/R model and detection of related index in vitroHuman umbilical vein endothelial cells(HUVECs)were divided into 4 groups at random:①control group;②H/R group;③H/R+miR-NC group;④H/R+miR-425-5p mimic group.miR-NC and miR-425-5p mimic were transfected by Lipofectamine 2000,respectively.Except for group ①,the cells were subjected to hypoxia for 6 h and then reoxygenated for 24 h to establish the hypoxia/reoxygenation(H/R)model of cells in vitro.The relative expression level of Mir-425-5p in the cells was detected by RT-qPCR.The proliferation and migration of the cells were detected by MTT and Transwell assay,and the apoptosis was detected by flow cytometry and Western blotting.At the same time,oxidative stress was detected by detecting NO level,MDA content and SOD activity,and the expression of inflammatory factors was detected by ELISA,RT-qPCR and Western blotting.2.Construction of AMI rat model and detection of related indexSD rats were selected and divided into 4 groups:①Sham group;②AMI group;③ agomir-NC group;④miR-agomir group.The AMI rat model was constructed by coronary artery ligation.In vivo transfection was completed by internal injection to regulate the expression level of miR-425-5p in rats.The expression level of miR-425-5p,changes of serum myocardial enzyme and cardiac function,oxidative stress level and inflammatory factors,and apoptotic proteins(Bax and Bcl-2)in myocardial tissue were detected.3.Prediction and verification of downstream target genes of miR-425-5pBased on bioinformatics analysis and luciferase report experiments,the relationship between miR-425-5p and the potential target gene PAI-1 was predicted and verified.RT-qPCR and Western Blot were used to determine the mRNA and protein levels of PAI-1.4.Effect of miR-425-5p and PAI-1 on cell function under H/R conditionsH/R endothelial cell model was constructed,and the levels of miR-425-5p and PAI-1 were regulated through transfection.The effect of miR-425-5p and PAI-1 on cell function was investigated,including cell proliferation and migration ability,cell apoptosis,oxidative stress level and inflammatory factor expression level.Results1.miR-425-5p attenuates H1 R-induced endothelial cell injury(1)Compared with the control group,the expression level of miR-425-5p in H/R group was significantly decreased(p<0.05),and miR-425-5p was significantly increased in the H/R+miR-425-5p mimic group(p<0.05).(2)The proliferation and migration of cardiomyocytes in H/R group were significantly decreased(p<0.05),and the apoptotic rate was significantly increased(p<0.05);Compared with the H/R+miR-NC group,the proliferation and migration ability of HUVECs in H/R+miR-425-5p mimic group were significantly increased(p<0.05),and the apoptosis of HUVECs was decreased(p<0.05).(3)The content of malondialdehyde(MDA)increased significantly,while the level of nitric oxide(NO)and the activity of superoxide dismutase(SOD)were decreased significantly(all p<0.05)in H/R group;after overexpression of miR-425-5p,the content of MDA was significantly decreased(p<0.05),while the level of NO and the activity of SOD were significantly increased(p<0.05),which indicated that miR-425-5p could ameliorate cell damage and inhibit oxidative stress response.(4)Under H/R condition,the expression levels of intercellular adhesion factor-1(ICAM-1),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)were significantly increased(p<0.05);after overexpression of miR-425-5p,the contents of various inflammatory factors were significantly decreased(p<0.05).2.miR-425-5p attenuates myocardial injury induced by AMI in rats(1)The expression level of miR-425-5p in serum of the AMI group was significantly decreased compared with sham group(p<0.05),and the change was time-dependent;After the miR-425-5p mimic was injected in vivo,the expression of miR-425-5p in AMI group was significantly increased(p<0.05).(2)Compared with the sham group,the left ventricular infarct area of the AMI group was significantly increased,while the myocardial infarct area of the AMI group was reduced after miR-agomir intervention.(3)Compared with the sham group,the contents of CK and LDH in AMI group were increased(p<0.05),and the cardiac function was decreased significantly.The overexpression of miR-425-5p can significantly reduce the expression levels of CK and LDH(p<0.05)and improve the cardiac function of AMI rats.(4)Compared with the sham group,the level of oxidative stress in AMI group was unbalanced,the contents of NO and SOD were significantly decreased,and the level of MDA was significantly increased(p<0.05).The injection of miR-425-5p mimics in vivo could significantly improve the oxidative stress of AMI rats.(5)Compared with the sham group,the levels of inflammatory factors in the myocardial tissue of AMI rats were increased,the level of pro-apoptotic protein Bax was increased,while the level of apoptosis inhibitor Bcl-2 was decreased.(p<0.05).Regulation of miR-425-5p overexpression could inhibit the release of inflammatory factors,inhibit the expression of Bax,and promote the expression of Bcl-2 in AMI rats.3.PAI-1 is the target gene of miR-425-5p(1)A complementary sequence of miR-425-5p was found in the 3’-UTR of PAI-1.The result of the luciferase report showed that the relative luciferase activity in the wild type(WT)group was significantly inhibited by the overexpression of miR-425-5p(p<0.05).However,no change in luciferase activity was observed in the mutant(MUT)group.(2)The mRNA and protein level of the target PAI-1 showed a trend of increased expression in the H/R cells and AMI rats.4.Overexpression of PAI-1 reverses the protective effect of miR-425-5p on H/R cells(1)Overexpression of miR-425-5p could significantly inhibit the expression of PAI-1,indicating a negative correlation between them.(2)After transfection of miR-425-5p and PAI-1 plasmids into H/R cells,the proliferation and migration levels of endothelial cells were significantly decreased,compared with those transfected with miR-425-5p mimic alone,and the rate of cell apoptosis was significantly increased.At the same time,the levels of oxidative stress and inflammation of endothelial cells were also significantly increased,indicating that overexpression of PAI-1 would reverse the protective effect of miR-425-5p on H/R cells.Conclusion1.The expression of miR-425-5p was low in the model of H/R cells and AMI rats.2.Upregulation of miR-425-5p could improve endothelial cell dysfunction,inflammation and oxidative stress induced by AMI.3.Overexpression of the target gene PAI-1 of miR-425-5p would reverse the protective effect of miR-425-5p on H/R cells.Part Ⅱ The predictive value of miR-425-5p combined with TIMI risk score,NT-proBNP and D-dimer in acute myocardial infarction with no-reflow phenomenon after emergency PCIBackgroundAMI is a common clinical critical and severe disease.Percutaneous coronary intervention(PCI)and surgical revascularization are commonly used in the treatment of AMI and have significantly improved the clinical outcome of AMI.However,there are still 5%-50%of patients with no-reflow after PCI,which has become the main cause of adverse prognostic events in AMI patients.Endothelial dysfunction is one of the main mechanisms of no-reflow after PCI.In the first part of this study,miR-425-5p was found to be closely related to endothelial dysfunction,suggesting that miR-425-5p may have a certain predictive value for no-reflow after PCI.In addition,previous studies suggest that TIMI risk score has predictive value for the prognosis of interventional therapy in patients with AMI.Preoperative N-terminal pro-brain natriuretic peptide(NT-proBNP)may be a powerful predictor of no-reflow in patients with AMI after PCI.Similarly,studies have shown that D-dimer is correlated with no-reflow phenomenon.However,the prediction of disease by a single biomarker has certain limitations.The second part of this study was explored the predictive value of serum miR-425-5p combined with the TIMI risk score,NT-proBNP and plasma D-dimer in the no-reflow phenomenon after emergency PCI,so as to promote the early warning and prevention of coronary reflow in STEMI patients after PCI,and improve the prognosis of patients.ObjectiveTo investigate the expression level of serum miR-425-5p in patients with normal blood flow group and no-reflow group.And to explore the predictive value of miR-425-5p combined with TIMI risk score,NT-proBNP,and D-dimer for the occurrence of no-reflow phenomenon in AMI patients after emergency PCI.MethodsPatients with STEMI who were hospitalized in Weifang Yidu Central Hospital from October 2019 to July 2021 and underwent emergency PCI were continuously included.According to the reflow phenomenon after PCI,the patients were divided into no-reflow group and normal reflow group.Basic clinical information,TIMI risk score,Killip classification,CK-MB peak,cTn I peak,LVEF and other indicators on admission were collected.Expression level of mir-425-5p in serum of all patients before emergency PCI was detected.The possible risk factors for no reflow after PCI obtained by univariate logistic analysis was used as the independent variables,and no-reflow phenomenon was used as the dependent variable.Multivariate logistic regression analysis was used to explore the independent influencing factors of no-reflow after PCI.ROC curve was used to evaluate the predictive value of mir-425-5p,TIMI risk score,serum NT-proBNP and D-dimer for no-reflow after PCI.Results1.Baseline characteristics analysis of research objectsA total of 280 AMI patients were included including 56 patients with no-reflow and 224 cases with normal reflow.Among the collected variables,there was no statistical difference between patients in terms of gender,age,BMI,hypertension,diabetes,smoking history and drinking history(p>0.05).The proportion of hypertension and hyperlipidemia in the no-reflow group was higher than that in the normal reflow group(p<0.05).Expression of miR-425-5p was lower in the serum of patients without reflow after PCI than in the normal reflow group(p<0.05).2.Logistic regression analysis of independent risk factors of no-reflowUnivariate logistic regression analysis showed that hypertension,hyperlipidemia,number of criminal vascular lesions,length of criminal vessels,time from onset to vascular opening of the criminal,D-dimer,NT-proBNP,Killip grading,TIMI risk score and miR-425-5p were related factors of the no-reflow phenomenon after PCI.The significant indexes in the univariate logistic regression analysis results were introduced into the multivariate logistic regression model,and the results showed that miR-425-5p(OR=0.02,95%CI=0.00-0.10),TIMI score(OR=1.21,95%CI=1.11-1.33),NT-proBNP(OR=1.63,95%CI=1.29-2.05),D-dimmer(OR=1,13,95%CI=1.06-1.20),and time from onset to vascular opening of the criminal(OR=3.23,95%CI=1.02-10.26)were independent risk factors for the patients of no-reflow after PCI in AMI.3.The predictive value of miR-425-5p levels,TIMI risk score,serum NT-proBNP and D-dimer in the absence of reflow after PCIThe ROC curve of miR-425-5p for predicting reflow was 0.89.When the cut-off value was 0.76,the sensitivity and specificity were 87.50%and 75.81%,respectively.The AUC of TIMI risk score,serum NT-proBNP and plasma D-dimer in predicting no reflow after PCI were 0.89 0.86 and 0.85,respectively.When the three indexes were combined with miR-425-5p,the AUC increased significantly(p<0.05).When the four indexes were combined,the predictive and diagnostic value of AMI patients without reflow after PCI was significantly improved,and the AUC value reached 0.98(95%CI=0.96-0.99).Conclusion1.MiR-425-5p was at a low expression in the serum of AMI patients with no-reflow after emergency PCI.2.MiR-425-5p,TIMI risk score,NT-proBNP,D-dimer and time from onset to vascular opening of the criminal were independent risk factors of no-reflow after PCI in AMI patients.3.The combination of miR-425-5p with TIMI risk score,serum NT-proBNP and D-dimer had a higher and more accurate predictive value for the phenomenon of no reflow after PCI. |
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