| With the improvement of prevention,diagnosis and treatment of cardiovascular diseases(CVDs),its morbidity and mortality have declined However,CVDs is still one of the most important causes of death in both developing and developed countries.In 2015,about 17.5 million people died from cardiovascular disease,accounting for 31 percent of all deaths worldwide.Cardiovascular morbidity in China is still increasing..Coronary artery occlusion during myocardial ischemia can lead to severe hypoxic damage.Rapid restoration of coronary blood flow through either thrombolytic therapy or percutaneous coronary intervention(PCI)is essential to limiting myocardial infarction(MI)size,preserving left ventricular(LV)ejection fraction and preventing LV remodeling.Revascularization via either thrombolytic therapy or PCI reduces the mortality rate of patients suffering a heart attack by almost50%.Paradoxically,restoring blood flow to the ischemic myocardium can also induce myocardium injury.This phenomenon was therefore called myocardial ischemia reperfusion injury.The concept of ischemia reperfusion put forward in 1960 by Jennings for the first time.Studies have shown that ischemia/reperfusion injury is associated with oxygen free radical release,intracellular calcium overload,myocardial energy metabolism disorder,neutrophil invasion,vascular endothelial cell injury and apoptosis.People have to seek new treatment strategies to protect the heart from I/R injury,thereby improving the prognosis of myocardial infarction.People have found that mi RNA is highly expressed in the cardiovascular system,it is involved in the pathophysiological processes such as heart development,myocardial remodeling,myocardial hypertrophy,myocardial cell apoptosis,arrhythmia and heart failure.In recent years,more attention has been paid to the regulatory role of mi RNA in cardiac I/RI.Studies found that mi R-1,mi R-21 and mi R-24 could reduce the range of myocardial infarction in mice induced cardiac I/RI.Ren et al.found that mi R-320 expression was significantly decreased in ischemia/reperfusion injury,Silencing of mi R-320 could increase the expression of heat-shock protein 20(Hsp20),thereby reducing the myocardial cell apoptosis induced by I/RI.In this study,primary myocardial cells and H9C2 myocardial cell lines were used as research objects.the methods involved in the experiment are as follows:real time fluorescence quantitative polymerase chain reaction(RT-PCR),protein imprinting method(Western Blot),MTS experiment,LDH,flow cytometry and immunocoprecipitation.We first contrasted the differences of mi R-199a-5p expression with or without ischemia-reperfusion stimulation.Then through experiments in vitro inhibition of mi R-199a-5p on myocardial cell sugar oxygen deprivation reperfusion injury of cell survival rate,and the apoptosis rate,mitochondrial membrane potential level.We further investigated the ways of mi R-199a-5p on myocardial ischemia-reperfusion injury by using RNA interference,so that we can provide a new therapeutic target for myocardial ischemia reperfusion injury.Part one The expression of mi R-199a-5p in patients of acute myocardial infarction and cell lines treated by OGD/RObjective: To investigate the expression of mi R-199a-5p in patients of acute myocardial infarction and cardiomyocytes.Methods:1.Expression levels of mi R-199a-5p in the plasma obtained from AMI patients(n = 19)and healthy subjects(n = 23)was determined by RT-PCR.2.Expression levels of mi R-199a-5p in the OGD/R-induced primary cardiomyocytes were examined by RT-PCR.3.Expression levels of mi R-199a-5p in the OGD/R-induced H9c2 cells were examined by RT-PCR.Results: mi R-199a-5p expression was significantly up-regulated in the plasma of AMI patients,and its expression was high in primary cardiomyocytes and H9c2 cells.1.Results of RT-PCR showed that mi R-199a-5p expression was significantly up-regulated in the plasma of AMI patients.2.Compared to the control group,the mi R-199a-5p expression in primary cardiomyocytes and H9c2 cells were significantly high.Summary: The expression of mi R-199a-5p was significantly upregulated in myocardial ischemia reperfusion injury.Part two The effects on apoptosis of mi R-199a-5p in OGD/R-induced H9c2 cell modelObjective: The effect of mi R-199a-5p on the biological behavior of OGD/R-induced H9c2 cell model.Methods:1.Establish the OGD/R-induced cell model with most effective mi R-199a-5p overexpression and knockdown.2.Cell viability and LDH leakage by overexpression of mi R-199a-5p was confirmed by LDH assay.Western Blot were used to further confirm effect of overexpression of mi R-199a-5p on the HIF-1??P-GSK3?/GSK3?.3.Cell viability and LDH leakage by knockdown of mi R-199a-5p was confirmed by MTS and LDH assay.4.The change of apoptosis by knockdown of mi R-199a-5p were investigated by TUNEL staining assay.5.Degree of MPTP opening by knockdown of mi R-199a-5p was assessed by flow cytometry.Results:1.H9c2 cells model with most effective mi R-199a-5p overexpression and knockdown were established.2.MTS results showed that mi R-199a-5p mimics significantly increased the OGD/R-injury in H9c2 cells.LDH results showed that mi R-199a-5p mimics group enhanced the LDH leakage than OGD/R group.Western Blot results showed that the mi R-199a-5p mimics could regular the expression of HIF-1??P-GSK3?/GSK3?.3.MTS results showed that mi R-199a-5p inhibitor significantly increased the OGD/R-injury in H9c2 cells.LDH results showed mi R-199a-5p inhibitor significantly decreased the OGD/R-injury in H9c2 cells.4.TUNEL staining result showed that the mi R-199a-5p inhibitor significantly decreased the OGD/R-injured apoptosis rate in H9c2 cells,compared to the OGD/R-treated group.5.Compared with the OGD/R-treated group,the degree of m PTP opening were significantly decreased in mi R-199a-5p inhibitor group.Summary:1.Overexpression of mi R-199a-5p significantly aggravated the extent of the OGD/R damage.2.Knockdown of mi R-199a-5p significantly alleviated the extent of the OGD/R damage.Part three Knockdown of mi R-199a-5p attenuates OGD/R-induced cytotoxicity in H9c2 cells by regulating the HIF-1?/PGSK3?/m PTP pathway.Objective: To study the effect of on the HIF-1?/P-GSK3?/m PTP pathway.Methods:1.Targetscan?MIRBase and MIRDB was used to predict the potential targets of mi R-199a-5p.2.Luciferase reporter assay were used to further confirm the direct binding between mi R-199a-5p and HIF-1?.3.Western Blot were used to further confirm effect of on the HIF-1?/PGSK3? /m PTP pathway.4.Cell immunofluorescence assays were used to further confirm effect of mi R-199a-5p on m PTP.5.Coimmunoprecipitation assays were used to further confirm effect of mi R-199a-5p on m PTP.Results:1.Luciferase reporter showed that cotransfection with mi R-199a-5p mimic and wild-type vector HIF-1?(WT)distinctively reduced the luciferase activity.However,cotransfection with mi R-199a-5p and mutant type HIF-1?(MUT)showed no obvious effect on the luciferase activity in 293 A cells.2.Western Blot results showed that the mi R-199a-5p could regular the HIF-1?/P-GSK3?/m PTP pathway.3.Cell immunofluorescence results showed the effect of mi R-199a-5p inhibitor on m PTP.4.Co-immunoprecipitation results showed the effect of mi R-199a-5p inhibitor on m PTP.Summary:1.Luciferase reporter showed that HIF-1? was a target of mi R-199a-5p.2.Knockdown of mi R-199a-5p attenuates OGD/R-induced cytotoxicity in H9c2 cells by regulating the HIF-1?/P-GSK3?/m PTP pathway.Conclusions:1.mi R-199a-5p expression was significantly up-regulated in myocardial ischemia reperfusion injury.2.Knockdown of mi R-199a-5p significantly alleviated myocardial ischemia reperfusion injury by regularing the HIF-1?/P-GSK3?/m PTP pathway. |
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