MiR-26a-5p Protects Against Myocardial Ischemia/Reperfusion Injury Through Regulating The PTEN/PI3K/AKT Signaling Pathway

BackgroundCardiovascular disease is one of the most common causes of death in both developing and developed countries in the world,and acute ST-segment elevation myocardial infarction（STEMI）is the most dangerous and fatal type.Although the prevention and treatment of cardiovascular disease in our country has greatly improved,the morbidity and mortality of acute myocardial infarction are still rising.Reperfusion therapy is the most effective treatment for STEMI patients.It can provide timely and effective energy to the ischemic area,thereby reducing the infarct size and maintaining cardiac contractile function.However,more and more basic and clinical studies have shown that ischemic myocardium can cause additional myocardial damage after recovery of blood and reperfusion,such as myocardial stunning,arrhythmia,no reflow,and myocardial necrosis,this phenomenon is called myocardial ischemia/reperfusion injury（MIRI）.MIRI has become a major factor affecting the outcome of reperfusion therapy and prognosis of STEMI patients.Previous researches on the mechanism of MIRI include the oxygen free radical theory,calcium overload theory,myocardial energy metabolism disorder theory,and apoptosis theory.The treatment strategies of MIRI include non-drug intervention methods such as ischemic preconditioning,post-ischemic adaptation,and remote ischemic preconditioning.Drug intervention methods include adenosine,atrial natriuretic peptide,cyclosporine A,GIK,glucagon-like peptide-1,etc.Although many cell experiments and animal experiments have proven that the above treatments are effective,the outcome of clinical trials is indeed disappointing.A single cardioprotective strategy is not effective in improving ischemia-reperfusion.Combining multiple protective strategies or synergistic multi-target therapy is very promising to treat ischemia-reperfusion injury.Therefore,it is very necessary for us to further study the molecular mechanism of MIRI.It may provide evidence for us to explore new diagnostic and therapeutic targets.More and more evidence indicates that an important pathophysiological mechanism of ischemia-reperfusion injury is apoptosis.Apoptosis is a process that requires energy,although the reperfusion process provides oxygen and energy for survival of ischemic myocardium,it also provides energy for apoptosis.The cysteine aspartic proteases（Caspase family）play a major role in the execution of apoptosis.Apoptosis is performed by a variety of Caspases.Caspase-3 is considered as a terminal cleaving enzyme in various apoptosis-stimulating factors to promote apoptosis.Studies have shown that Caspsase-3 plays an important role in the apoptosis of myocardial cells caused by ischemia-reperfusion.MiRNA is an endogenous non-coding single-stranded small-molecule RNA discovered in recent years.The target mRNA is degraded or its translation is inhibited by binding to the 3’UTR,thereby achieving the goal of regulating gene expression.miRNAs play important roles in the regulation of various cellular functions,such as embryonic development,cell apoptosis,cell growth and differentiation,and tumorigenesis.Studies have shown that in terms of cardiovascular system,miRNA is closely related to the pathogenesis of various heart diseases such as myocardial infarction,cardiac hypertrophy,myocardial remodeling,heart failure and arrhythmia.miRNAs are cell and tissue specific,have rapid release kinetics,and are relatively stable in serum.Therefore,circulating miRNAs are expected to be used for early diagnosis,risk stratification,prognosis and even treatment of cardiovascular disease.There are many studies on miRNA,some studies have shown that miRNAs play an important regulatory role in myocardial cell apoptosis,but research on myocardial ischemia-reperfusion injury is still immature.As a newly discovered miRNA,miR-26a-5p has differential expression in various cardiovascular diseases.Studies have shown that the miR-26a-5p inhibitor transfected mouse model of coronary heart disease,the viability of the isolated endothelial cells decreased significantly and the apoptosis rate increased significantly.Another study showed that overexpression of miR-26a-5p can alleviate no-reflow and myocardial necrosis caused by coronary microcirculation embolism.Given the close relationship between MIRI and cardiomyocyte apoptosis or coronary microcirculation embolism,we speculate that miR-26a-5p may be involved in reperfusion injury by regulating apoptosis.However,the role of miR-26a-5p in myocardial infarction ischemia-reperfusion injury has not been reported.We established a model of myocardial ischemia-reperfusion injury in vitro and in vivo by a hypoxia/reoxygenation model of myocardial cell and a ischemia-reperfusion model of mice.Then,we use biological experiments combined with bioinformatics methods to explore the expression and regulation of miR-26a-5p in myocardial ischemia-reperfusion injury,and further explore the possible effects and pathways of miR-26a-5p on ischemia-reperfusion injury.Therefore,it lays the experimental foundation for better explaining the mechanism of myocardial ischemia reperfusion injury and providing possible therapeutic strategies.Chapter 1 Expression and Regulation of miR-26a-5p in Hypoxic/Reoxygenated CardiomyocytesPurposeThe molecular mechanism of myocardial ischemia-reperfusion injury remains unclear,and the role of miR-26a-5p in MIRI is unclear.The purpose of the first chapter is to determine the expression level of miR-26a-5p in hypoxic/reoxygenated cells and analyze the effect of miR-26a-5p overexpression or inhibition on the degree of ischemia-reperfusion injury.Methods1.Bioinformatics analysis:Big data of miRNAs related to myocardial ischemia/reperfusion were searched from GEO database to compare and analyze differentially expressed miRNAs.2.Isolation and culture of primary cardiomyocytes:2 days old C57BL/6 embryonic mice were taken,the heart was removed after disinfection,isolated ventricles were minced and trypsinized.The cardiomyocytes were purified by differential adherence centrifugation.DMEM medium was added and cultured in 37℃incubator.3.Cell transfection:miR-26a-5p mimics,miR-26a-5p inhibitors and corresponding controls were transfected into primary cardiomyocytes using Lipofectamine█ RNAiMAX Reagent.4.Establish cardiomyocyte hypoxia/reoxygenation model:When the cardiomyocyte culture fusion reaches about 80%,it is replaced with serum-free and sugar-free DMEM medium,and placed at 37℃ with 5%CO2 and 95%N2 hypoxia incubator for 4 hours,then replace with DMEM medium containing sugar and FBS and placed in a 37 ℃ incubator containing 95%air and 5%CO2 for 2 hours.The hypoxia/reoxygenation（H/R）model was successfully established.5.Cell Experiment Grouping5.1 Experiment one grouping:1.Control group.2.Hypoxia/reoxygenation group（H/R group）.5.2 Experiment two grouping:1.miRNA control group.2.miR-26a-5p mimic group.3.Inhibitor control group.4.miR-26a-5p inhibitor group.6.MTT assay for cardiomyocyte viability:after adding MTT and dimethyl sulfoxide to the culture wells of each group of cardiomyocytes,the survival rate of cardiomyocytes was measured.7.Flow cytometry was used to determine the cardiomyocyte apoptosis rate in cell experiments.8.Detection of miR-26a-5p in cardiomyocytes of each group by real-time fluorescent quantitative PCR.9.Western Blot assay was used to detect the expression of cleaved caspase-3.Results1.The GEO website is https://www.ncbi.nlm.nih.gov/geo/,which identifies the biochip data of miRNAs related to myocardial ischemia-reperfusion and compares it with normal tissues to make a difference analysis.It was found that miR-26a-5p was down-regulated in myocardial ischemia-reperfusion tissue,and the difference was significant（P=0.0004）.2.The flow cytometry technique to detect the apoptosis rate of H/R cardiomyocytes was 12.65%,and the control group apoptosis rate was 0.71%,the difference was obvious.Western Blot test results showed that the expression of the apoptotic protein cleaved caspase-3 in H/R cardiomyocytes was significantly higher than that in the control group（P=0.0003）..3.The results of qRT-PCR showed that the content of miR-26a-5p in H/R cardiomyocytes was significantly lower than that in the control group（P=0.0003）.4.The level of miR-26a-5p in cardiomyocytes increased significantly after transfection of miR-26a-5p mimic;but the content of miR-26a-5p in cardiomyocytes decreased significantly after transfection of miR-26a-5p inhibitor（P<0.0001）.MTT test results showed that compared with the control group,overexpression of miR-26a-5p significantly increased the cell viability of cardiomyocytes after hypoxia/reoxygenation.Inhibition of miR-26a-5p expression significantly reduced myocardial cell viability after hypoxia/reoxygenation（P<0.01）.5.The results of flow cytometry showed that the apoptosis rate of H/R cardiomyocytes in the overexpressed miR-26a-5p group was significantly lower than that in the control group.The apoptosis rate of miR-26a-5p mimic group was 7.54%,and that of mimic control group was 20.86%,indicating a significant difference.The apoptosis rate of H/R cardiomyocytes in the inhibited miR-26a-5p group was significantly higher than that in the control group,with the apoptosis rate of 35.89%in the miR-26a-5p inhibitor group and 23.25%in the inhibitor control group,indicating a significant difference.Western Blot results showed that the content of cleaved caspase-3 in H/R cardiomyocytes in the overexpressed miR-26a-5p group was significantly lower than that in the control group.The content of cleaved caspase-3 in H/R cardiomyocytes of the inhibited miR-26a-5p group was significantly higher than that of the control group（P<0.001）.Conclusions and significance1.Compared with the normal control group,the expression of miR-26a-5p in hypoxic/reoxygenated cells decreased significantly;the myocardial cell apoptosis rate in the ischemia-reperfusion group increased significantly.2.In hypoxic/reoxygenated cardiomyocyte models,overexpression of miR-26a-5p can reduce myocardial ischemia-reperfusion injury by reducing the rate of cardiomyocyte apoptosis;while low expression of miR-26a-5p can increase myocardial cell apoptosis and aggravates myocardial ischemia-reperfusion injury.3.Our results may help to discover new mechanisms that occur and progress in myocardial ischemia-reperfusion injury,and help to develop new early diagnostic biomarkers or new treatments for MIRI.Chapter 2 miR-26a-5p Improves Myocardial Ischemia-Reperfusion Injury by Regulating PTEN/PI3K/AKT Signaling PathwayPurposemiRNA is a type of endogenous non-coding single-stranded small molecule RNA,which binds to the 3 ’untranslated region of the target gene mRNA to degrade or inhibit the target gene.miRNA has the characteristics of functional diversity,and the study of target gene functions of miRNA is more complicated.In recent years,with the development of large-scale miRNA analysis databases such as TargetScan and miRBase,scientists have been able to predict miRNA target genes.Our findings in Chapter 1 show that miR-26a-5p is down-regulated in myocardial ischemia-reperfusion cells,and overexpression of miR-26a-5p can reduce myocardial ischemia-reperfusion injury,but the specific mechanism is not clear.The purpose of this chapter is to predict the target genes of miR-26a-5p,to study the possible mechanism of miR-26a-5p regulating myocardial ischemia-reperfusion injury and to further verify.Methods1.Bioinformatics analysis:Targetscan database predicting the target genes of miR-26a-5p（http://www.targetscan.org/vert₇2/）.2.Double luciferase reporter gene test to verify whether miR-26a-5p and 3’UTR of PTEN have complementary binding relationship.We constructed the dual fluorescent reporter vectors pMIR-REPORTM-PTEN-WT and pMIR-REPORTTM-PTEN-Mut,together with miR-26a-5p mimic and negative control were transfected into myocardial cells,and the changes of luciferase activity in each group were observed 24 hours later to verify whether PTEN is a target gene of miR-26a-5p.3.Cell culture,transfection of miRNA and simulated hypoxia/reoxygenation model（H/R）:same as the first chapter.4.Cell experiment grouping:4.1 Experiment one grouping:detection of luciferase reporter gene.1.Transfect pMIR-REPORTTM-PTEN-WT+miR-26a-5p mimics group.2.Transfect pMIR-REPORTTM-PTEN-WT+miRNA control group.3.Transfect pMIR-REPORTTM-PTEN-Mut+miR-26a-5p mimics group.4.Transfect pMIR-REPORTTM-PTEN-Mut+miRNA control group4.2 Experiment two grouping:1.Control group;2.Hypoxia/reoxygenation group（H/R group）.4.3 Experiment three grouping:1.miRNA control group.2.miR-26a-5p mimic group.3.Inhibitor control group.4.miR-26a-5p inhibitor group.5.qRT-PCR technology was used to detect the content of PTEN gene;Western Blot technology was used to detect the expression of PTEN/PI3K/AKT protein,the method is similar to the first chapter.Results1.Targetscan predicts PTEN as a target gene for miR-26a-5p.2.The double luciferase reporting experiment showed that compared with the control group,the luciferase activity of wild-type PTEN-3’UTR and miR-26a-5p mimics co-transfection was significantly reduced（P=0.0003）.While there was no significant difference in luciferase activity between co-transfection of mutant PTEN-3’UTR and miR-26a-5p mimics groups compared with the control group（P>0.9999）.The results indicate that miR-26a-5p can directly interact with the 3 ’UTR of PTEN.3.The content of PTEN gene in H/R cardiomyocytes was detected by qRT-PCR.The results showed that the content of PTEN gene in H/R group was significantly higher than that in normal control group（P=0.0038）.Western Blot detected the expression level of PTEN protein in H/R cardiomyocytes.The results showed that the expression level of PTEN protein in H/R group was significantly higher than that in control group（P=0.0001）.4.Transfection of cardiomyocytes were used to construct the over-expressing miR-26a-5p group,knockdown miR-26a-5p group,and the corresponding control group.Western Blot test showed that over-expressing miR-26a-5p significantly down-regulated PTEN protein expression level,at the same time significantly increased the expression levels of PI3K and AKT.On the contrary,knocking down miR-26a-5p significantly increased the expression level of PTEN protein,while significantly reducing the expression levels of PI3K and AKT（P<0.001）.Conclusions and significance1.PTEN is a target gene of miR-26a-5p during myocardial ischemia-reperfusion.2.miR-26a-5p negatively regulates PTEN protein expression by binding to 3’UTR of PTEN.After PTEN is inhibited,PI3K/AKT pathway is reversely activated to achieve anti-cardiac cell apoptosis,thereby ameliorating myocardial ischemia-reperfusion injury.damage.3.Studying the specific regulatory mechanism of miR-26a-5p will help us to better understand the mechanism of ischemia-reperfusion injury,and help to develop new drug or non-drug treatment methods for myocardial ischemia-reperfusion injury.Chapter 3 Expression and Regulation of miR-26a-5p in Ischemia-Reperfusion MicePurposeIn previous studies,we established a model of cardiomyocyte hypoxia/reoxygenation（H/R）through in vitro experiments and found that the apoptosis rate of cardiomyocytes in the H/R group increased,and the expression of miR-26a-5p decreased in the H/R group.Overexpression of miR-26a-5p reduced myocardial cell ischemia-reperfusion injury.It was also found that PTEN is a target gene of miR-26a-5p.miR-26a-5p improves myocardial ischemia-reperfusion injury through the PTEN/PI3K/AKT pathway.In this chapter,in mouse heart experiments in vivo,we further verify whether miR-26a-5p has the same effect on myocardial ischemia-reperfusion injury.Methods1.Establishment of mice model of ischemia-reperfusion injury:C57BL/6 mice were placed in a supine position after anesthesia,connected to an animal ventilator after intubation,and the limbs of the mice were connected to an electrocardiograph via electrodes.After iodine disinfection of the chest area,thoracotomy was performed on the third and fourth intercostal space.The left anterior descending coronary artery was ligated 1-2mm from the lower edge of the left atrial appendage,and a significant ST segment elevation was found to indicate successful ligation.30 minutes after the blood flow was blocked,the suture was released,the coronary arteries regained blood flow,and the mice model of myocardial ischemia-reperfusion was successfully established.In the sham operation group,sutures only passed through the heart of the mice,and no ligation was performed.2.Myocardial transfection of mice:Construction of adeno-associated virus AAV9-miRNA vector.8-week-old C57BL/6 mice were randomly divided into groups and injected with AAV9-miRNA-26a-5p or AAV9-control via tail vein to construct a transfection model.3.Animal experiment grouping3.1 Experiment one grouping:1.Sham group;2.I/R group.3.2 Grouping of experiment two:1.miRNA control+I/R group;2.miR-26a-5p mimic+I/R group;3.inhibitor control+I/R group;4.miR-26a-5p inhibitor+I/R group.4.TTC-Evans blue double staining method to detect myocardial infarction area of mice in each group.5.Flow cytometry was used to determine the apoptosis rate of myocardial cells in each group of mice in animal experiments.6.Real-time fluorescent quantitative PCR was used to detect miR-26a-5p and PTEN content in myocardial tissue of mice in each group.7.Western Blot assay was used to detect the expression of cleaved caspase-3,PTEN,PI3K and AKT.Results1.Evans blue/TTC staining showed that the area of myocardial infarction in the I/R group was larger than that in the control group（P=0.0058）.Flow cytometry showed that the apoptosis rate of myocardial cells in the I/R group was significantly higher than that in the control group.The apoptosis rate of myocardial cells in the I/R group was 40.24%,while that in the control group was 8.83%,showing a significant difference.The Wesetern Blot test showed that the myocardial cleaved caspase-3 protein content in the I/R group was significantly higher than that in the control group（P=0.0015）.2.The results of qRT-PCR showed that the expression of miR-26a-5p in cardiomyocytes of mice in the I/R group was significantly lower than that in the normal control group（P<0.0001）.3.qRT-PCR was used to detect the expression of miR-26a-5p in the myocardium of the transfected mice in each group,and the results showed that the level of miR-26a-5p in the myocardium of mice transfected with miR-26a-5p mimic was significantly increased.The content of miR-26a-5p in the myocardium of mice transfected with miR-26a-5p inhibitor was significantly reduced（P<0.01）.Evans blue/TTC staining showed that over-expression of miR-26a-5p significantly reduced the infarct size after myocardial I/R in the mice,and the infarct area after myocardial I/R in the miR-26a-5p inhibitor group was significantly larger than that in the control group（P<0.05）.4.qRT-PCR showed that the content of myocardial PTEN gene in the I/R group was significantly higher than that in the normal control group（P=0.0080）.Western Blot test results showed that the expression of PTEN protein in the myocardial tissue of the I/R group was significantly higher than that of the control group（P<0.0001）.5.Western Blot test showed that in the mice I/R injury model,compared with the control group,overexpression of miR-26a-5p significantly reduced the expression level of PTEN protein,while significantly increasing the protein expression levels of PI3K and AKT.In contrast,the miR-26a-5p inhibited group significantly increased the expression level of PTEN protein and significantly reduced the protein expression levels of PI3K and AKT.The differences between the groups were statistically significant（P<0.001）.Conclusions and significance1.Compared with normal control,in ischemia-reperfusion mice model,cardiomyocute apoptosis and caspase-3 expression were increased,and miR-26a-5p expression was significantly decreased in ischemia-reperfusion tissues.Overexpression of miR-26a-5p reduced myocardial ischemia-reperfusion injury;low expression of miR-26a-5p could increase myocardial apoptosis and aggravate myocardial ischemia-reperfusion injury.2.In vivo experiments we have further verified that the expression of PTEN gene and protein is significantly increased in ischemia-reperfusion tissues.PTEN is a target gene of miR-26a-5p;miR-26a-5p can regulate myocardial ischemia-reperfusion injury through the PTEN/PI3K/AKT pathway.3.The results of in vivo experiments further validate the regulatory role and mechanism of miR-26a-5p on myocardial ischemia-reperfusion injury,and provide an experimental basis for the development of new early diagnostic biomarkers or new treatments for myocardial ischemia-reperfision injury.