Transcriptome Analysis And Functional Study Of Myocardial Ischemia-reperfusion Injury In Different Time Series

The extensive development of revascularization for acute myocardial infarction has greatly reduced the area of myocardial infaction and improved the prognosis.However,Myocardial Ischemia Reperfusion injury(MIRI)occurs while the recovery of blood perfusion,which aggravates the myocardial injury and leads to myocardial cell apoptosis,cardiac arrest and other adverse events.As a result,patients after revascularization are still at risk of heart failure and cardiac arrest.The mechanism and evaluation of MIRI have received extensive attention and research limited to the aspects of hiopathology,myocardial injury markers and oxidative stress.Noncoding RNAs have great potential as molecular markers of pathophysiological processes and therapeutic targets of diseases.We explored the post-transcriptional regulation mechanism of myocardial ischemia/reperfusion injury from the perspective of non-coding RNAs.In this study,we estabilised the MIRI model and analysed the RNAs by high-throughput sequencing.Then,we used quantitative real-time PCR(q PCR)and Western blot(WB)to verify the 11 molecules with the most significant changes in the sequencing results in the myocardial tissues of MIRI and the Hypoxia /Reoxygenation(H/R)induced apoptosis model.The third part is to explore the function of lnc RNA MAPKAPK5 and mi R-124-3p by using gene interference technique.Part Ⅰ.Transcriptome Analysis in myocardial tissue of rats with different reperfusion time seriesObjective:To construct MIRI model at the animal level and identify the non-coding RNAs regulatory molecules that change with the time of reperfusion by sequencing and analyzing the tissues of MIRI at different times.Methods:We established the MIRI animal model by ligating the left descending anterior.The ischemia time was 30 minutes.Then we solved ligature to build reperfusion injury model in reperfusion for 30 minutes,60 minutes,120 minutes.The rats in each group were sacrificed for heart tissue and blood sampling tissue.Hematoxylin eosin staining and immunohistochemical staining were used to evaluate the MIRI;Serum myocardial injury markers were detected by enzyme-linked immunoassay(ELISA).After the evaluation,myocardial tissues with the same trend were selected to extract RNA and protein.After the quality control of RNA,high-throughput sequencing technology was used to sequence the myocardial tissues.The differentially expressed m RNA,lnc RNA and mi RNA in each group were analyzed,and the timing sequence and trend prediction analysis were conducted to construct possible post-transcriptional regulatory chains.Results:1.In the early stage of MIRI(Reperfution 30mins),there were 177 differentially expressed m RNAs,11 lnc RNAs and 2 mi RNAs,and the differentially expressed RNAs were mainly concentrated in the pathophysiological processes such as inflammation and oxidative stress.2.In the late stage of MIRI(Reperfution 120mins),there were 143 differentially expressed m RNAs,330 lnc RNAs and 115 mi RNAs,and the differentially expressed RNAs were concentrated in the pathophysiological processes such as apoptosis and cell cycle regulation.3.During the whole process of MIRI,there were 11 regulatory molecules with significant differences with reperfusion time,including LOC10369223,MAPKAPK5,LOC102553854,LOC103690547,mi R-124-3p,mi R-21,mi R-615,mi R-125-5p,E2F3,PTEN,and PI3 K.Part Ⅱ.Transcriptional molecule expression after myocardial ischemia with time of reperfusion Objective:To verify the accuracy of sequencing results,we constructed and validated MIRI model in vivo and in vitro experiments.Methods:11 molecules with the most significant changes in the sequencing results were verified by q PCR and WB detection in the myocardial tissue of MIRI and the H/R-induced apoptosis model.Results:1.q PCR and WB results of cell H/R model and MIRI myocardial tissue showed that the quantitative analysis of the 11 regulatory molecules with the most significant changes over time was consistent with the high-throughput sequencing results.2.In the process of MIRI,the expression levels of mi R-124-3p and LOC103690547 gradually decreased with the reperfusion time,while the expression levels of mi R-615 and lnc RNA MAPKAPK5 gradually increased with the reperfusion time,suggesting that they may be involved in the whole process of the occurrence and development of MIRI.Part Ⅲ.Exploring the mechanism of MAPKAPK5/ mi R-124-3p/E2F3 axis Objective:After verifying the sequencing results,bioinformatics analysis and mechanism speculated that MAPKAPK5,mi R-124-3p and E2F3 might regulate each oher.To explore the mechanism by which MAPKAPK5 and mi R-124-3p aggravate MIRI.Methods:The changes of plasmid vector and interfering RNA and the expression level of MAPKAPK5 in H9C2 cardiomyocytes were used to construct H/R model.The physicochemistry of apoptosis moleculars and the expression changes of mi R-124-3p and E2F3 were detected by q PCR and WB.The dual luciferase reporting system verified the target relationship of MAPKAPK5,mi R-124-3p and E2F3.Results:1.After the overexpression of lnc RNA MAPKAPK5-AS1,the expression of anti-apoptotic proteins decreased and the expression of pro-apoptotic proteins increased,indicating that the overexpression of lnc RNA MAPKAPK5-AS1 can aggravate the apoptosis of cardiomyocytes induced by hypoxia/reoxygenation.2.After the overexpression of lnc RNA MAPKAPK5-AS1,the expression of mi R-124-3p decreased and the expression of E2F3 increased;after the silencing of lnc RNA MAPKAPK5-AS1,the expression of mi R-124-3p increased and the expression of E2F3 decreased.Meanwhile,the expression of E2F3 returned to normal level after the silencing of lnc RNA MAPKAPK5-AS1 and mi R-124-3p.It shows that there is a certain regulatory relationship among the three.3.The target relationship of lnc RNA MAPKAPK5-AS1/mi R-124-3p/E2F3 was confirmed by dual luciferase reporting system.Conclusion:1.Different genes are expressed in different time sequences during the process of ischemia reperfusion injury.The low expression of mi R-124-3p and LOC103690547 and the high expression of mi R-615 and lnc RNA MAPKAPK5-AS1 during the whole process of MIRI are expected to become time-sensitive molecular markers of myocardial ischemia reperfusion injury.2.Downregulation of the expression of lnc RNA MAPKAPK5-AS1 can improve myocardial apoptosis after myocardial hypoxia/reoxygenation and may become a therapeutic target for improving MIRI.3.Downregulation of the expression of lnc RNA MAPKAPK5-AS1 can up-regulate the expression of mi R-124-3p and down-regulate the expression of E2F3,thus alleviating myocardial ischemia reperfusion injury.