The Differential Expression And Functional Analysis Of LncRNA In Acute Coronary Syndrome With Coronary Microvascular Dysfunction

Coronary microvascular dysfunction is a structural and functional change of coronary vessels with a diameter of less than 500μm,which seriously affects myocardial perfusion and cardiac function.A significant proportion of acute coronary syndromes patients with normal epicardial coronary flow after primary percutaneous coronary intervention have diminished tissue-level perfusion in the myocardial regions,especially in ST-elevation myocardial infarction(STEMI).Recently,the autopsy studies of sudden death coursed by STEMI have shown that plaque erosion and coronary micro-embolism are found in the coronary epicardial vessels and Microvascular.The coronary microvascular dysfunction often result in persistent ST-segment elevation and impact long-term prognostic.The pathological changes of coronary microvascular dysfunction include abnormal microvascular endothelial swelling and compression of microvascular structures,and microembolization.It affects the outcome of patients and has a high fatality rate and more heart failure,which has become one of the hot topic in cardiovascular medicine.Previous animal and clinical studies have shown that the higher expression of inflammatory mediators are associated with more myocardial cell apoptosis in coronary microembolization.In animal models of coronary microembolization,the myocardial minor infarction is mismatch with sever impair global cardiac function,in which apoptosis in the peri-infarct area plays an important role.The activation of apoptotic molecules is affected by a variety of factors,including inflammation,ischemia and hypoxia,and reperfusion injury.However,the key regulatory signals and regulatory networks underlying the pathogenesis of microvascular dysfunction have not been fully elucidated.RNA-seq,as the main analysis method of genomics research,has promoted the research progress of genes including protein-coding mRNA,non-coding RNA,tRNA,mtRNA and so on.Compared with microarray technology,RNA-seq has considerable advantages in many aspects,such as novel transcript identification by de novo assembly,splice junction identification and allele-specific expression analysis.For the genomics study of these regulatory key molecules,RNA-seq provides us with powerful data analysis capabilities.Long non-coding RNAs(lncRNA)are a class of non-coding RNAs with a length of more than 200 nucleotides,which modulate target gene’s pre-transcript or post-transcriptional translation and their expression is specific in time and tissues.It participates in cardiac-related gene expression and various biological regulation processes of the heart in various ways.However,we still lack research on the expression levels of lncRNA and functions of regulating apoptosis in patients with coronary microvascular dysfunctions。The main purposes of this study are as follows:1.To study the expression profile of lncRNA in acute coronary syndrome patients with coronary microvascular dysfunction(CMD);2.To analyze the effect of specific lncRNA on the risk of CMD,the diagnostic value with clinical pathological parameters。3.To explore the biological functions of regulating apoptosis mechanism in endothelial cells Therefore,in this study,we used RNA-seq method to extract RNA from coronary blood of ACS patients with microvascular dysfunction,and analyzed the expression of long non-coding lncRNA and coding RNA.Quantitative PCR validation of specific lncRNA in coronary blood sample and human umbilical venous cells,establishment of vitro OGD model,and detection of apoptosis-related pathway signals.Our in vivo and in vitro studies provide important evidence for the expression and potential function of LncRNA in coronary micro vascular dysfunction。Part I Study on the Gene Expression Level of LncRNA in Coronary plasma in Patients with Acute Coronary Syndrome and Coronary Micro vascular DysfunctionObjective To establish the lncRNA and mRNA expression profiles of acute coronary syndrome patients in coronary plasma between low perfusion groups and normal perfusion groups,to study the functions and pathways involved in the differentially expressed lncRNA,and to analyze the functions between the expression differences lncRNA and enrichment pathways of lncRNA.Methods Microvascular perfusion assessed by myocardial blush grade(MBG)was evaluated in patients with acute coronary syndrome.Coronary blood plasma was collected from the patients status post-coronary intervention and then patients were divided into low MBG(0 and 1)group and high MBG(2 and 3)group.Total RNA was extracted by RNA Kit,and RNA-seq sequencing and data analysis were performed.Differentially expressed lncRNA were defined as p<0.05 and fold change value ≥1.5.Bioinformatics methods were used to predict the function and the pathways among the selected lncRNA.To verify the expressed level of these selected lncRNA,Selected lncRNA perform quantitative PCR verification on the 260 patients with acute coronary syndrome which low MBG group vs high MBG group;Binary logistic regression analysis to study risk factors and differentially clinical characteristics Correlation with Microvascular perfusion.Results A total of 3016 differentially expressed lncRNA gene and 11293 differentially expressed mRNA gene were identified by RNA-seq between low MBG group and high MBG group.There are differentially expressed 13377 lncRNA transcripts and 16130 mRNA transcripts.GO analysis showed that the differential mRNAs gene related to low microvascular perfusion were mainly involved in lipid metabolism molecules,regulation of transcription factor activity,cytoskeleton and metabolism regulation and other biological functions.KEGG analysis showed that the differentially expressed mRNA enrichment pathways mainly involved Hippo pathway,autophagy,lipid metabolism signals.The results of quantitative PCR verification showed that the expression levels of two lncRNA including AC145207 and LINC00294 increased,while the expression of AC005332.7 decreased in the low MBG group.Univariate analysis of clinical baseline data showed that there were differences in left ventricular ejection fraction,thrombus burden score,and absolute value of neutrophils.Multivariate regression analysis model showed that only LVEF remained statistically significant between groups(p<0.05).Conclusions:1.There are differentially expressed lncRNA in patients with acute coronary syndrome microcirculation disorder and normal blood flow group.The differentially gene expressed mRNA and lncRNA are mainly up-regulated,and their functions involve cytoskeleton and lipid metabolism,and are mostly enriched in signaling pathways such as Hippo pathway and autophagy.2.Quantitative PCR showed that the expressions of LINC00294,AC145207 and AC005332.7 were significantly different between ACS patients with coronary microvascular dysfunction which was consistent with the prior results of bioinformation.3.Logistic regression analysis showed that LVEF can be used as a predictor of low microvascular perfusion in acute coronary syndrome.Part Ⅱ Expression Levels of LncRNA in Human Umbilical Vein Endothelial Cells under Hypoxia and HypoglycemiaObjective The expression of the selected lncRNA genes were testified in human umbilical vein blood endothelial cells.The selected lncRNA and the possible apoptosis signaling pathway were studied in the hypoxic and hypoglycemic environment mimicking coronary microvascular dysfunction.Methods Human umbilical vein endothelial cells were cultured in vitro and divided into normal culture group and hypoxia-hypoglycemia deprivation persisting 4hours group(OGD4h).The morphological changes of human blood endothelial cells were observed under an inverted microscope;the cell viability was detected by CCK8 method,and the gene expression of AC145207,LINC00294,and AC005332.7 were detected by real-time quantitative PCR;A western blot method was used to determine the expression of apoptosis related proteins including p-PI3K,AKT,Bax,Bcl-2,and Caspase3.ROC curve prediction of lncRNA AC005332.7.Results 1.Human umbilical vein endothelial cells were treated with hypoglycemia and hypoxia for 4 hours,the expressional level of AC145207 and LINC00294 was not significantly increased,while the expression of AC005332.7 was decreased,and the difference was statistically significant.2.The cell morphology of this cell line changed after modeling,and the cell viability decreased and the cell morphology changed in OGD groups,indicating that the modeling was successful at the functional level.3.The expression of AC005332.7 decreased in ODG group.The protein expression rate of p-PI3K/AKT was also decreased,by contrast the protein expression rate of Bax/Bcl-2 was increased,and the caspase3 was also significantly increased.The differences were all statistically significant.4.In the low MBG group,the ROC curve results of AC005332.7 showed that its AUC for microvascular dysfunction diagnosing was 0.729,with 95%confidence intervals of 0.598 and 0.860.(p=0.004)Conclusions 1,In the OGD group,the gene expression of AC005332.7in human umbilical vein endothelial cells decreased,cell viability inhibited and cell apoptosis increased.2,Part of its mechanism maybe related to the decreased activity of phosphorylated PI3K-AKT protein and the increased expression of Caspase3 apoptotic protein 3,The lncRNA AC005332.7 has the ability to diagnose acute coronary syndrome with poor microcirculation perfusion.