The Expression Profile And Potential Regulation Mechanism Of Circular RNAs In Human Thoracic Aortic Dissection

BackgroundAcute Stanford type A thoracic aortic dissection（TAD）is a catastrophic vascular disease.Despite advances in open or endovascular surgical techniques and medical therapy over the years,Strict control of risk factors such as hypertension,atherosclerosis,smoking,the overall morbidity and mortality of TAD remain high.Owing to such challenging clinical dilemma,the molecular mechanisms responsible for initiating the dissection are not yet elucidated.Hitherto,studies are primarily focused on mutation in several categories of Syndromic TAD-associated protein-coding genes such as ECM genes and SMCs genes,which partially elucidated the general pathological processes of TAD.Therefore,excluding the protein functions,efforts should be emphasized on understanding the non-protein modulator functions in the progression of sporadic TAD.Non-coding RNA（ncRNAs）are emerging as new fundamental regulators of gene expression.The small microRNAs have opened the field of ncRNAs capturing the attention of basic and clinical scientists for their potential to become new therapeutic targets and clinical biomarkers for TAD.More recently,Circular RNAs（CircRNAs）have started to be actively investigated,leading to first exciting reports,which further suggest their important and yet largely unexplored contribution to vascular physiology and disease.Nowadays,with the rapid development of molecular biological techniques and biochip technology,researchers could reveal the pathogenesis of disease at the genetic level.This study is aimed to discuss the pathogenesis of acute Stanford type A TAD at CircRNA expression levels.Methods and materials1.Ascending aorta specimens were collected from Six Stanford A type TAD patients undergoing aortic replacement in the intimal tear position at the Guangzhou General Hospital of Guangzhou Military Region and Zhongshan Hospital in 2015.Normal ascending aorta tissues（NA）were obtained from 6 age-and gender-matched organ donors without aortic diseases.HE staining and immunohistochemistry were used to investigate the strucral characteristics of blood vessles in TAD and NA.2.NanoDrop ND-1000 was utilized to test the quality of the six specimens in the microarray test experiment.The differentially expressed circRNAs profiles of the aortic segments between human type A TAD patients（n=3）and NA（n=3）were analyzed using the Array star human circRNAs microarray.Agilent Feature Extraction software was included to extract the information contained in the biochip images obtained.GeneSpring GX software was utilized to further screen the obtained original expression information of CircRNA.3.Two Class Diff method was utilized to select the different CircRNAs.Volcano Plots and hierarchical cluster diagram showed the expression of those different CircRNAs in acute TAD vascular vessel wall tissues.Gene ontologyical（Go）analysis was made for parental genes of the differentially expressed CircRNAs,including biological process（BP）,cellular component（CC）and molecular function（MF）.The circRNA/microRNA interaction was predicted with Arraystar’s home-made miRNA target prediction software based on TargetScan&miRanda.KEGG database was used to analyze the biological signaling pathways of validated target genes of TAD-associated miRNAs.Target genes of TAD-associated miRNAs were predicted by TargetScan and miRanda.The circRNA-miRNA interaction network and the circRNA-miRNA-mRNA network of hsa_circ₁01238 were illustrated by Cytoscape.4.The dysregulation circRNAs of hsa_circRNA₁01238,hsa_circRNA₁04634,hsa_circRNA₀02271,hsa-circRNA₁02771,hsa_circRNA₁04349,hsa_circRNA₁02683,hsa_circRNA₀05525,hsa_circRNA₁03458 and parental genes of hsa_circRNA₁02121,hsa_circRNA₀58819 and hsa_circRNA₁05039,hsa_circRN A 105038,hsa_circRNA₀92022,hsa_circRNA₁05040:COL1A1,COL6A3,FLNA and hsa-miR-320a were validated by Quantitative real-time PCR（qRT-PCR）with additional samples,meanwhile,Matrix Metalloproteinase 9（MMP9）was confirmed by Western blotting（WB）with additional samples.Results1.HE show that the vascular wall of normal ascending aorae consists of intima,tunica media and adventitia.TAD ascending aorae usually do not have complete three layers of intima,tunica media and adventitia in gross observation.HE show that blood cells infiltration.Immunohistochemistry showed MMP9 were expressed in the TAD group in all levels,obviously in tunica media.2.The six selected specimens of the ascending aortic vascular vessel wall tissues of TAD and NA all complied with requirements of circRNA spectrum biochips.The RNA OD 260/280 ratios in three TAD vascular vessel wall tissues（TAD1,TAD2,TAD3）were 1.9,1.89,1.87,respectively,and those in the three NA vascular vessel wall tissues（NA1,NA2,NA3）were 1.89,1.89,1.93,respectively.All the OD 260/280 ratios were greater than 1.8 and between 1.8 and 2.1,meeting the experimental requirements.Cy3 fluorescent dyes were used mark the RNA in the tissue specimens,and the specific mark efficiencies of TAD 1,TAD2 and TAD3 were 23.2,22.5,23.5,respectively,and those of NA1,NA2 and NA3 were 23.6,22.5,22.3,respectively,they all meeting the experimental requirements.3.The circRNA expression spectrum biochips intial data results after filtering low expression messages and standardizing show that:in above-mentioned tissue specimens,the total number of detectable circRNAs was 8,173.The quality assessment of standard data show that:the Box-plot analyzed the average expression level of circRNAs in each group was in very closely level,and about 50%data met high consistency.Scatter diagram indicated that there were a large number of differentially expressed circRNAs in the TAD tissues comparing with NA tissues.4.Volcano Plots was used to analyzed the detected circRNAs,and the number of obtained differentially expressed circRNAs was 262 in patients with TAD as compared to the NA tissue（fold-change≥1.5,P-value≤0.05）,among which 106 circRNAs were downregulated while 156 were upregulated.According to the gene ontological analysis results,the top ten significantly enchiched GO terms of upregulated circRNAs gene symbols were negative regulation of cell proliferation,blood vessel development,vasculature development,regulation of synapse structure and activity,cell adhesion,biological adhesion,cell motion,cell migration,protein localization,extrcellular matrix organization;the top ten significantly enchiched GO terms of downregulated circRNAs gene symbols were actomyosin structure organization,translational elongation,nuclear migration,protein ubiquitination,cytoskeleton organization,cell cycle process,actin cytoskeleton organization,protein modification by small protein conjugation,establishment of nucleus localization,actin filament-based prosess.5.From the circRNA-miRNA interaction network,we found that a total of 1286 miRNAs could be combined with differentially expressed circRNAs,among which 28 miRNAs were associated with TAD.According to KEGG pathways analysis,the top ten enriched KEGG pathways of validated target genes of 28 TAD-associated miRNAs were focal adhension,regulation of actin cytoskeleton,vascular smooth muscle contraction,dilated cardiomyopathy,hypertrophic cardiomyopathy,axon guidance,arrhythmogenic right ventricular cardiomyopathy,adherens junction,calcium signaling pathway,valine,lenucine and isoleucine degradation.6.From the circRNA-miRNA interaction network,we found that hsa_circ₁01238 could be combined with most TAD-associated miRNAs,such as hsa-miR-320a,hsa-miR-320b,hsa-miR-320c.From the circRNA-miRNA-miRNA co expression network,we found that 46 TAD-associated target genes,such as MMP9 of hsa-miR-320a.7.The results of qRT-PCR showed that the expression levels of hsa_circRNA₁01238,hsa_circRNA₁04634,hsa_circRNA₀02271,hsa_circRNA₁02771,hsa_circRNA₁04349,COL1A1,COL6A3 was increased in patients with TAD as compared to the NA tissue 3.41,2.96,2.11,2.67,2.6,3.11,2.03,respectively;hsa_circRNA₁02683,hsa_circRNA₀05525,hsa_circRNA₁03458,FLNA,hsa-miR-320a was decreased in patients with TAD as compared to the NA tissue 5.88,5.0,2.94,2.38,2.0,respectively;WB showed that the expression levels of MMP9 was increased 1.8 in patients with TAD as compared to the NA tissue.Conclusion1.Compared with normal ascending aortae,TAD ascending aortae showed the seperaration of tunica media and characterized by extracellular matrix degradation.2.The differential expression of circRNAs between human TAD tissues and normal control tissues was universal and associated with extracellular matrix degradation and smooth muscle cell depletion.3.Dysregulated circRNAs in human TAD tissue may play a major role in the pathological processes of TAD by regulating the expression of parental genes or acting as miRNA sponges.