| Background and Objective Aortic valve disease is one of the most common disease.As The economy develops,more and more caleific aortic valve diseases and bicuspid aortic valve diseases aortic require surgical intervention,without findings of effective prevention or cutting the progression of the diseases yet.Owingto suchchallengingclinicaldilemma,themolecular mechanismsresponsibleforinitiatingthe diseases attracted more investigators' attention.Recently,Circular RNA(circRNAs),a unique type ofRNA with covalently closed continuous rings,has become a new research hotspot.Species richness and evolutionary conservatism suggest that circRNAs may play a special role in cell physiology.The purpose of this study was to analyze the transcriptome of aortic valve and explore ways to prevent the occurrence and progression of aortic valve disease.Materials and methods We collected two calcified human aortic valve samples during operation obtained form department of cardiothoracic surgery of the First Affiliated Hospital of Nanjing Medical University and Jiangsu Province People's Hospital,one from a BAV patient and the other from a TAV patient.First,library preparation and high-throughput sequencing was established.Then we performed RNA-Seq data analysis,tissue specific circRNA identification,sequence conservation analysis and gene Ontology and KEGG enrichment analysis of circRNAs host genes.Results All the RNA-Seq data were extracted from the two aortic valve samples,and a gene library was constructed for each sample with removing rRNAs.The two libraries contained a mean of 112 million pair-end reads for each sample.In order to obtain the high reliable circRNAs,we constructed a stringent set of human aortic valve cir-cRNAs,including 5476 high confidence circRNAs in aortic valve using in-house pipeline.After the comparison between circRNAs identified in aortic valve and all other circRNAs downloaded from circNet,circBase and TSCD database,1412(25.79%,out of 5476)aortic valve specific circRNAs was obtained.Then the full length of 4132 mature aortic valve specific circRNA isoforms,including 1561 full exonic circRNA isoforms,2378 with partial intronflank isoforms,115 full intron isoforms and 78 in-tergenic circRNAs isoforms,were obtained by in-house scripts.The results also showed that there were 194,471 miRNA response elements(MREs)in 3060 circRNA isoforms(64 miRNA target sites percircRNA isoform).About 512(16.7%)of the circRNA isoforms had more than one hundred of target sites,and all these circRNA isoforms were come from intergenic region or with intron flanks.It is interested that the host gene of chr10:61,815,416|61,898,845 was ankyrin 3(ANK3),which encodes the ankyrin family protein ankyrin-G,and it was also proved to associate with the cardiac sodium channel gene sodium voltage-gated channel alpha subunit 5(SCN5A).Both proteins are highly expressed at ventricular intercalated disc and T-tubule membranes in cardiomyo-cytes.These results revealed that chr10:61,815,416|61,898,845 might involve in the process of cardiac conduction and this circRNA might act as a miRNA sponge.We downloaded CLIP-Seq data sets from STARBASE V2.0 which includes 37 RBPs.Computational analysis of the RBPs revealed that there were 734 cir-cRNA isoforms have complementary with RBP sequences,among them the RBP EIF4 AIII displayed the most abundant.We also observed that the Argonaute(AGO)family member AGO2 had abundant binding sites in these tissue specific circ RNAs.Conclusion 1,5476 circRNAs were detected in the human aortic valve,including 1,412(25.79%)aortic valve specific circRNAs.2,Most of the aortic valve specific circRNAs are derived from the exon region of their host genes,and most of the host genes contain less than three circRNAs.3,Most of the aortic valve specific circRNAs have abundant miRNA reaction elements(MREs),and some of the aortic valve specific circRNAs may tend to bind RBPs.Functional analysis showed that these aortic valve specific circRNAs could be used as post-transcriptional regulatory factors. |
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