Long Noncoding RNA Expression Profile And The Effect Of HOTAIR In Sporadic Thoracic Aortic Aneurysm

Background:Thoracic aortic aneurysm(TAA)is a high-risk of sudden death disease duo to thoracic aortic structure damage caused by a variety of factors.Majority of TAA patients are asymptomatic,until imaging examination are made or fatal complications occur,such as rupture or aortic dissection.Although the aorta dilates slowly,there is no effective drugs to inhibit the progression of TAAs.TAAs can be classified into three types,including syndromatic,family and sporadic TAAs.Many studies have discovered specific pathogenic gene loci related to syndrome and family TAAs,but the pathogenesis of sporadic thoracic aortic aneurysm(STAA)which is the largest group of TAAs remains unclear.Whether reliable targeted therapies can be found is depend on understanding of the pathogenesis.As a recently discovered noncoding RNA,long noncoding RNA(IncRNA)can regulate gene expression on both transcriptional and post-transcriptional level.LncRNAs have also been reported to participate in cardiac development and many cardiovascular diseases.However,their roles in STAA are unknown.Therefore,the objective of this study is to quantify the expression profile of lncRNA in STAA,to observe the effect of IncRNA HOTAIR on human aortic smooth muscle cells and extracellular matrix,to explore the mechanism of its biological functions,and to present a new clue for the pathogenesis of STAA.We describe our work in following two pats.PART ?:The Expression Profile of Long Noncoding RNA in Sporadic Thoracic Aortic AneurysmObjective:To discover the difference of IncRNA expression profile in STAA through microarray and to trace target lncRNAs may be associated with STAA through bioinformatics analysis.Methods:Ascending aortic specimens were obtained from STAA patients at the time of aneurysm repair(STAA group)and from coronary artery disease patients at coronary bypass graft(control group)at Fuwai Hospital between July 2013 and June 2015.Differentially expressed lncRNAs and mRNAs between four pairs of age-,sex-,blood pressure-,blood fat-and blood glucose-matched samples were identified by microarray analysis.Quantitative real-time polymerase chain reaction(qRT-PCR)was performed to verify the validity of the microarray analysis results.Then Gene Ontology enrichment,lncRNA-mRNA correlation analysis and target prediction were implemented with differentially expressed genes.Results:Compared with the control group,1105(5%)lncRNAs were significantly differentially expressed(DEG)(P<0.05 and fold change>2),in which 151(1%)were upregulated and 954(4%)were downregulated in STAA group.Among all DEG lncRNAs,antisense lncRNAs account for 58%of upregulated lncRNAs,whereas majority of downregulated lncRNAs was intergenic lncRNAs(68%).Chromosome 2 had most DEG lncRNAs(upregulated:11;downregulated:82)followed by chromosome 1(upregulated:18;downregulated:68).Significantly differentially expressed mRNAs accounted for 5%(1,568)of total mRNA,in which 4%(1,175)were upregulated and 1%(393)were downregulated.Those DEG mRNAs were significantly enriched in following four biological processes:angiogenesis,extracellular structure organization,extracellular matrix organization,and leukocyte migration.The extracellular matrix-related genes were selected to further construct lncRNA-mRNA interaction networks which include 277 lncRNAs and 50 mRNAs.Almost all lncRNAs in the network were novel,except for HOTAIR.Conclusion:This sections have proved that the expression profile of IncRNA is distinct between STAAs and control tissues,and bioinformatics analysis showed HOTAIR was related with pathogenesis of STAAs.PART ?:The expression and the Potential Role of HOTAIR in Sporadic Thoracic Aortic AneurysmObjective:Transcriptomic and lncRNA-mRNA correlation analysis revealed HOTAIR was downregulated in STAA and associated with genes involved in extracellular matrix remodeling.In this part,we investigated expression of HOTAIR in a larger group,and tested the effect of lncRNA HOTAIR on human aortic smooth muscle cells and extracellular matrix in vitro.Methods:HOTAIR expression was examined by qRT-PCR in STAA group(n=24)and controls group(n=24:coronary artery bypass graft(n=22),heart transplant donors(n =2)).Knocking down HOTAIR expression with siRNA in human aortic smooth muscle cell was performed to assay its impact on apoptosis,cell proliferation and expression of collagen type ? and ?,transforming growth factor-? 1(TGF-?)and matrix metalloproteinase-2(MMP-2)though an Annexin V-APC Apoptosis Detection Kit,Cell Counting Kit-8(CCK-8),qRT-PCR,Western Blot(WB),enzyme linked immunosorbent assay(ELISA).Then RNA Sequencing was performed to examine the mRNA expression profile after knocking down HOTAIR.The functional and pathway enrichment analysis of DEG was processed by Kyoto Encyclopedia of Genes and Genomes(KEGG).Results:There was significantly decreased expression of HOTAIR in STAAs than in controls(P = 0.0147).In addition,the expression of HOTAIR was inversely correlated with the aortic diameter(r =-0.47,P = 0.0218)in STAAs,but not to patients' ages.After transfecting with siRNA for 24 h,HASMCs showed more early(P<0.0001)and late(P = 0.0001)apoptotic cells than the control group.The proliferation of HASMCs was suppressed by 28%after 24 h,38%after 48 h,and 44%after 72 h.Knocking down HOTIAR downregulated the expression of BCL-2(p=0.0485,=0.0389),collagen type?(p=0.0004,=0.0313)and ?(p=0.0003,= p=0.00286)and upregulated MMP-2(p<0.0001,=0.0113 = and TGF-?1(p=0.011,=0.0153)at both mRNA and protein level.620 mRNAs were significantly differentially expressed,in which 53 were upregulated and 567 were downregulated.The most significantly functional enrichment item was vascular smooth muscle contraction,including following genes differentially expressed NPR1,ACTA2,GUCY1B3,GUCY1A2,ITPR1,GNA11,PPP1R14A,MYLK,ADCY6,PLA2G2A,MRVI1,CALML6,CALD1.KEGG Pathway enrichment-revealed many signaling pathways had been altered,including MAPK,Wnt,GnRH,.TGF-beta signaling pathways.Conclusion:HOTAIR could affect contractile function of smooth muscle cells and remodeling of extracellular matrix which may cause thoracic aortic aneurysm.Summary:The expression profile of lncRNA is distinct between sporadic thoracic aortic aneurysm and control tissues.HOTAIR,one differentially expressed lncRNA in sporadic thoracic aortic aneurysm,could affect contractile function of smooth muscle cells and remodeling of extracellular matrix.