Role Of Circ₀022920 In The Pathogenesis Of Aortic Dissection Via Controlling The MiR-650/TGFβR1 Axis As A Competing Endogenous RNA

【Background】Aortic dissection（AD）is a lethal vascular disease with a high mortality.In the treatment of AD,including surgery,medication and endovascular repair,have been made,however,AD is still a severely life-threatening disease and needs to be intervened as quickly as possible.Prompt effective treatment could reduce the mortality and improve the life quality of AD patients.Up to now,how to effectively manage the AD patients remain a challenge to surgeons for the.To establish a better treatment for AD patients,the molecular regulatory mechanisms of AD need to be clarified.VSMCs exist as a synthetic or contractile phenotype.They switch from a proliferative and synthetic phenotype in embryonic tissue to a quiescent and contractile phenotype in adult blood vessels.In healthy blood vessels,most VSMCs are contractile phenotype,however,in response to the pathological injury,contractile VSMCs can switch-back to synthetic phenotype.Abnormal phenotypic switch of vascular smooth muscle cells（VSMCs）is regarded as iconic pathological change of AD.CircRNAs form a closed loop via the covalent linkage between the 3’ and 5’ ends.As a research hotpot since 2012,growing evidence has proved that circRNAs are intensively involved in the pathogenesis and progression of diseases.Similar with lncRNAs,circRNAs exert its role via acting as competing endogenous RNAs（ceRNAs）to sponge miRNAs.According to the previous reports,dysregulated circRNA expression was found in thoracic AD patients and circRNA₁01238 inhibited miR-320 a expression as a sponge to enhance MMP9 expression,which might be associated with the pathogenesis of AD.However,the specific circRNA mediating the pathogenesis and progression of AD is still not identified.【Objective】Identified the differential expression profiles of circRNA,miRNA,and mRNA between normal aortic wall and aortic dissection aortic wall tissue by high-throughput gene chip,and applying bioinformatics analysis to analyze the differential expression profiles of circRNAs,miRNAs,and mRNAs,then the circRNA-miRNA-mRNA regulatory network in aortic dissection was integrated according to the mechanism of competing Endogenous RNA（ceRNA）.Validation experiment at the molecular level,HASMC and aortic tissue levels were then performed.The aims of this study are to provide new biomarkers and molecular targets for the early diagnosis and treatment of AD.【Method】1.Differential expression profiles of circRNA,miRNA,and mRNA in AD.Identified the differential expression profiles of circRNA,miRNA,and mRNA between normal aortic wall（n = 6）and aortic dissection aortic wall tissue（n = 6）by high-throughput gene chip,then the accuracy of the chip was verified by RT-qPCR.2.Bioinformatics analysis of the differentially expressed circRNA,miRNA,and mRNA in AD.Appling Bioinformatics analysis to analyze the differential expression profiles of circRNAs,miRNAs,and mRNAs,then the circRNA-mediated ceRNA regulatory network significantly extremely relevant to AD was integrated.3.Role of circ₀022920 in the pathogenesis of aortic dissection via controlling the miR-650/TGFβR1 axis as a ceRNA.Angiotensin II（Ang II）was adopted to induce AD model in HASMC.Ad-circ0022920,sh-circ0022920,miR-650 mimic,anti-miR-650 and their negative controls were applied for cell transfection to assess their biological functions.CCK-8 and EdU assays were subjected to determine cell proliferation.Transwell and wound healing assays were conducted to assess cell migration.Besides,western blot was used to analyze the expression of phenotypic switching markers（α-SMA,SM22α and OPN）.Moreover,the interaction of circ₀022920 and miR-650 was verified using RNA pull-down,FISH and luciferase reporter assay.【Results】1.A total of 881 significantly dysregulated circRNAs including 285 up-regulated and 596 down-regulated circRNAs were identified.185 significantly dysregulated miRNAs including 170 up-regulated and 15 down-regulated miRNAs were found to be differently expressed by high-throughput gene chip.The verification results of RT-qPCR were consistent with the high-throughput gene chip results,which indicated that the results of high-throughput gene chip were reliable.2.Construction of ceRNA networks.A circRNA-miRNA-mRNA ceRNA network was generated that comprised 105 nodes,including 25 circRNAs,17 miRNAs,and 72 mRNAs.Based on the degree of node connection,the top 5 miRNAs were miR-1285-3p,miR-637,miR-650,miR-485-5p,and miR-509-5p,respectively;the top 5 circRNAs were circRNA₄04522,circRNA₀22920,circRNA₀75881,circRNA₁03147,and circRNA₀000536,respectively;and the top 5 mRNAs were TINAGL1,JPH4,PLXNA2,TGFBR1,and THSD4,respectively.These genes were extremely relevant to AD disease.Moreover,we predicted that circ₀022920/miR-650/TGFβR1axis as a ceRNA may play an important role in the regulatory mechanism of AD disease.3.Role of circ₀022920 in the pathogenesis of aortic dissection via controlling the miR-650/TGFβR1 axis as a ceRNA.We verified the expression of circ₀022920,miR-650 and TGFβR1 by RT-qPCR and found decreased expression of circ₀022920 and TGFβR1 and increased expression of miR-650 in AD patients,the Pearson correlation analysis indicated the negative correlation between miR-650 and circ₀022920 or TGFβR1,and the positive correlation of circ₀022920 and TGFβR1.Ang II treatment also induced down-regulation of circ₀022920 and up-regulation of miR-650.Enhanced expression of circ₀022920 dramatically inhibited HASMC proliferation,migration and phenotypic switch,while miR-650 exerted the opposite effects.Moreover,circ₀022920-mediated proliferation,migration and phenotypic switch of HASMC was restored by miR-650 overexpression.Circ₀022920 could directly interact with miR-650 were verified using RNA pull-down,FISH and luciferase reporter assay.Furthermore,TGFβR1 was up-regulated in miR-650 knockdown HASMC but down-regulated in miR-650 over-expressed HASMC.Knockdown of miR-650 inhibited the proliferation,migration and phenotypic switch of Ang II-treated HASMC,but the inhibitory effect was reversed by the knockdown of TGFβR1.【Conclusion】1.The differential expression profiles of circRNA,miRNA,and mRNA in AD tissue was obtained.A total of 881 circRNAs,185 miRNAs,and 2834 mRNAs were found to be differently expressed.2.According to the biological information analysis,a circRNA-miRNA-mRNA ceRNA network was generated that comprised 105 nodes,including 25 circRNAs,17 miRNAs,and 72 mRNAs.These RNAs may be key genes in the molecular regulatory mechanism of AD disease.3.We found decreased expression of circ₀022920 and TGFβR1 and increased expression of miR-650 in AD patients.Circ₀022920 maintains the contractile phenotype of HASMC,and miR-650 impairs the contractile phenotype of HASMC in AD.Circ₀022920 directly targets miR-650.Circ₀022920 regulates the phenotype switch of HASMC and suppresses aortic dissection via miR-650/TGFβR1 axis.Taken together,our study helps elucidate the role of circ₀022920 in AD.More importantly,our study might lay a solid foundation of a promising diagnostic biomarker and therapeutic target for AD.