Study Of Transcriptomic Profile Of Intracranial Arteries And The Mechanisms Of Circular RNA CircSATB2 In Moyamoya Disease

Moyamoya disease(MMD)is a rare progressively steno-occlusive cerebrovascular disease.Although there has been some progress in the study of epidemiology,clinical features,and treatment of MMD in recent decades,the etiology and pathogenesis still remain largely unknown.The differences in the incidence of MMD in different ethnic populations suggests genetic factors have been implicated in the development of this disease.In addition,numerous genome wide associated studies revealed that ring finger protein 213(RNF213)p.R4810 K mutation was an important susceptibility gene for MMD through whole blood samples.However,RNF213 p.R4810 K mutation showed marked frequency variation across ethnic populations,suggesting that mutation of this gene may not be solely responsible for MMD,and provide hints that other factors may also be involved in the development of MMD.Gene expression profiling based on RNA sequencing has been widely utilized to investigate a variety of vascular diseases,facilitating the discovery of effective disease biomarkers for earlier diagnosis and accurate classification of disease subtypes.In addition,more and more evidence have found that circular RNA(circ RNA)plays an important role in a variety of cerebrovascular diseases including ischemic stroke,cerebral aneurysm and cerebral hemorrhage.However,there are few reports on gene expression profiling of intracranial blood vessels in MMD patients and the pathogenesis of circ RNAs in MMD.First part: Study of transcriptomic profile of intracranial arteriesObjective: The samples investigated in genome-wide or RNA-sequencing studies were mainly obtained from the MMD patients’ peripheral blood.Studies for elucidating the genome-wide molecular characteristics of intracranial arteries in MMD are rarely reported.This study was implemented to revealed the genome-wide transcriptomic profile of intracranial arteries in MMD.Materials and methods: Sixteen adult patients with MMD and five with atherosclerosis associated intra-cranial artery stenosis-occlusion(AS-ICASO)were enrolled in this study.The cortical segments of middle cerebral artery(MCA)specimens were intraoperatively collected during superficial temporal artery(STA)–to–MCA bypass surgery.Total RNA was extracted from MCA samples and applied to RNA library construction and high-throughput RNA-sequencing.Then,bioinformatic analysis was used to uncover the gene expression profile of MCA in MMD.RT-q PCR was conducted to validate the gene expression level.Results: We identified 556 differentially expressed genes(DEGs)for MMD,including 449 and 107 significantly up-or down-regulated genes.Comparing with AS-ICASO controls,up-regulated genes were mainly involved in extracellular matrix(ECM)organization,whereas down-regulated genes were primarily associated with mitochondrial function and oxidative phosphorylation in MMD.We identified 133 and 439 sex-specific DEGs for males or females in MMD,respectively.About 95.6% of sex-specific DEGs were protein-coding genes and 3% genes belonged to long noncoding RNA(lnc RNA).Sex-specific DEGs were observed in all chromosomes,of which 95.49% and 96.59% were autosomal genes in males or females respectively.These sex-specific DEGs,such as aquaporin-4(AQP4),superoxide dismutase 3(SOD3)and nuclear receptor subfamily 4 group A member 1(NR4A1),may contribute to the sex difference in MMD.Conclusion: This transcriptomic study indicated ECM and mitochondrial function as central molecular mechanisms underlying MMD,and revealed sex difference of gene expression in intracranial arteries,thereby providing new insights into the study of pathogenesis of MMD.Second part: Circular RNA circ SATB2 involved in moyamoya disease by regulating the function of vascular endothelial cells via mi R-6997-3p/STRN axisObjective: In recent years,a large number of circ RNAs have been found to play important roles in a variety of cerebrovascular diseases.However,there are few studies focus on the role of circ RNAs in the pathogenesis of MMD.Two previous circ RNA microarray sequencing studies in MMD patients and our sequencing data found that circ SATB2 derived from SATB2 gene was abnormally expressed in MMD patients.This study was implemented to investigate the potential molecular mechanism of circSATB2 in the pathogenesis of MMD.Materials and methods: The expression of circ SATB2 was detected in artery samples obtained from MMD patients and oxygen-glucose deprivation(OGD)induced brain microvascular endothelial cells(BMVECs)by RT-q PCR.Interaction among circ SATB2,micro RNA-6997-3p(mi R-6997-3p),and STRN was analyzed by dual luciferase reporter assay.The BMVECs were transfected with overexpressing plasmid or si RNA to study the effects of circ SATB2/mi R-6997-3p/STRN on cell proliferation and apoptosis in vitro.Results: By performing intersection analysis of our sequencing data with previous circ RNA microarrary sequencing data in MMD patients,a total of 2704 circ RNAs weredetected in MMD patients,of which 72 circ RNAs were significantly expressed differently(log2FC>1,P<0.05),including circ SATB2 derived from the SATB2 gene.Reduction of circ SATB2 occurred in artery of MMD and OGD treated BMVECs.Overexpression of circ SATB2 promoted cell proliferation and inhibited cell apoptosis in BMVECs,whereas knockdown of circ SATB2 suppressed cell proliferation and increased cell apoptosis in BMVECs.Functionally,we confirmed that circ SATB2 could negatively regulate the expression of mi R-6997-3p through its mi RNA sponging.Moreover,Overexpression of mi R-6997-3p inhibited cell proliferation and promoted cell apoptosis in BMVECs,while inhibition of mi R-6997-3p promoted cell proliferation and inhibited cell apoptosis in BMVECs.The dual-luciferase reporter gene assay confirmed that STRN was the target gene of mi R-6997-3p.The expression of STRN was positively regulated by circ SATB2 and negatively regulated by mi R-6997-3p.In vascular samples from MMD patients,we detected significant downregulation of circ SATB2 and STRN,while upregulation of mi R-6997-3p,a result consistent with the expression pattern in OGD treated BMVECs.Conclusion: Circ SATB2 may regulate the proliferation and apoptosis of BMVECs through the mi R-6997-3p/STRN axis,thereby playing an important role in the pathogenesis of MMD.