The Diagnostic Value Of Exosomal Micro RNA In LAA Stroke

1.Background and aimsIschemic stroke is one of the common causes of long-term disability and death,and its global burden is increasing.Large artery atherosclerosis(LAA)stroke is the most common subtype of ischemic stroke,which has the characteristics of high incidence,high disability rate,high mortality,high recurrence rate,and high economic burden.It is well accepted that atherosclerosis is a chronic inflammatory disease.The increase of plaque volume and the rupture of unstable plaque caused by the continuous progression of atherosclerosis are the most common causes of ischemic stroke.Exosomes are endosomal-derived phospholipid bilayer vesicles,which not only play a very important role in physiological processes such as intercellular communication,material transport,and immune response,but also in pathological processes such as cardiovascular and cerebrovascular diseases.Exosomes contain a variety of substances,including noncoding RNA such as miRNA,m RNA,and protein.In recent years,it has been found that exosomal micro RNAs(miRNAs)can be used in the diagnosis and treatment of diseases.Recent studies have found that the bilayer structure of exosomes can protect miRNAs from degradation by RNase in plasma and maintain high stability in peripheral blood.Therefore,exosomal miRNAs are promising biomarkers for diagnosis and prognosis.?However,studies focusing on the diagnostic efficiency of exosomal miRNAs in LAA stroke are rare.Our study aimed to identify the exosomal miRNAs associated with LAA stroke and further validate their diagnostic efficiency to obtain promising biomarkers.2.Methods1 Extraction and identification of exosomes: Invitrogen plasma exosomes extraction kit was used to extract exosomes from plasma.And exosomes were identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western Bloting.2 Profiles and characteristics of differentially expressed exosomal miRNAs:High-throughput sequencing technology was used to analyze the differential expression profiles of plasma exosomal miRNAs in 10 subjects(LAA: control = 5: 5).Through comprehensive analysis of the expression level,fold change of differentially expressed miRNAs,and functional enrichment analysis results,the exosomal miRNAs related to LAA ischemic stroke were preliminarily selected.3 Clinical value of exosomal miRNAs in Discovery phase: Detect the expressed level of selected exosomal miRNAs in the discovery phase(LAA: control = 33: 33)by q RT-PCR,then perform difference analysis between groups,and preliminarily evaluate the diagnostic value of exosomal miRNAs for LAA ischemic stroke.4 Potential clinical diagnostic value of exosomal miRNAs in Validation phase:The exosomal miRNAs whose differential expression in the discovery phase was consistent with the trend of sequencing results and which performed well in the difference analysis between groups were verified in the validation phase.To better evaluate the diagnostic performance of exosomal miRNAs for LAA stroke,we added a small artery occlusion(SAO)ischemic stroke group and asymptomatic atherosclerosis(AS)group to the validation phase.Firstly,the expression levels of exosomal miRNAs in the validation phase(LAA: SAO: AS: control = 155: 155: 105: 105)was analyzed by q RT-PCR,and then the potential clinical value of exosomal miRNAs for the diagnosis of LAA stroke was explored by regression analysis and ROC analysis.We also further analyzed the relationship between the selected exosomal miRNAs and the severity of stroke.5 Comparison between exosomal miRNAs and plasma miRNAs: The diagnostic efficiency of plasma miRNAs for LAA stroke was evaluated by q RT-PCR and receiver operating characteristic(ROC)analysis was performed.And further to evaluate the correlation between the two.Furthermore,the diagnostic performance of exosomal miRNAs was compared with that of plasma miRNAs.3.Result1 Characteristics of exosomes: The extracted exosomes were bilayer phospholipid vesicles with a double-concave oval shape and sizes ranging from 30 to 150 nm.The positive markers of exosomes,namely CD9,CD63 and TSG101,were all verified in the extracted exosomes.However,the negative marker of exosomes,GRP94,was absent.2 Screening differentially expressed exo-miRNAs: The results of high-throughput sequencing indicated that there were 18 differentially expressed exo-miRNAs in LAA stroke,of which 2 were up-regulated and 16 were down-regulated.Functional enrichment analysis showed that target genes of differentially expressed exo-miRNAs were involved in atherosclerotic pathways,including metabolic process,cell adhesion molecules(CAMs),and insulin signaling pathway.3 Diagnostic efficiency of exosomal miRNAs in LAA stroke: In discovery phase,we selected 5 differentially expressed candidate exo-miRNAs(miR-369-3p,miR-493-3p,miR-379-5p,miR-1296-5,and miR-1277-5p)and 4 miRNAs(miR-369-3p,miR-493-3p,miR-379-5p,and miR-1296-5p)were selected for further verification.In validation phase,q RT-PCR and ROC analysis showed that the area under the curve(AUC)of miR-369-3p,miR-493-3p,miR-379-5p,and miR-1296-5p were 0.841,0.852,0.857 and 0.838 respectively.Compared with a single diagnostic biomarker,the combined diagnostic panel(miR-369-3p + miR-493-3p + miR-379-5p)showed higher diagnostic performance(AUC=0.872).4 The diagnostic efficiencies of exosomal miRNAs were better than that in plasma:We evaluated the expression levels of plasma miRNAs in the validation phase and performed correlation analysis between the expression of plasma and exosomal miRNAs.The results showed that there was no correlation between the expression of exosomal miRNAs and their counterparts in plasma.Plasma miR-369-3p,miR-493-3p,and miR-379-5p failed to distinguish between the LAA group and the control group.In addition,miR-1296-5p in plasma exhibited an AUC of 0.611,while the exosomal miRNA exhibited an AUC of 0.838.Thus,the diagnostic efficiency of miRNAs in exosomes is better than that of miRNAs in plasma.5 Correlation between exosomal miRNAs and the severity of LAA ischemic stroke:Spearman rank correlation analysis indicated that miR-493-3p and miR-1296-5p were negatively correlated with the severity score of stroke(National Institute of Health Stroke Scale,NIHSS)(P < 0.05).4.Conclusion1 Exosomal miRNAs(miR-369-3p,miR-493-3p,miR-379-5p,and miR-1296-5p)are potential biomarkers for LAA stroke,and the diagnostic efficiencies of exosomal miRNAs were higher than plasma miRNAs.And composite diagnostic biomarkers achieve higher diagnostic efficiency than single biomarkers.2 The exosomal miRNAs expression is negatively correlated with NIHSS score,which indicates that exosomal miRNAs could be used to indicate the severity of disease.