Differential Expression Profile Of Serum Exosome MiRNA In Patients With Large Atherosclerotic Cerebral Infarction

Objective:Large artery artherosas(LAA)cerebral infarction is one of the most common types of ischemic stroke and also helps relieve headaches and is a major cause of LAA cerebral infarction.Laa-type cerebral infarction can cause changes in peripheral blood circulating Exosome(Exo)miRNA(microRNA).By analyzing the differential expression profile of serum exosome miRNA genes in patients with LAA type cerebral infarction,the differential exosome miRNA closely related to LAA type cerebral infarction and related pathways were excavated,and combined with bioinformatics analysis technology,the target of differential genes was predicted.In order to search for potential new biomarkers to predict the occurrence and development of LAA type cerebral infarction and AS,and to explore their possible involvement in the occurrence and development of LAA type cerebral infarction.Method:A total of 106 patients admitted to the Department of Neurology of Qianfoshan Hospital of Shandong Province from January 2021 to December 2022 were included,including 40 patients with LAA cerebral infarction(LAA group),30 patients with atherosclerosis without cerebral infarction(AS group),and 36 patients without atherosclerosis during the same period(NC group).1.After peripheral blood was collected and pretreated,serum exosomes were extracted using qEV column.The size,concentration and quality of exosomes were determined by TEM,NTA and WB.2.Total RNA was extracted from Exo,and the primary screening cohort of 5 LAA patients and 6 controls was constructed by Illumina’s Tru Seq RNA method,and small RNA sequencing was performed.The differential expression of miRNA was analyzed,and the differential expression of Exo miRNA was screened(| log2FC |>1、p<0.05).3.The quantitative analysis of the significant differences in expression of targets in the validation of 95 people in the queue,using quantitative Real □Time PCR(qRT-PCR)in detection of micrornas subjects.4.Receiver Operating Characteristic(ROC)curves are used to evaluate the effectiveness of diagnostic miRNAs.The area under the ROC curve(AUC)is used to assess the optimal threshold and predictive power of the selected variable.Results:1.The vesicles extracted and isolated in this study were observed to be sphere-like particles with diameters ranging from 90-150nm under transmission electron microscopy.Exosome concentration was 107～1011 Particles/mL.VGsicles-specific proteins marker TSG101、CD9 and CD63.2.In high-throughput sequencing analysis,172 cases with significant differences were identified(p<0.05)expression level of exosome miRNA.Compared with the control group,40 exosome mirnas were significantly up-regulated and 132 were down-regulated in LAA patients.miR-144-3p、miR146a-5p、miR-199-5p、miR-320b and miR-660-5p were selected as target genes after further screening by cluster heat map,volcano map and scatter map.3.Statistical analysis was conducted on the expression levels of miR-144-3p,miR146a-5p,miR-199-5p,miR-320b and miR-660-5p in serum exosomes of patients in LAA group,AS group and NC group by qRT-PCR.The results showed that:Compared with NC group,the expression levels of miR-144-3p,miR-320b and miR-660-5p in serum exosomes in LAA group were up-regulated(p<0.05),while the expression levels of miR-199-5p were down-regulated(p<0.05).The expression level of miR-144-3p in serum exosomes in AS group was up-regulated compared with that in NC group(p<0.05).The expression level of miR-146a-5p in serum exosomes in LAA group had no significant difference compared with AS group and NC group(p>0.05).4.In order to evaluate the predictive ability of miR-144-3p,miR-199-5p,miR-320b and miR-660-5p on the occurrence of LAA in serum exosomes,ROC analysis was performed.The results show that:In the LAA and NC groups,the AUC of exosome miR-144-3p,miR-320b and miR-660-5p was 0.8611,0.8582 and 0.8433,respectively.The results showed that exosome miR-144-3p,miR-320b and miR-660-5p might be independent diagnostic indicators of LAA.In the AS group and the NC group,the AUC of miR-144-3p was 0.7209,indicating that exosome miR-144-3p may be an independent diagnostic indicator of AS.The AUC of miR-144-3p+miR-320b was 0.9267;The AUC of miR-144-3p+miR-660-5p was 0.9395,suggesting that it could be used as a collective diagnostic indicator for LAA.Conclusions:Through the analysis of serum exosome miRNA expression profiles specific to LAA patients,the selected exosomes miR-144-3p,miR-320b and miR-660-5p are related to the occurrence and development of LAA and AS,which has certain guiding significance for the diagnosis of LAA and AS,and can be used as potential biomarkers of LAA.