| Objective: Ischemic stroke is a common acute cerebrovascular accident with a complicated pathogenesis and is affected by environmental and genetic factors.At present,several studies have showed the role of non-coding RNA in cardiovascular pathogenesis,and more of them involve long non-coding RNA(lnc RNA)and micro RNA(mi RNA);while circRNA are existing as covalently closed loops,the stability of this structure is higher than that of a linear RNA,and has the potential to become a disease biomarker.Based on high-throughput sequencing,this study screened the differential expression profile of circRNA in peripheral blood samples of patients with ischemic stroke,and further replicated and verified some differentially expressed circRNA,aiming to explore the pathophysiology of circRNA in ischemic stroke expression changes and effects in the process.Constructed of the circRNA-mi RNA-m RNA biological information regulatory network preliminarily,predicted the molecular mechanism and key pathways involved in the regulation of ischemic stroke by circRNA,provides new molecular targets for the diagnosis and treatment of diseases.Methods: The method of case-control study was used in this study,in the first stage,10 cases with first-diagnosed ischemic stroke were collected in two tertiary hospitals in Jiangsu Province.Matched by age and gender,20 subjects were included in the control group from the local or neighboring natural population cohort,including 10 hypertension controls and 10 healthy controls.High-throughput sequencing was used to identify the expression levels of circRNA in peripheral blood of ischemic stroke cases and control groups,and circRNA differential expression profiles were screened.In the second stage,145 patients with ischemic stroke and 150 control were included,and peripheral blood samples,general demographic information,biochemical indicators were collected.The ten circRNAs were screened in the circRNA differential expression profile(|Log2Fold Change|≥1 and the test levels of P<0.05)were quantified by quantitative real time polymerase chain reaction(q RT-PCR).The levels of circRNA expression in cases and controls and among different toast types were analyzed by Mann–Whitney U nonparametric test or K-sample nonparametric test.The receiver operator characteristic curve(ROC curve)was used to evaluate the diagnostic value of the target circRNA in ischemic stroke.The correlation between the target circRNA and biochemical indicators was analyzed using spearman rank correlation.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to explore the possible molecular mechanism of candidate circRNA in the progression of ischemic stroke and the signal pathways that the target circRNA may participate in the pathogenesis of ischemic stroke.Results: The results of high-throughput sequencing showed that the differentially expressed circRNA existed in the peripheral blood samples of patients with ischemic stroke and the controls.A total of 734 circRNAs were found to be differentially expressed in ischemic stroke(P<0.05 and∣log2Fold Change∣≥1).430 circRNAs were up-regulated and 304 circRNAs were down-regulated.The results of q RT-PCR showed that the 5 up-regulated and 5 down-regulated significantly different circRNAs were selected in the preliminary screening in the expanded population verification,the expression levels of hsa＿circ＿0118748,hsa＿circ＿0095288,hsa＿circ＿0125195 were down-regulated significantly,and the expression level of hsa＿circ＿0064555 increased in stroke cases(P<0.05),which are consistent with the sequencing results.However,the expression levels of hsa＿circ＿0112125,hsa＿circ＿0085440,hsa＿circ＿0100946,hsa＿circ＿0070935,hsa＿circ＿0135590,hsa＿circ＿0125366 were not significantly different between the cases and the control group(P>0.05).The results of stratified analysis showed that in male subgroup,the expression levels of hsa＿circ＿0118748and hsa＿circ＿0135590 in the case group [0.644(0.323,1.075),0.546(0.326,1.263)]were lower than those in the control group significantly[0.930(0.495,1.704),0.984(0.449,2.328)],and the P values were 0.005 and 0.045 respectively.The expression level of hsa＿circ＿0125195 was down-regulated with ischemic stroke between men and women(P<0.05).In the subgroup ≤ 65 years of age,the expression levels of hsa＿circ＿0118748,hsa＿circ＿0095288,and hsa＿circ＿0125195 in the stroke case group were 0.793(0.327,1.433),0.752(0.410,1.022),0.661(0.365,1.069),which were significantly lower than those in the controls [1.200(0.762,2.099),0.999(0.442),1.488),1.106(0.602,2.107)],P<0.05.Based on Toast type of ischemic stroke,in the comparison between the Large Artery Atherosclerosis stroke(LAA)group and the controls,the expression level of hsa＿circ＿0064555 was 1.274(0.693,2.203)in the LAA group was significantly higher than 0.919(0.591,1.573)in the control group,P<0.05;hsa＿circ＿0118748 and hsa＿circ＿0125195 were significantly down-regulated in the LAA group(P<0.05).In the small artery occlusive stroke(SAA)group and the control group,the expression levels of hsa＿circ＿0118748,hsa＿circ＿0095288,hsa＿circ＿0125195,hsa＿circ＿0135590 were statistically different(P<0.05),and all showed down-regulation in the case group.ROC curve analysis results showed that the area under the curve(AUC)of hsa＿circ＿0064555 was 0.687(95%CI:0.626-0.747),with a sensitivity of 83.45%,and a specificity of 46.67%;and the AUC of hsa＿circ＿0118748 was 0.702(95%CI:0.643-0.761),with a sensitivity of 74.82% and a specificity was 56.00%;the AUC of hsa＿circ＿0095288 was 0.695(95%CI :0.636-0.755),the sensitivity and specificity were 84.89% and 46.00%;the AUC of hsa＿circ＿0125195 was 0.708(95%CI:0.649-0.768),the sensitivity and specificity were 75.54% and 60.00% respectively.When hsa＿circ＿0064555,hsa＿circ＿0118748,hsa＿circ＿0095288 and hsa＿circ＿0125195 were combined,the AUC was0.765(95%CI:0.711-0.819),the sensitivity and specificity were 84.17% and 56.67%.The results of correlation analysis showed that the expression levels of hsa＿circ＿0118748 and hsa＿circ＿0095288 were positive correlated with total cholesterol(TC)and high-density lipoprotein cholesterol(HDL-C)(P<0.05),and the expression level of hsa＿circ＿0125195 and glucose(GLU),TC,HDL-C were all positive correlated(P<0.05).hsa＿circ＿0064555 and other biochemical indicators were not statistically significant(P>0.05).Function prediction analysis showed that these differential circRNAs have multiple mi RNA binding sites;through GO analysis,it was found that differential expression of circRNA-related target genes were involved in transcription regulation,axon guidance and other biological processes.KEGG signaling pathway enrichment analysis shows that differential expression of circRNA may participate in the occurrence and development of ischemic stroke through c AMP signaling pathway,Ras signaling pathway,and so on.Conclusion: There was a differential expression profile of circRNA in the peripheral blood of patients with ischemic stroke and the control group,and the q RT-PCR replication verification results showed that hsa＿circ＿0064555,hsa＿circ＿0118748,hsa＿circ＿0095288,and hsa＿circ＿0125195 are dysregulated in the peripheral blood of patients with ischemic stroke,which has a certain value for the diagnosis of ischemic stroke and can be used to diagnose ischemic stroke.The function prediction showed that these circRNAs were involved in the pathophysiological process of ischemic stroke and have certain value for the diagnosis,treatment and prognosis of the disease,and can be considered as a new target for ischemic stroke intervention. |
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