Comprehensive Analysis Of Differentially Expressed Profiles Of Circular RNAs In Ischemic Stroke Patients With Wind-phlegm Blocking Collaterals Symptom

Objective: Circular RNA?circRNA?is a noncoding RNA.CircRNAs are existing as covalently closed loops,which have covalently joint ends of neither 5?caps nor 3?tails formed by back-splice events.The stability of this structure is higher than that of a linear transcript and is unaffected by RNase R and RNA exonuclease,which makes circRNAs perfect clinical diagnostic biomarker.Study has revealed that circRNA is implicated in cerebral ischemia–reperfusion injury in mouse,but their role in the development of traditional Chinese medical syndrome of ischemic stroke?IS?remains unclear.This study aimed to explore circRNAs expression profiling of IS patients with wind-phlegm blocking collaterals symptom.Methods:In this study,20 patients with IS and 20 healthy subjects were included.All study subjects are from Chinese Han.Five blood samples of IS patients with wind-phlegm blocking collaterals symptom and 5 blood samples of healthy subjects were randomly selected for microarray studies.Four dysregulated circRNAs were validated by quantitative real-time reverse transcription polymerase chain reaction?qRT-PCR?in 20 IS patients with wind-phlegm blocking collaterals symptom and 20 healthy subjects.A coding–noncoding co-expression?CNC?network with upregulated circRNAs and a competing endogenous RNA?ceRNA?network with validated circRNAs were constructed through bioinformatics.Results: 1.A total of 9,241 circRNAs were detected by microarray probes.After microarray scanning and normalization were completed,288 circRNAs comprising 102 upregulated circRNAs and 186 downregulated circRNAs were detected to be differentially expressed in IS patients with wind-phlegm blocking collaterals symptom compared with those in the healthy controls?FC > 1.5 and P < 0.05?.Of these dysregulated circRNAs,14 circRNAs with false discovery rate?FDR?< 0.05,FC ? 1.5,and P < 0.05 were upregulated and 7 circRNAs were downregulated.2.These 288 dysregulated circRNAs were widely distributed among all chromosomes,including sex chromosomes.The distribution of upregulated circRNAs was balanced.The top five chromosomes were chr 12?7.84%?,chr 1?6.86%?,chr 3?6.86%?,chr 9?6.86%?,and chr 16?6.86%?,whereas the percentages of upregulated circRNAs from any other chromosome were less than 6%.Among the downregulated circRNAs,approximately 10.29% came from chromosome 2?chr 2?,9.71% from chr 17,7.73% from chr 1,and 6.84% from chr 14.For genomic positions,there were 1 antisense,6 sense-overlapping,93 exonic,2 intronic circRNAs of upregulated circRNAs and 4 sense-overlapping,174 exonic,8 intronic circRNAs of downregulated circRNAs.3.The expression levels of hsa_circRNA₁02687?P=0.003?,hsa_circRNA₁04600?P=0.017?,and hsa_circRNA₁05035?P < 0.001?were significantly higher in IS patients with wind-phlegm blocking collaterals symptom than in the healthy controls.By contrast,the expression level of hsa_circRNA₄06494?P=0.674?was not statistically different between the two groups.4.Hsa_circRNA₁05035 had the highest diagnostic accuracy,with an area under the curve?AUC?of 0.83?95%CI: 0.69-0.96?,while has-circRNA₁02687 and hsa_circRNA₁04600 are with an AUC of 0.79?95% CI: 0.61-0.91?and 0.75?95% CI: 0.60-0.91?.5.We further determined that miRNAs are predicted to be regulated by three verified circRNAs involved in possible pathways.The data showed that candidate miRNAs were implicated in IS-related pathways,such as TGF-beta signaling pathway,Wnt signaling pathway,PI3K–Akt and ErbB signaling pathways.6.On the basis of the microarray results,we built the circRNA–mRNA co-expression network with 102 upregulated circRNAs and 972 mRNAs in IS patients with wind-phlegm blocking collaterals symptom according to the degree of correlation.CircRNA–mRNA co-expression network was constucted with three validated and significantly expressed circRNAs in IS.Conclusions: 1.This study identified the differentially expressed profiles of circRNA in the IS patients with wind-phlegm blocking collaterals symptom.2.The dysregulated circRNAs were derived from different region of the gene and widely distributed among all chromosomes,including sex chromosomes.These findings greatly expand our vision on circRNAs and splicing.3.The constuction of circRNA-miRNA-mRNA networks and circRNA-mRNA co-expression networks with hsa_circRNA₁02687,hsa_circRNA₁04600,hsa_circRNA₁05035 provides basic data for further research on traditional Chinese medical syndrome mechanism of IS.5.Hsa_circRNA₁02687,hsa_circRNA₁04600,hsa_circRNA₁05035 may be served as perfect clinical diagnostic biomarkers of of IS.