The Study Of CircRNA,lncRNA And MRNA Expression Profiles Of Acute Ischemic Stroke With Combination Of Phlegm,Blood Stasis And Heat Syndrome

Objective:This study aims to explore circular RNA（circRNA）,long noncoding RNA（lncRNA）and Messenger RNA（mRNA）expression profiles between acute ischemic stroke（AIS）patients with combination of phlegm,blood stasis and heat syndrome and AIS patients with phlegm and blood stasis syndrome,and to investigate the potential mechanism of the differential expressed circRNA,lncRNA and mRNA on the AIS traditional Chinese medicine syndrome.Method:A total of 40 AIS patients,including 20 AIS patients with combination of phlegm,blood stasis and heat syndrome and 20 AIS patients with phlegm and blood stasis syndrome,and 20 non-AIS controls were recruited in this study.High-throughput RNA sequencing（RNA-Seq）was used to analyze the circRNA,lncRNA and mRNA expression levels in 5 AIS patients with combination of phlegm,blood stasis and heat syndrome,5 AIS patients with phlegm and blood stasis syndrome,and 5 non-AIS controls.Five differentially expressed genes were validated in 20 AIS patients with combination of phlegm,blood stasis and heat syndrome,20 AIS patients with phlegm and blood stasis syndrome,and 20 non-AIS controls using real-time quantitative PCR（qRT-PCR）.Receiver operating characteristic（ROC）curve with area under curve（AUC）was conducted to assess the potential diagnostic value of the validated circRNAs,lncRNA and mRNA for AIS.GO and KEGG pathway analysis were used for functional annotation and pathway enrichment of differentially expressed circRNAs,lncRNAs and mRNAs.Competitive endogenous RNA（ceRNA）network was conducted for qRT-PCR validated molecules.Result:1.There were 96 circRNAs,135 lncRNAs,and 184 mRNAs differentially expressed in between AIS and non-AIS controls.2.There were 79 circRNAs,135 lncRNAs and 221 mRNAs differentially expressed between AIS patients with combination of phlegm,blood stasis and heat syndrome and non-AIS controls.There were 96 circRNAs,211 lncRNAs and 286 mRNAs differentially expressed between AIS patients with phlegm and blood stasis syndrome and non-AIS controls.3.There were 105 circRNAs,162 lncRNAs and 113 mRNAs differentially expressed between AIS patients with combination of phlegm,blood stasis and heat syndrome and AIS patients with phlegm and blood stasis syndrome.Among them,hsa_circ₀000997,hsa_circ₀105101,hsa_circ₀000620,lnc-FCGR3A-5:1,LINC02397:24,FCGR3B,VNN1,ZNF876P and P2RY2 may be can serve as diagnostic candidates for AIS with combination of phlegm,blood stasis and heat syndrome diagnosis,while hsa_circ₀005946,lnc-PRKACG-1:1,MIR17HG:20 and HIST1H4B for AIS with phlegm and blood stasis syndrome diagnosis.4.In qRT-PCR analysis,results showed that hsa_circ₀005946 expression levels in the AIS group were lower than that in the non-AIS control group（P=0.007）,The hsa_circ₀005946 expression levels in AIS with phlegm and blood stasis syndrome group were higher than that in AIS with combination of phlegm,blood stasis and heat syndrome group（P=0.048）,moreover hsa_circ₀005946 expression levels in AIS with phlegm and blood stasis syndrome group were lower than that in non-AIS control group（P<0.001）.But there was no difference in hsa_circ₀005946 expression level between AIS with combination of phlegm,blood stasis and heat syndrome group and non-AIS control group（P=0.256）.P2RY2 expression levels in AIS than that in non-AIS control group（P=0.003）.The expression levels of P2RY2 in AIS with combination of phlegm,blood stasis and heat syndrome group were lower than that in non-AIS control group（P=0.035）.The P2RY2 expression level in AIS with phlegm and blood stasis syndrome group was lower than that in non-AIS control group（P=0.003）.But there was no difference in the expression level of P2RY2 between AIS with combination of phlegm,blood stasis and heat syndrome group and AIS with phlegm and blood stasis syndrome group（P=0.433）.There was no significant differences in hsa_circ₀000620,LINC02397:24 and HIST1H4B expression between different groups（all P>0.05）.5.The cut-off value of hsa_circ₀00594 expression between AIS and non-AIS controls was 0.609,which yielded a sensitivity of 90%and a specificity of 55%with the area under curve（AUC）at 0.716（P<0.05）.The cut-off value of P2RY2 expression between AIS and non-AIS controls was 1.216,which yielded a sensitivity of 60%and a specificity of 85%with the area under curve（AUC）at 0.734（P<0.05）.The cut-off value of hsa_circ₀00594 expression between AIS with combination of phlegm,blood stasis and heat syndrome group and AIS phlegm and AIS with phlegm and blood stasis syndrome group was 0.782,which yielded a sensitivity of 60%and a specificity of 80%with the area under curve（AUC）at 0.683（P<0.05）.6.The GO and KEGG analysis results showed that differentially expressed circRNA between AIS and non-AIS have molecular functions,such as thiol-dependent ubiquitinyl hydrolase activity,involved in biological processes,such as protein deubiquitination,and mainly regulates thyroid hormone signaling pathway,FoxO signaling pathway and Notch signaling pathway.Differentially expressed lncRNA have molecular functions,such as monosaccharide binding,protein heterodimerization activity,involved in biological processes,such as cardiac septum morphogenesis,cardiac septum development,phospholipase C-activating G protein-coupled receptor signaling pathway,and mainly regulates pathways,such as inflammatory mediator regulation of TRP channels,nitrogen metabolism pathway.Differentially expressed mRNA have molecular functions,such as immunoglobulin binding,cytokine receptor activity,involved in biological processes,such as regulation of inflammatory response,neutrophil degranulation,and mainly regulates pathways,such as hematopoietic cell lineage,osteoclast differentiation,staphylococcus aureus infection.7.The GO and KEGG analysis results showed that differentially expressed circRNA between AIS with combination of phlegm,blood stasis and heat syndrome group and AIS phlegm and AIS with phlegm and blood stasis syndrome group have molecular functions,such as methylated histone binding,GDP binding,involved in biological processes,such as G1/S transition of mitotic cell cycle,cell cycle G1/S phase transition,cerebellar cortex development,cerebellar Purkinje cell layer development,and mainly regulates pathways,such as AMPK signaling pathway and drug metabolism.Differentially expressed lncRNA have molecular functions,such as amyloid-beta binding,peptide binding,involved in biological processes,such as phosphatidic acid biosynthetic process,phosphatidic acid metabolic process,regulation of carbohydrate metabolic process,and mainly regulates pathways,such as regulation of lipolysis in adipocytes,glycerolipid metabolism,GABAergic synapse,calcium signaling pathway.Differentially expressed mRNA have molecular functions,such as immunoglobulin binding,phospholipid binding,modified amino acid binding,involved in biological processes,such as neuron maturation,negative regulation of cation channel activity,protein heterotetramerization,and mainly regulates pathways,such as drug metabolism,neuroactive ligand-receptor interaction,regulation of lipolysis in adipocytes.8.Bioinformatics analyses results showed that hsa_circ₀005946 associated ceRNA network includes 8 miRNAs and 26 mRNAs.Hsa_circ₀005946-miR-432-5p-IGF1R axis and hsa_circ₀005946-miR-523-3p-VEGFA axis may affect the occurrence and development of AIS TCM syndromes by regulating the PI3K-Akt signal pathway.Conclusion:There is a different expression profiles of circRNA,lncRNA and mRNA between AIS with phlegm and blood stasis syndrome and non-AIS controls.CircRNA,lncRNA and mRNA expression levels are different between AIS with combination of phlegm,blood stasis and heat syndrome group and AIS phlegm and AIS with phlegm and blood stasis syndrome group.Hsa_circ₀005946 and P2RY2 may serve as diagnostic biomarker for AIS.Moreover hsa_circ₀005946 may serve as diagnostic biomarker for AIS with phlegm and blood stasis syndrome group.Hsa_circ₀005946-miR-432-5p-IGF1R axis and hsa_circ₀005946-miR-523-3p-VEGFA axis may affect the occurrence and development of AIS TCM syndromes by regulating the PI3K-Akt signal pathway,which provides a clue for further exploring the mechanism of TCM syndromatology.The differential expressed circRNAs between AIS with combination of phlegm,blood stasis and heat syndrome group and AIS phlegm and AIS with phlegm and blood stasis syndrome group are mainly related to biological processes,such as cell cycle,cellular oxidative stress response,cerebellar development.The differential expressed lncRNAs are mainly involved in substance metabolism,while differential expressed mRNAs are related to biological processes,such as cell stress response,cell differentiation,glycosylation metabolism.The signaling pathways,such AMPK signaling pathway,drug metabolism pathway,calcium signaling pathway and FoxO signaling pathway may be associated with the development of AIS with combination of phlegm,blood stasis and heat syndrome.