Study On Circulating MiRNA Fingerprint And Network Construction Of Chronic Blood Stasis Syndrome In Coronary Heart Disease

ObjectiveCoronary heart disease(CHD)with blood stasis syndrome(BSS)is one of the most common Tranditional Chinese Medicine(TCM)syndromes in coronary heart disease.At present,researches have been carried out from many aspects,such as imaging examination,routine biochemistry examination,gene polymorphism detection,transcriptomics detection,proteomics detection and metabonomics detection.In order to enrich the understanding of microcosmic syndrome differentiation of CHD with BSS,we studied the influence of miRNAs on coronary heart disease with blood stasis syndrome.Through detected the miRNA expression profile in BSS of stable angina patients,this study tried to construct the miRNA regulatory network and fingerprint map of blood stasis syndrome in coronary heart disease under the chronic ischemia pathology.Methods(1)Established chronic myocardial ischemia rat model with Blood Stasis Syndrome(BSS)through “high-fat feeding & isoprenaline injection” method.Extracted the total RNAs from rat hearts and plasmas respectively,then used Agilent miRNA arrays to screen the differently expressed miRNAs based on the Sanger microRNA Database which including 719 rat miRNAs.Analyzed differently expressed miRNAs by bioinformatics methods,such as Gene Ontology(GO)analysis and Pathway analysis.(2)Enrolled stable angina patients with Blood Stasis Syndrome and collected their plasmas.Extracted the total RNAs,then used Agilent miRNA arrays to screen the differently expressed miRNAs based on the Sanger microRNA Database which including 2006 human miRNAs.Analyzed differently expressed miRNAs by bioinformatics methods,such as Gene Ontology(GO)analysis and Pathway analysis.(3)Compared the miRNAs profiles of rat heart/plasma and human plasma.Constructed the CHD chronic blood stasis syndrome fingerprint and miRNAs-target genes/pathways regulatory network.(4)Validated the significant differently expressed miRNA by RT-qPCR.Explored the possibility of using miRNAs as biomarker of CHD with chronic blood stasis syndrome.Results(1)miRNA array screening results showed that the expression of 21 miRNAs in rat hearts had significantly changed,15 up-regulated,6 down-regulated.Meanwhile,the expression of 31 miRNAs in rat plasmas had significant changes,18 up-regulated,13 down-regulated.(2)miRNA array screening results showed that the expression of 22 miRNAs in human plasma had significantly changed,9 up-regulated,13 down-regulated.(3)GO enrichment analysis and pathway enrichment analysis were used to analyze differently expressed miRNA target genes in rat hearts,rat plasmas and human plasmas.The mainly changed biologic processes in CHD with chronic BSS included signal transduction,cell communication,metabolism,transport,energy pathway,cell growth and maintenance,apoptosis,immune response.The mainly changed pathway included proteoglycan syndecan-mediated signaling events,glypican pathway,ErbB receptor signaling network,integrin family cell surface interactions,TRAIL signaling pathway,LKB1 signaling network,endothelins,thrombin/proteotease-activated recptor pathway,signaling events mediated by Hepatocyte Growth Factor Receptor,VEGF and VEGFR signaling network,S1 P pathway,plasma membrane estrogen receptor signaling.These changes have close relationship with pathological/physiological process such as vascular endothelial injury,energy metabolism,cell proliferation/apoptosis,cell signal transduction,stem cells growth and differentiation,inflammation,angiogenesis,immune response,platelet aggregation,autophagy.these results are highly consistent with the results obtained from previous transcriptome,proteomics and metabolomics studies.(4)Our research also constructed a miRNAs-target genes network and a miRNAs-pathways network.We found that the mainly changed pathways include fatty acid metabolism,Signaling pathways regulating pluripotency of stem cells,TGF-beta signaling pathway,PI3K-Akt-mTOR signaling pathway,Adrenergic signaling in cardiomyocytes,and Platelet activation.We also found pathways in cancer,PI3K-Akt signaling pathway,MAPK signaling pathway,Proteoglycans in cancer,Ras signaling pathway are five important pathways,which have the most abundant degreement in the network and are the most critical nodes in the network.And these results are also consistent with previous studies’ results.(5)Two most stable endogenous controls(ECs)were identified as EC candidates,which can be used in qPCR in CHD with chronic BSS patients.And miR-223-3p,miR-24-3p,miR-106b-5p were detected a significant expression changed in CHD with chronic BSS patients,which maybe biomarkers for CHD with chronic BSS.ConclusionDifferent with CHD with acute BSS,the biologic processes changed in CHD with chronic BSS first mainly include vascular endothelial function,energy metabolism,cell proliferation/ apoptosis,signal transduction.In the later period of chronic BSS,the biologic processes including cardiomyto injury and fibrosis were changed subsequently.We proposed the pathological mechanism of CHD with chronic BSS is the blood stasis state results a disorder of energy metabolism,cell proliferation/apoptosis,immune response,and inflammation,then develops into a cardiomyto injury,fatty acid metabolism disorder,fibrosis in the later period.