Influenza-miRNA-mRNA Regulatory Network Related To Coronary Heart Disease And Blood Stasis Syndrome

Background and PurposeWith the rapid development of society and economy,and the acceleration of population aging process,the risk factors for cardiovascular diseases have been increased continuously.Coronary heart disease(CHD)has become one of the most lethal chronic diseases.The morbidity and unsatisfied secondary prevention of CHD have caused social and economic burden to China and worldwide.It is warrant that we constantly explore the pathological mechanisms of CHD and optimize the treating strategies.In the modern theory of Traditional Chinese medicine(TCM),CHD is reckoned as "chest pain",which is caused by Blood Stasis.Blood Stasis is the major syndrome element of CHD angina.Exploring the pathogenesis of CHD from the perspective of Blood Stasis can benefit the profound understanding of CHD Blood Stasis syndrome.With the rapid development of omics and bioinformatics technology in the past decade,the researches on the essence of CHD Blood Stasis syndrome have reached the level of genomics and transcriptomics.The massive information,objective and profound indexes,network-correlated characteristics of genomics can be contributed to excavating the syndrome essence of CHD Blood Stasis syndrome in multiple levels.This research is based on previous researches to explore the pathological mechanisms and syndrome essence of CHD Blood Stasis syndrome.Screen the specific gene expression profiles of CHD Blood Stasis via high-throughput sequencing,and validate the gene-gene interactions by shRNA transfection,to construct the lncRNA-miRNA-mRNA interaction network and locate the pivot nodes in this network.In that can this research provide objective and scientific evidences for the development of biomarkers and intervention targets of CHD Blood Stasis syndrome.Methods1 Literature researchSearch the CNKI,VIP,Wanfang,Pubmed and Web of Science databases,acquire the genomic researches on TCM syndrome without limitation of diseases,syndrome differentiation and intervention,systematically review the research subjects,technologies,methodology and characteristics of these researches.Study contents include clinical trials,animal and cell experiments,via microarray or high-throughput sequencing technologies to explore SNP,DNA,mRNA,ncRNA(including miRNA,lncRNA and circular RNA).Summarize the trend of these researches and provide suggestions for this research.2 Experimental researchUse high-throughput technologies to detect the IncRNA,miRNA and mRNA expression in peripheral blood nucleated cells of CHD with Blood Stasis syndrome(A),CHD without Blood Stasis syndrome(B)and healthy control(N).Screen the differentially expressed genes(DEGs)in A vs N and A vs B,obtain the CHD Blood Stasis specific expressed IncRNA,miRNA and mRNA profiles.Perform Hierarch clustering,Principle component analysis(PCA),Gene Ontology and KEGG enrichment analysis with the DEGs.Through the combination of starbase prediction and Pearson correlation analysis,obtain the gene-gene interactions and construct the CHD Blood Stasis syndrome specific IncRNA-miRNA-mRNA interaction network.Validate the key nodes of this network in another cohort with qRT-PCR.Use lentivirus introduced shRNA infection to interfere the pivot nodes in lncRNA-miRNA-mRNA interaction network,detect the expression of their up and down stream nodes,in order to validate the gene-gene interactions and network.Results1 Systematic analysis of genomic researches on TCM syndromesThe systematic analysis of 30 included researches showed that there have been 13 diseases/conditions and 11 syndromes in these researches.The earliest publication was in 2005,and the latest was in 2016.The applied technologies included microarray and high-throughput sequencing of DNDA/cDNA,mRNA,miRNA,lncRNA and SNP.The most studied diseases were CHD,stroke,hepatitis B,diabetes and aging.The most studied syndromes were Blood Stasis syndrome and Kidney-Yang deficiency(8 articles respectively),followed by damp turbidity obstruction and liver depression and spleen deficiency(4 articles respectively).mRNA was the most focused research index.The combination of miRNA-mRNA and lncRNA-mRNA were gradually increased.From the perspective of methodology and outcome,genomic researches on TCM syndrome are still in the developing phase.Lack of unified and well-reckoned diagnostic criteria and modeling methods,superficial analysis which only identify DEGs without functional research,absence of PCR validation,all of these have hampered the deeper discovery of the included researches.The genomic researches on TCM syndrome have achieved certain positive results,yet the study design,methodology and long-term scheme should be improved and more rigorous and scientific.The result interpretation should be more profound.In that can it provide scientific evidence and new research perspective for TCM syndrome objectification.2 CHD Blood Stasis syndrome related lncRNA-miRNA-mRNA interaction network researchUsed high-throughput sequencing and bioinformatics analysis,this research identified 39 lncRNAs(3 upregulated and 36 downregulated),229 miRNAs(138 upregulated and 91 downregulated)and 221 mRNAs(105 upregulated and 116 downregulated)in CHD Blood Stasis syndrome.Hierarch clustering and PCA results showed that A,B and C group can be distinguished by IncRNA,miRNA and mRNA respectively.All the samples were categorized orderly.Functional and pathway enrichment analysis showed that the DEGs were mainly related to immune and inflammation,such as immune response-activating signal transduction,interferon-gamma-mediated signaling pathway,T cell costimulation,leukocyte mediated immunity,leukocyte activation and Toll-like receptor signaling pathway.The gene-gene interaction analysis returned 76 edges,included 9 lncRNAs(downregulated),24 miRNAs(14 upregulated and 10 downregulated)and 31 mRNA(11 upregulated and 20 downregulated).The IncRNA-miRNA-mRNA interaction network was constructed.Topological analysis and Venn diagram were performed,CTA-384D8.35,CTB-114C7.4,RP11-567M16.6 and hsa-miR-3158-3p were considered the pivotal nodes in this interaction network.PCR experiment validated and supported these results.The lentivirus introduced shRNA infection in vivo experiment has validated the gene-gene interaction in CHD Blood Stasis syndrome network.There are mainly 6 interaction mode within this network,including IncRNA upregulates mRNA,IncRNA downregulates mRNA,miRNA downregulates mRNA,miRNA upregulates mRNA,IncRNA and miRNA mutually interfere with each other,and lncRNA-miRNA-mRNA co-expression.ConclusionThere exist CHD Blood Stasis syndrome specific IncRNA,miRNA,mRNA expression profiles.The related functions and pathways were involved in immune and inflammation.Based on the gene-gene interactions a IncRNA-miRNA-mRNA interaction network was constructed.RNAi experiment has validated the multi-direction,overlapped and complicated interaction mode between lncRNA,miRNA and mRNA,and the authenticity of this network.The exploration of CHD Blood Stasis syndrome lncRNA-miRNA-mRNA interaction network and the research on the essence of Blood Stasis have provided solid scientific foundation and new perspective for genomic and transcriptomic research on TCM syndrome.