Methylation Regulation Mechanism And Biomarker Of MiRNA Expression Related To Blood Stasis Syndrome In Coronary Heart Disease

Background and purposeCoronary heart disease(CHD)is the leading cause of death worldwide.Fifty-six million people worldwide died of CHD.With the development of the social economy and the accelerated aging of the population,risk factors for cardiovascular diseases will further increase,and it has become a very serious public health problem.Individualized treatment of patients with CHD can improve efficacy and prognosis.The essence of TCM syndromes is to determine the different subtypes of the disease from the macroscopic syndromes and to treat patients individually.For modern medicine,the individualized treatment focuses on searching for suitable biomarkers to distinguish different subtypes of patients with CHD.By modern medical methods,to objectify CHD blood stasis syndrome is conducive to understand deeply the CHD blood stasis syndrome.DNA methylation,miRNA,and mRNA can be cascaded to form a network.DNA methylation affects transcription.Hypomethylation of a gene promoter can upregulate gene expression,whereas methylation of a gene promoter can downregulate gene expression.Changes of DNA methylation in the promoter region of miRNAs affect miRNA production,whereas miRNAs inhibit mRNA expression by incomplete binding.Through this regulatory relationship,DNA methylation-miRNA-mRNA will form a gene regulatory network.Method1 Literature reviewRecent medical philosophy has begun to subdivide patients based on gene expression in order to optimize treatment.Genetic testing,the concept of individualized treatment for the more accurate treatment of diseases,resembles with traditional Chinese medicine differentiation of syndromes and treatment.By collecting the latest epigenetic data,we further analyzed the internal relationship between TCM constitution and syndrome differentiation.CHD has both genetic and environmental factors.In the past two decades,the epigenetic understanding has developed rapidly.It has been confirmed that epigenetic modifications are related to the occurrence and development of CHD.Search Pubmed,Web of Science,China Knowledge Network,Weipu Network,Wanfang Data Database to obtain epigenetic studies related to CHD and epigenetic progress related to CHD syndrome of TCM.The aim is to raise awareness of epigenetic mechanisms closely related to CHD and CHD syndromes and provide a new perspective for the development of CHD.In the study of CHD drugs,traditional Chinese medicine Panax notoginseng(Sanqi)has shown great potential,and has been widely used in the treatment of cardiovascular diseases due to its anti-inflammatory,lipid-lowering and anticoagulant effects,especially in the treatment of CHD.A significant effect.The main component of Panax notoginseng,Panax notoginseng saponins(PNS),reduced the degree of aortic endometrial lesions,and significantly changed blood lipids,inflammation,and hemorheology,which is an important Chinese medicine for inhibiting atherosclerosis.The assessment of the efficacy of PNS on CHD was conducted according to the systematic evaluation and the recommended report item of the Meta-analysis statement.Search data from the database to February 2017,including the following databases:CENTRAL,MEDLINE,EMBASE,WHO ICTRP,China Knowledge Network,Wanfang,Weipu and SinoMed.All randomized controlled trials(RCTs)for PNS-based unstable angina(UA)compliance were included.With reference to the Cochrane Handbook,we assessed the risk of bias in each study.The meta-analysis was performed using RevMan 5.3 software.2 Experimental studyHigh-throughput sequencing technology was used to detect DNA methylation,miRNA and mRNA expression in peripheral blood nucleated cells of CHD patients with blood stasis syndrome,non-blood stasis syndrome of CHD,blood stasis syndrome of non-CHD and normal control subjects.The bioinformatics methods were used to screen for CHD blood stasis syndrome,CHD non-blood stasis syndrome,non-CHD blood stasis syndrome and normal control group of differential genes.Taking the differential gene of CHD blood stasis group and normal control group as the main body,the correlation analysis was conducted to construct a DNA methylation-miRNA-mRNA gene regulatory network.The clustering analysis,principal component analysis,GO function enrichment,and KEGG signaling pathway analysis were performed with genes obtained.qPCR verification is performed on key nodes in the gene network in another matching queue.Eighty cases of CHD patients with blood stasis were collected and randomized.The treatment group took Xuesaitong soft capsules and the control group took soft capsule simulator.The treatment period was 4 weeks.The outcomes included 90 days of cardiovascular events,blood stasis syndrome score,blood lipids and adverse reactions during the use of patients and liver and kidney function.The methylation level of CpG islands in the miRNA promoter region,miRNAs,mRNA and signal pathways were measured in patients with CHD and blood stasis syndrome before and after treatment.Result1 Epigenetic Mechanism of Blood Stasis Syndrome in CHDMany studies have found that epigenetic changes such as DNA methylation,histone modification,miRNA expression,and IncRNA expression play an important role in cardiovascular disease,and are related to environmental factors,age factors,and heredity.In the epigenetic study of CAD,the study of miRNA expression is more in-depth,and researches on DNA methylation and histone modification are increasing,and IncRNA is also an interesting subject.DNA methylation is stable;however,it is strongly influenced by the environment.The DNA methylation of Hcy,Alu,LINE-1 and specific gene promoters has diagnostic value and is closely related to sex,race and age.DNA methylation and histone acetylation participate in the process of mutual stimulation.miRNAs play an important role in the development of CAD.Knockout and overexpression of individual miRNAs are suitable topics for exploring the diversity of cardiovascular events,such as vascular injury.In addition,expression profiling and biological methods for predicting miRNA target genes are beginning to provide many clues for cardiovascular function.Biomarker studies have also found some heart-specific miRNAs.Some miRNAs may be the main potential target for the treatment of myocardial ischemic injury.However,there are few therapeutic options for epigenetic mechanisms other than miRNAs.Epigenetic studies have found that certain DNA methylation sites and differentially expressed miRNAs are present in CHD with blood stasis syndrome,including the extent of DNA methylation in the promoter region of ER-?,DNA methylation of DES and CTNNB1 genes,miR-384,hsa-miR-199a-5p,hsa-miR-146b-5p,hsa-miR-3158-3p,miR-208a-3p,miR-222-3p and miR-198.These studies indicate that epigenetic methylation sites or miRNAs are potential biomarkers of CHD blood stasis syndrome and provide new ideas for the pathological mechanism of CHD blood stasis syndrome.2 Systematic Assessment and Mechanism of PNS Intervene in CHDA total of 17 studies totaling 2,315 UA patients were included in this systematic review.The results of the included studies indicated that PNS had therapeutic effect in reducing the primary endpoint,the improvement of symptoms,including the frequency and duration of angina pectoris,and the use of nitroglycerin.The primary end point was 21(3.0%)of the 680 patients who took oral PNS plus conventional medicine(PPCM),and 53 of the 680 patients(7.8%)with only conventional therapy.PPCM had a duration of angina attack(decreasing 1.88 min),nitroglycerin use(decreasing 1.13 mg),TC(decreasing 0.79 mmol/L),TG(decreasing 0.23 mmol/L),LDL(decreasing 0.77 mmol/L),HDL(increasing 0.30 mmol/L).Compared with the traditional medicine group,oral adverse reactions of PNS are rare.However,due to significant heterogeneity and poor quality of results,results should be treated with caution.PNS has a variety of positive effects in the key processes of CAD,including anti-inflammatory,regulating lipid metabolism and coagulation system,anti-apoptosis,angiogenesis,anti-atherosclerosis and anti-myocardial ischemia.Inflammation runs through the occurrence,formation,and onset of CAD.Extensive inflammation produces major features of vulnerable plaques.PNS exerts anti-inflammatory effects through several signaling pathways.PNS can modulate proinflammatory cytokines by inhibiting ROS,TNA?,NF-?B and promoting SOD.Interestingly,PNS has dual functions for COX-2 at different times.PNS alone,NR1 can inhibit inflammatory cytokine production by activating PPAR? and inhibiting TNF-? in vitro and in vivo by inhibiting ERK and PKB.Inflammation plays an important role in the entire CAD process,allowing long-term use of PNS with less side effects.At the same time,the long-term application of PNS can reduce the endpoint of CAD.3 DNA Methylation-miRNA-mRNA Regulatory Networks in Blood Stasis Syndrome ofCHDHigh-throughput sequencing technology and bioinformatics analysis were used to compare the blood stasis syndrome of CHD,non-blood stasis syndrome of CHD,blood stasis syndrome of non-CHD and normal control group.Differential methylation sites,miRNA and mRNA were screened out from the UA blood group and the normal control group.The results showed that a total of 28,461 methylation sites were different(methylation of 4,498 sites were down-regulated,methylation of 23963 sites were up-regulated),and 295 miRNAs were differentially expressed(171 miRNAs were down-regulated,and 124 miRNAs were up-regulated).Upregulated),470 mRNA differentially expressed(220 mRNAs were down-regulated,250 mRNAs were up-regulated).Hierarchical cluster analysis and principal component analysis showed that DNA methylation,miRNA,and mRNA could distinguish the four groups of samples between the CHD syndrome group,the CHD non-blood stasis syndrome group,and the normal control group.Through further analysis of related genes and signal pathways,the differentially expressed genes related to blood stasis syndrome in CHD are mainly related to immunity and inflammation,such as immune response,I-kappaB kinase/Positive regulation of NF-kappaB cascade,B cell receptor signaling pathway,Toll-like receptor signaling pathway,T cell receptor signaling pathway,etc.Using the UCSC database,MiRanda and correlation analysis revealed that 46 DNA methylation sites,45 miRNAs,and 109 DNA methylation sites were involved in the DNA methylation-miRNA-mRNA regulatory network associated with CHD blood stasis syndrome.According to network topology analysis and database comparison,it was determined that the key nodes include three DNA methylation sites,four miRNAs and six mRNAs.Pyrosequencing and qPCR detection methods found that the trend of changes at the key nodes was basically the same as the sequencing results.4 Changes in key nodes in DNA methylation-miRNA-mRNA regulatory network when changes in blood stasis syndrome occur in CHDIn a randomized controlled clinical trial of 84 patients with CHD and blood stasis syndrome,four weeks of intervention with Xuesaitong soft capsule and placebo showed that Xuesaitong intervention can reduce the frequency of angina attacks and reduce blood stasis syndrome in patients with CHD,regulate blood lipids with no significant adverse reactions.Before and after treatment,DNA methylation,miRNA,and mRNA in the key nodes of the Xuesaitong group and the control group were detected.The above-mentioned key nodes all showed changes,and there was no significant difference,but there were already obvious tendency.When the blood stasis syndrome of CHD changed,the miR-194 promoter region,miR-194,FOS,TNFR,and MRAS all changed at the key nodes,which was consistent with the previous biomarker screening results.ConclusionThere are DNA methylation sites,differential gene expression profiles of miRNA and mRNA in CHD with blood stasis syndrome.And related gene functions involve immune and inflammatory response pathways.There are mutual regulatory relationships among the above-mentioned differential genes,and a DNA methylation-miRNA-mRNA regulatory network related to blood stasis syndrome in CHD can be constructed.Randomized controlled trials have confirmed that after the improvement of CHD blood stasis syndrome,the key nodes of the regulatory network will change.The study of DNA methylation-miRNA-mRNA regulatory network and its key nodes in CHD blood stasis syndrome provides a scientific basis for finding the material basis and diagnostic biomarkers of CHD blood stasis syndrome.