| Objective1.To study the relationship between differential expression of microRNA and coronary heart disease,Qi-deficiency and blood-stasis syndrome,qi deficiency phlegm and blood stasis syndrome,mainly to find the two types of specific miRNAs in coronary heart disease,and to understand the regulation mechanism of coronary syndromes,Dialectical classification to provide an objective basis for the promotion of coronary heart disease syndrome diagnosis and standardization of the construction.2.To explore the dynamic relationship between specific microRNA and related syndromes after dialectical treatment of Chinese medicine,to further reveal the essence of TCM syndromes,and to find a new target for TCM treatment from microRNA level,and to do a related mechanism for the treatment of coronary heart disease New exploration,enriched the connotation of modern Chinese medicine theory.Methods1.Two patients with coronary heart disease and qi deficiency and blood stasis syndrome,two coronary heart disease phlegm stasis syndrome and one healthy volunteers,collected peripheral blood,and microRNA serum kit to extract microRNA,the use of high-throughput sequencing platform to detect normal human peripheral blood And the miRNA expression profiles of peripheral blood in patients with qi deficiency and blood stasis and qi deficiency phlegm and blood stasis,and analyzed the differentially expressed miRNAs.The target gene was predicted by miRanda-3.3a,and the target gene was enriched by miranda database,and the differential gene enrichment of KEGG pathway was analyzed by DAVID 6.7.2.Fluorescence quantitative PCR was used to validate some differences miRNA: 10 patients with coronary heart disease and blood stasis syndrome,14 patients with coronary heart disease and phlegm and blood stasis syndrome,5 healthy volunteers were collected peripheral blood,qRT-PCR method to verify the high Flux sequencing of miRNA results,analysis of miRNA expression differences and significance.3.In this study,patients with coronary heart disease and blood stasis syndrome were randomly divided into control group and treatment group.The control group was treated with anticoagulation,anti-poly and other conventional treatment,the treatment group on the basis of conventional Western medicine treatment,plus Tongguan capsule intervention in the first half of the treatment of patients with microRNA levels.Thirty patients with coronary heart disease and qi deficiency and blood stasis were randomly divided into treatment group(n =10),control group(n = 10)and healthy control group(n = 15).The expression of miR-17-5p.miR-320 c was detected by qRT-PCR Results: 1.High-throughput sequencing to construct the differential microRNA expression profiles of coronary heart disease with ACS deficiency and phlegm and blood stasis.(1)Compared with the normal group,127 differentially expressed microRNAs were screened,0 were up-regulated and 127 were down-regulated(P <0.05).Among them,there were 46 miRNAs with significant difference(P <0.01).The major down-regulated genes were miR-17-5P,miR-320 a,miR-320 c and so on.(2)Compared with the normal group,72 differentially expressed microRNAs were screened,0 were up-regulated and 72 were down-regulated(P <0.05).There were 39 microRNAs with significant differences(P <O.O1),and the major down-regulated genes were miR-4433-3P,miR-363-5P and so on.(P <O.O5),among which 4 were up-regulated and 4 were down-regulated,and coronary heart disease(ACS)was obtained from Qi-deficiency and blood-stasis group and Qi-deficiency and phlegm-blood stasis group.The difference of microRNA expression spectrum was miR-17-5P,miR-320 a,miR-320 c and miR-320 d.(4)Predicting the gene of Qi deficiency and blood stasis and phlegm and phlegm stasis(1)Compared with the healthy volunteers group,8597 differentially expressed microRNA target genes were screened and the corresponding target genes were analyzed for CO function.The difference between the miRNAs and the genes in the biological process was mainly related to the cell process(cellular process),metabolic process,single organism biological process,and cell components and components and cells and cell parts(cell,cell apart)function is closely linked,and finally in the molecular function of binding and the catalytic process is Most target gene enrichment function.(2)Compared with the healthy volunteers group,the gene microRNAs were screened to predict the target gene,and the target gene was obtained by 9832,and the corresponding target gene was analyzed by CO function.The difference between miRNA target gene This part of the process is mainly related to cellular processes,metabolic processes,and cell components and components and cells and cell parts(cell,cell apart)function is closely linked,and finally in the molecular function of binding(binding)As well as the catalytic process is considered to be the most target gene enrichment function.(5)Analysis of KEGG pathway of qi deficiency and blood stasis and deficiency phlegm and blood stasis gene(1)DAVID software was used to analyze the differentially expressed miRNA gene-enriched KEGG pathways.The expression of miRNAs in the patients with coronary heart disease and qi deficiency and blood stasis was down-regulated,and the expression of miRNAs was up-regulated.The up-regulated target genes were analyzed by enrichment of KEGG pathway and found that MAPK signal pathway was closely related to coronary heart disease,Calcium signaling pathway.(2)DAVID software was used to analyze the differentially expressed miRNA gene-enriched KEGG pathways.The expression of miRNAs was down-regulated in miRNA-regulated phlegm-stasis patients and healthy subjects.The up-regulated target genes were up-regulated by KEGG pathway and found that there was a close relationship with the relationship between coronary artery disease and T cells Body signal pathway,wnT signal pathway,MAPK signal pathway,calcium signal pathway.2.The second part of the qPCR validation(1)Compared with the healthy control group,the miR-320 a normal group in the ACS deficiency group was not amplified and could be compared with the gene The expression of miR-320 c and miR-17-5P was down-regulated,the difference was statistically significant(P <0.05)(2)Compared with the healthy control group,the expression of miR-668 and miR-320 d in qi deficiency phlegm and blood stasis group was significantly lower than that in healthy control group(P <0.05),and the difference was statistically significant(P <0.05).The expression of miR-363-5P and miR-4284 was down-regulated,but the difference was not statistically significant.The miR-4433-3P was not able to participate in the expression of some fragments.(3)The qRT-PCR demonstrated that there were two common miRNAs in the plasma of patients with qi deficiency and blood stasis syndrome and qi deficiency phlegm and blood stasis,namely miR-320 c and miR-320 d.The miR-320 c and miR-320 d were down-regulated in the two syndromes by qRT-PCR,but there was no significant difference between them.3.Effect of Tongguan Capsule on MicroRNA of Coronary Heart Disease and Qi Deficiency and Blood Stasis Syndrome.Compared with normal subjects,the expression of miR-320 c was decreased in patients with coronary heart disease and Qi deficiency and blood stasis syndrome.After treatment with Tongguan Capsule,the expression of miR-320 c in both groups was higher than that before treatment.There was no significant difference in miR-320 c expression between the treatment group and the control group(P = 0.41> 0.05).After treatment,the expression of miR-320 c in the treatment group was significantly higher than that before treatment(P =(P = 0.22> 0.05).Compared with normal subjects,the expression of miR-17-5P in coronary heart disease was lower than that in normal control group(P <0.05).After treatment,the expression of miR-17-5P in both groups was higher than that before treatment.There was no significant difference in miR-17-5P expression between the treatment group and the control group(P = 0.62> 0.05).After treatment,the expression level of miR-17-5P in the treatment group was significantly higher than that before treatment(P = 0.016 <0.05).There was no significant difference in the expression of miR-17-5P before and after treatment(P = 0.34> 0.05).Conclusions 1.Coronary heart disease Qi deficiency and blood stasis related microRNA for miR-320 c,miR-17-5P,related target gene prediction,differential miRNA target gene in the biological process This part is mainly with the cellular process(cellular process),Metabolic processes,single organism biological processes,and cell components and components and cells and cell parts(cell,cell apart)function is closely linked,and finally in the molecular function of binding and the catalytic process is the most target gene rich Set of functions.The main target gene KEGG pathway is MAPK signal pathway,calcium signal pathway.2.MiR-3208,miR-320 d,related target gene prediction,differential miRNA-targeted genes in the biological process of this part of the most important with the cellular process(cellular process),metabolism(Cell,cell apart)function is closely linked,and finally in the molecular function of binding(binding)and the catalytic process is considered to be the most target gene enrichment function.The main target gene KEGG pathway is T cell receptor signaling pathway,wnT signal pathway,MAPK signal pathway,calcium signaling pathway.3.The miRNAs of miR-320 c and miR-320 d were significantly different from those in the normal group,and there was no difference between the two syndromes.The pathophysiology of ACS The regulation of the process plays an important role in the pathogenesis of ACS and can be further studied as a potential biomarker of ACS.4.MiR-320 c and miR-17-5P were associated with the disease progression of coronary heart disease and qi deficiency and blood stasis syndrome after Tongguan capsule intervention.It was further proved that miR-320 c and miR-17-5P could be used as coronary heart disease and qi deficiency and blood stasis syndrome And to lay the foundation for the study of the target and mechanism of Tongguan Capsule in the treatment of coronary heart disease. |
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