Epidemiological Characteristics Of TCM Syndrome Of Very Elderly Hypertensive Patients And Long Non Coding RNA Expression Profile Study

Essential hypertension is a series of syndrome with high blood pressure as the main clinical manifestations with or without multiple cardiovascular risk factors. It is an important cause and risk factors of a variety of cardiovascular and cerebrovascular diseases, effect structure and function of important organs such as heart, brain and kidney, finally lead to failure of them. So it is the leading cause of the death of Cardiovascular disease and its pathogenesis is very complicated. The incidence of hypertension is increased with age significantly, epidemiological data showed that:hypertension incidence of the population 80 years old or elder was over 70%. Currently we call them very elderly people whose age is 80 years old or elder, and then we call it very elderly hypertension of these people.Based on evidence based medicine, effective treatment of very elderly hypertensive patients is of significant meaning for reducing the risk of cardiovascular disease and prolong life and improve quality of life and reduce rate of death.In recent years, correlation between TCM syndrome type of essential hypertension and objective physical and chemical indicators had been studied, and acquired certain results. However, TCM syndrome types of hypertension had not yet been unified. Objective study of TCM syndromes are also at the exploratory stage,and need further study in depth. Micro-array technology has high throughput, miniaturization and automation features, provides a powerful tool for the study of the nature syndrome, and had consistency with holistic concept of TCM, thus the gene chip is now widely used in many areas of medical research.LncRNA are a class of transcript longer than RNA 200bp, which itself lacks significant open reading frame, located in the nucleus or cytoplasm, has no protein-coding function, but control expression of gene in the form of RNA at the level of epigenetic, transcriptional and post transcriptional etc. In this study, we use LncRNA and mRNA microarray technology to detect very elderly hypertensive patients’ LncRNAs and mRNA expression characteristics and find the difference of different TCM syndrome. LncRNA study is difficult because of its uncertainty, no existing database to find its function, to solve this problem, so we study the LncRNA related mRNA research to detect the function of LncRNA indirectly. This study we use GO and KEGG analysis, use bioinformatics analysis method, combining the networks and pathways, analyze the regulation function of different expressed genes in the process of disease.At present, the study of TCM syndrome research of very elderly patients with hypertension was lacking. In this study we use epidemiological methods to collect TCM clinical symptoms of very elderly hypertensive patients, in order to further understand the syndrome distribution of them. Provide evidence for clinical diagnosis and treatment of TCM of very elderly hypertensive patients, Lay the foundation for the study of TCM syndromes biological nature of hypertension.1. ObjectStudy TCM syndromes distribution characteristic of very elderly essential hypertensive patients and differences of physical and chemical indicators of and different TCM syndromes. Detect LncRNAs and mRNA expression differences in very elderly hypertensive patients of each TCM syndrome. Provide more biological evidence for very elderly hypertensive patients’ diagnosis and treatment of TCM syndrome.To initially exploration biological basis of the pathogenesis of very elderly hypertension of different TCM syndromes in LncRNA level.2. Method2.1 Clinical data collectionWe used epidemiological survey methods, collected clinical symptoms of very elderly hypertensive patients comprehensively.Selected the symptoms which frequency> 10%, and use factor analysis to extracted syndrome elements. Then use clustering analysis to extract main syndrome type of these factors. Compare indicators of ambulatory blood pressure monitoring, Holter, neck ultrasound and biochemical, find their difference and explore it.2.2 Experimental Research MethodsSelected very elderly hypertensive patients with typical TCM syndrome above randomly.5 cases in each group, a total of 25 cases,5 cases of healthy people, collect venous blood, total RNA of peripheral mononuclear cells was extracted. LncRNA+mRNA expression microarray Chip were applicated.Detect differences of LncRNA+mRNA expression between very elderly hypertensive patients group and the healthy group. Simultaneous detect LncRNA+mRNA expression differences between each group of very elderly hypertensive patients with different syndromes. GO and KEGG analysis were applicated to analyze the function of differentially expressed genes preliminary. Use gene co-expression and target gene prediction methods, to selection of key LncRNAs of very elderly hypertensive patients and key LncRNAs of different TCM syndrome of them.2.3 statistic methodsSPSS20.0 statistical software were used to analyze data, TCM symptom frequency statistics using frequency analysis;TCM elements, syndromes distribution of cases using factor analysis and cluster analysis statistical method. Measurement data expressed as mean ± standard deviation （x±SD）; Among groups when comparing the measurement data, Failure to comply with the normal distribution, Using Kruskal. Wallis test, If they meet the mindfulness distribution, Levene’S determine whether the use of test homogeneity of variance, Meet the homogeneity of variance using ANOVA, If heterogeneity of variance is used Kruskal. Wallis test; Count data using x2 test;when p≤0.05 Statistical differences were significant. Using excel-2013 software to draw chart. Analysis of genetic differences using Agilent Feature Extraction （v10.7） image analysis software for hybridization and data extraction. Then use the Agilent GeneSpring software for data normalization and variance analysis. Cluster3.0 software were average-linkage hierarchical clustering operation According to the clustering results in a Java environment, run the TreeView software to draw heat map. GO analysis, KEGG analysis system using mas system of bio-capital company.3. Results3.1 TCM Syndrome Characteristics of very elderly hypertensive patientsA total of 357 cases very elderly patients with hypertension were included, after epidemiological investigation, factor analysis is divided into nine common factors, namely F1-F9; disease location Syndrome elements:heart and liver with kidney （9%）, heart and liver （4%）; heart （10%）; kidney （8%）; spleen and kidney （31%）; liver and kidney （33%）; liver （5%）. property of Syndrome elements:Qi and Yin deficiency （8.68%）; Phlegm+ Qi stagnation+Blood stasis+ Yang excessive （16.25%）; Yang deficiency+ Blood stasis （10.08%）; Qi deficiency+ Phlegm+ Dampness （6.16%）; Qi stagnation+ Blood stasis （7.56%）; Phlegm+ Blood stasis （22.69%）; water retention+ Phlegm（14.85%）; Blood stasis+ Qi stagnation （9.52%）; Phlegm+ Blood stasis+ Qi stagnation （4.21%）. cluster analyze these nine factor above, The very elderly hypertensive patients can be grouped into five categories, That first category:Yin deficiency of Liver and kidney and Qi deficiency and Blood stasis（19.05%）; the second category:hyperactivity and phlegm Dampness resistance （24.93%）; the third category:Qi deficiency and Blood stasis （9.52%）;fourth class:yin and yang deficiency and qi stagnation Phlegm （38.94%）; fifth category: phlegm and blood stasis （7.56%）. Comparison these five different kinds of patients with clinical syndromes of physical and chemical indicators, we find were differences between the syndromes of ambulatory blood pressure, heart rate variability, carotid artery thickness, biochemical index, blood indicators of thyroid function, renin levels, homocysteine, etc.3.2 LncRNA expression profiling analysis of very elderly hypertensive patientsLncRNA+mRNA expression microarray analysis revealed:LncRNA and mRNA expression were significantly different in" very elderly hypertensive patients " and "healthy" group," very elderly hypertensive patients" and "healthy" group of differentially expressed mRNA widely enriched in cell components, biological processes, many functional pathway analysis;KEGG analysis revealed: "very elderly hypertensive patients" group vs "healthy" group upregulate the expression of mRNA enriched in Epithelial cell signaling in Helicobacter pylori infection^ Toll-like receptor signaling pathway% Asthma% Allograft rejection, Graft-versus-host disease、Bladder cancer Type I diabetes mellitus、 Cytokine-cytokine receptor interaction paths and so on. Down-regulated genes enriched in six paths:Systemic lupus erythematosus、Antigen processing and presentation、Natural killer cell mediated cytotoxicity、 Graft-versus-host disease、Glycine, serine and threonine metabolism、 ABC transporters-General. There is a significant link these pathways associated with inflammation, immune response mechanisms.3.3 TCM syndromes target gene prediction of very elderly hypertensive patients with differentcomparison between the various syndromes of very elderly hypertensive patients, existed a large number of differentially expressed LncRNA and mRNA. Extensive enrichment of differentially expressed mRNA in the cell components, biological processes, analytical functions. KEGG pathway compare, there were also a lot of difference. All these provided objective basis for the very elderly hypertensive patients with different syndromes, and simultaneously pointed out the research direction. The use of differentially expressed genes, the gene coexpression of the target gene prediction analysis found that different target genes TCM syndrome as follows:CTD-2369P2.8, CTB-61M7.2 crucial for the very elderly hypertension up regulate LncRNA;ENST00000424181.1 critical down-regulated genes. "Yin deficiency of Liver and kidney and Qi deficiency andBlood stasis"syndromes:ENST00000505155.1 , ENST00000420417.3, CTD-2369P2.8, TCONS₀0010443, ENST00000435554.1, ENST 00000457303.3, ENST00000505564.2, CTB-61M7.2, ENST00000435554.1, ENST0000 0505564.2, ENST00000453832.2 were critical up-regulated LncRNA,uc021wqv.1, CTD-2012I17.1, ZNF718, TCONS₀0026226 ,TCONS₀0022737, ENST00000424181.1 were key down-regulated LncRNA;"hyperactivity and phlegm Dampness resistance" Syndrome: CTD-2369P2.8, XXbac-BPG254F23.6 were critical up-regulated LncRNA, ENST00000424181.1 were key down-regulated LncRNA; "QDBS" card in: ENST00000589217. lcritical raised LncRNA, ENST00000424181.1 critical downward LncRNA, "yin and yang deficiency and qi stagnation Phlegm" syndromes:CTD-2369P2.8, HIT000092395₀3,HIT000095414₀4 critical raisedLncRNA;XR₁71093.1,TC0NS₀0010987,ENST00000595 309.1.ENST00000513626.1, ENST00000424181. lfor key down LncRNA; "Phlegm and blood stasis" Syndrome:ENST00000457239.1, ENST00000455707.1 , CTD-2369P2.8, ENST00000432452.1, ENST00000449954.1, CTB-61M7.2critical raised LncRNA, HIT000326149, ENST00000424181.1 were critical down regulated LncRNA.4. Conclusion4.1 very elderly hypertensive patients had more concomitant diseases, clinical symptoms are very complex, its syndromes characterized mainly mixed with the actual situation, a single syndrome elements were rare, most complex syndrome consisting of multiple certificates prime candidates, through factor analysis and cluster analysis combined results: liver and kidney and blood stasis, hyperactivity and phlegm, blood stasis, yin and yang and qi stagnation phlegm, phlegm and blood stasis syndrome were mainly TCM syndrome of very elderly hypertensive.4.2 There are differences between the candidates ambulatory blood pressure, heart rate variability, carotid artery thickness, biochemical indicates, blood indicators of thyroid function, renin levels and other physical and chemical indicators old elderly hypertensive patients of different syndromes, these all provided biological basis for very elderly hypertensive patients.4.3 Through the study of LncRNA and mRNA between very elderly hypertensive patients and healthy control group,we find LncRNA and mRNA differential expression in very elderly hypertensive patients and healthy controls and different TCM syndromes, by bioinformatics analysis of differential expression gene, we found a significant link between hypertension and inflammation, immune response mechanism, which pointed out the direction for subsequent study of the internal mechanism of hypertension.4.4 In this study, gene co-expression of the target gene prediction method to establish the link between LncRNA and mRNA, initially established hypertension of various syndromes coexpression network, and a series of preliminary findings of LncRNAs and mRNAs of very elderly hypertension-related key genes, which essentially follow-up study of hypertensive syndrome foundation.