脂代谢相关miRNA靶基因结合区域多态性与代谢综合征及其组分的分子流行病学研究

  本文关键词：脂代谢相关miRNA靶基因结合区域多态性与代谢综合征及其组分的分子流行病学研究，由笔耕文化传播整理发布。

        代谢综合征(metabolic syndrome, MetS)是一组以肥胖、高血糖(糖尿病(DM)或糖调节异常(IGR))、血脂异常(高甘油三酯(TG)和(或)低高密度脂蛋白胆固醇(HDL-C))以及高血压等聚集发病，严重影响人类健康的临床征候群。2005年国际糖尿病联盟(International Diabetes Federation, IDF)估计全球约1/4的人患有MetS。MetS是2型糖尿病和心血管疾病患病率及死亡率增高的重要危险因素之一。近年来，随着经济高速发展，人们的饮食、生活方式发生了巨大改变，MetS的发病率快速增长，已成为严重威胁人类健康的公共卫生问题。因此，有关MetS的病因学机制及其防治研究成为研究热点。MetS是一种环境因素与遗传因素共同作用引起的疾病，由于它本身为多种因素的集合，遗传特点复杂，其主要机制为机体热量过剩导致脂肪在脂肪组织和非脂肪组织中过度蓄积及其相关的胰岛素抵抗。其中脂肪代谢紊乱是MetS发病的重要环节。研究表明microRNA (miRNA)在多种生物进程中发挥着重要的作用，其中包括脂肪细胞的增殖分化、代谢整合、胰岛素抵抗及食欲的调节。MiRNA在代谢综合征发展的各个阶段也起到不容忽视的调控作用，在与MetS发病机理有关的脂代谢和脂肪形成中, miRNA不但参与动物调控脂肪细胞的分化，还参与脂类代谢及与脂类代谢相关的内分泌激素调节，有的甚至与肥胖、T2DM、MetS等脂类代谢病的基本病理相关。越来越多的证据表明，，miRNA靶基因3’UTRs(untranslated regions)或5’UTRs内的单核苷酸多态性(single-nucleotide polymorphism, SNP)可以影响miRNA与靶区域的结合效率，造成miRNA调控作用发生改变，最终导致相应表型—疾病状态。因此，探讨脂代谢相关miRNA靶基因结合区域SNPs与MetS发病风险间的联系，将进一步丰富MetS分子病因学内容。此外，近年来，生物信息学方面提供了一系列基于miRNA序列及靶基因的数据库()，同时可获取多种miRNA靶基因预测的方法和生物信息学软件，这为系统深入地研究miRNA的作用机制提供了便利条件。本研究拟以脂代谢相关miRNAs靶基因结合区域的单核苷酸多态性为切入点，采用病例-对照研究，选择中国东南方汉族人群，利用全基因组和分子生物遗传学的研究成果，结合MetS发生机制路径，采用生物信息学技术，筛选候选脂代谢相关miRNAs靶基因结合区域的SNP位点，结合环境因素，探讨其与MetS遗传易感性的关联，并进一步分析其与代谢综合征组分的相关性。系统探讨脂代谢相关miRNAs靶基因结合区域的多态性与中国东南方汉族人群MetS遗传易感性的关系，为MetS的病因学一级预防、高危险人群筛查提供科学依据。第一部分脂代谢相关miRNA靶基因结合区域多态的筛选及与代谢综合征易感性的关系本文采用病例-对照研究设计，利用国际人类基因组miRBase公共数据库()提供的miRNA信息，结合PUBMED等数据库收录的分子生物遗传学方面的文献，基于miRNA靶基因预测软件和NCBI( nih.gov/)的SNP数据库，筛选出脂代谢相关的miRNA靶基因结合区域SNP位点，应用Taqman-MGB探针的技术，检测全部研究对象(MetS患者1026例，正常对照1032例) SNP位点的多态性，分析其不同基因型及组成的单倍型在中国东南方汉族人群中频率的分布及其与MetS的关联。应用Logistic回归模型计算不同基因型人群患MetS的风险(OR)及其95%可信区间(95%CI)。主要结果如下：1.脂代谢相关的miRNA靶基因结合区域SNP位点筛选利用生物信息学方法共筛选出7个脂代谢相关的miRNA，及其靶基因结合区域SNP位点11个，分别为rs1042094，rs741191，rs5750146，rs11724758，rs6710015，rs4597342，rs2292899，rs5999924。2.单个脂代谢相关的miRNA靶基因结合区域SNP位点与MetS的关系在对照组中，对11个SNPs位点三种基因型的频率进行遗传平衡检验，结果显示实际频数与理论频数分布的差别均无统计学意义，说明该对照组人群11个位点基因型频率分布均符合遗传平衡法则，具有人群代表性。11个脂代谢相关的miRNA靶基因结合区域SNP位点与MetS易感性的关联性分析显示，调整年龄、性别、吸烟、饮酒和体力活动因素后，APOL6基因的rs5750146(G>A)和rs5999924(T>A)位点变异与MetS的发生呈正关联，而FABP2基因的rs11724758(A>G)的位点变异与MetS的发生呈负关联。与野生型rs5750146GG相比，杂合型rs5750146GA和rs5750146GA/AA可以增加MetS的发病风险[调整ORs(95%CIs)分别为1.31(1.08-1.58)和1.24(1.03-1.49)]。Rs5999924AT杂合型以及rs5999924AT/TT型的患病风险与rs5999924AA相比分别升高了27%、22%[调整ORs (95%CIs)分别为1.27(1.05-1.54)、1.22(1.01-1.46)]。此外，rs11724758位点的突变纯合型AA与野生型GG相比可以显著降低MetS的发病风险[调整ORs(95%CIs)为0.65(0.50-0.86)]。经5000次置换检验(Permutation test)方法校正后，这3个位点的基因型频率分布在病例组与对照组间仍存在统计学差异。未发现其他8个位点的多态性与MetS存在统计学关联(P>0.05)。3.脂代谢相关的miRNA靶基因结合区域多态性与MetS的性别分层分析在女性中，与野生型rs5750146GG相比，携带杂合型rs5750146GA和GA/AA可增加患MetS的风险[调整OR及其95%CI为1.37(1.06-1.72)、1.33(1.03-1.71)]；Rs5999924AT杂合型以及rs5999924AT/TT型与野生型rs5999924AA相比也可增加患MetS的风险[调整OR及其95%CI为1.36(1.05-1.77)、1.33(1.04-1.72)]。在男性中，与野生型rs11724758GG相比，携带突变型rs11724758AA可降低患MetS的风险[调整OR及其95%CI为0.52(0.34-0.80)]。对其他位点多态性的性别分层分析显示各层病例对照间的基因型频率分布差异无统计学意义(P>0.05)。4.单倍型分析对APOL6基因2个SNP位点(rs5750146,rs5999924)构成的4种单倍型分析中，未发现其单倍型在病例与对照组间的频率分布差异具有统计学意义(P>0.05)。5. rs5750146和rs11724758与MetS易感性的联合分析对rs5750146和rs11724758两位点与MetS易感性的联合分析显示，在显性模型下，随着携带危险等位基因个数的增加，MetS的发病风险也增加，呈剂量—反应关系(趋势性检验P值=5.4×10-5)。第二部分脂代谢相关miRNA靶基因结合区域单核苷酸多态性与代谢综合征组分的相关性基于第一部分脂代谢相关miRNA靶基因结合区域单核苷酸多态性与MetS易感性的关联研究，并且，考虑到多态性位点可能在病例组与对照组中对组分作用效应存在差异。本研究选取与MetS的发生有相关性的2个SNPs位点，进一步分别在病例组和对照组中分析两位点与MetS各组分的关联性，并对两位点与MetS各组分的关系进行联合分析。主要结果如下：1.两候选基因多态性位点基因型与甘油三酯水平的相关性在对照组中，调整年龄、性别、高密度脂蛋白胆固醇、空腹血糖、血压、腰围、吸烟、饮酒和体力活动因素后，携带突变型rs5750146AA者的甘油三酯平均水平(1.48±0.95mmol/L)高于携带野生型GG者(1.23±0.77mmol/L)，差异具有统计学意义(P=0.034)。未发现rs11724758位点不同基因型与甘油三酯平均水平间的差别存在统计学关联(P＞0.05)。2.两候选基因多态性位点基因型与空腹血糖水平的相关性在病例组中，调整年龄、性别、甘油三酯、高密度脂蛋白胆固醇、腰围、吸烟、饮酒和体力活动因素后，携带突变型rs11724758AA者的空腹血糖平均水平(7.66±3.46mmol/L)低于携带野生型GG者(8.99±3.93mmol/L)，差异具有统计学意义(P=0.002)。未发现位点rs5750146不同基因型的空腹血糖平均水平间的差别存在统计学意义(P＞0.05)。3. rs5750146和rs11724758两位点与血糖水平的联合分析调整年龄、性别、甘油三脂、高密度脂蛋白、血压、腰围、吸烟、饮酒和体力活动因素后。在病例组中，空腹血糖与危险等位基因的增加存在相关性，回归系数为0.432(95%CI=0.043-0.820, P=0.030)，即显性模型下，每增加一个危险等位基因的突变，病例组中个体空腹血糖水平将增加0.432mmol/L。

    Metabolic syndrome (MetS) is a common, multicomponent condition characterizedby abdominal obesity, insulin resistance, dyslipidemia (elevated plasma triglyceridesand low high-density cholesterol) and hypertension that seriously impact to humanhealth. In2005, the International Diabetes Federation (IDF) estimated that there wereabout a quarter of people in the world with MetS. MetS is an important modifiablerisk factor for type2diabetes mellitus (T2DM) and cardiovscular disease. As a resultof economic growth, changes in lifestyle and diet have led to an increased burden ofMetS, which become a major public health concern. Therefore, the pathogenesis andpreventive measure of MetS are concerned widely.MetS is a complex, multifactorial disease, and its development is determined bya combination of genetic susceptibility and environmental factors. Severalmechanisms have been implicated in the pathogenesis of MetS: excess calories leadto the fat accumulated unduly in adipose tissue or non-fatty tissues and correlativeinsulin resistance. Mi(cro)RNAs play important regulatory roles in a variety ofbiological processes including adipocyte differentiation, metabolic integration, insulinresistance and appetite regulation. A multitude of studies have demonstratedassociations between MetS and specific miRNAs as microRNAs play a key role in thepathological development of MetS by affecting adipocyte differentiation, endocrine hormone.Increasingly evidence confirms that SNPs in3’untranslated regions (3’UTRs)targeted by miRNAs alter the strength of miRNA binding, with consequences onregulation of target genes thereby affecting the individual’s diseases. Therefore, wewill better understand the pathogenesis of MetS by investigate the relationshipbetween SNPs in miRNA binding sites and MetS. Furthermore, there are a number ofalgorithms and bioinformatics data banks to help identify putative miRNA bindingsites.In this study, we conducted a case-control study of the Chinese southeast Hanpopulation, and analyzed the SNPs in lipid metabolism related miRNA binding sitesand haplotype analysis, combined with environmental factors, to find whether thepolymorphisms and its phenotype are associated with the MetS. We furtherresearched the association between these SNPs and the components of MetS. A betterunderstanding of the functional role of polymorphisms in miRNA binding sites willbe a key step to allow us to fully exploit these new data to provide the scientificevidence for the prevention of MetS and screening of high-risk population.Part ⅠScreening microRNAs target SNPs and investigatingtheir associations with MetS susceptibilityWe conducted a case-control study of the Chinese southeast Han population. PotentialmiRNA binding sites in genomic sequences for the list of miRNAs were assessed bybioinformatics data bank () and NCBI data bank( nih.gov/) using4bioinformatics software. Eventually,8SNPswere selected for subsequent genotyping and association analyses. By utilizingTaqMan assay detection technology, we detect all subjects (1026MetS patients,1032health controls) and analyzed the association between these11SNPs and MetS in the Chinese Han population. Associations between genotypes and MetS risk (ORsand95%CI) were estimated by logistic regression. The main results were as follows:1. Lipid metabolism related miRNA target SNP selectionWe selected7miRNAs which were previously identified related to lipidmetabolism,11target SNPs were assessed by bioinformatics data bank includingrs1042094,rs741191,rs5750146,rs11724758,rs6710015,rs4597342,rs2292899,rs5999924.2. The distribution in cases and controls of SNPs in miRNA binding sitesThe genotype distribution for all the11SNPs did not show any deviation fromthe Hardy-Weinberg equilibrium (all P>0.05in controls). Compared with thegenotypes GG homozygote, the variant genotypes GA and the combined genotypeGA/AA of rs5750146were all associated with a significant risk effect for MetS[Adjusted ORs(95%CIs)=1.31(1.08-1.58),1.24(1.03-1.49)]. Similarly, the variantgenotypes AT and the combined genotype AT/TT of rs5999924compared with the AAgenotype were all associated with a significantly increased risk of MetS [AdjustedORs(95%CIs)=1.27(1.05-1.54),1.22(1.01-1.46)]. The rs11724758AA genotypecarriers were significantly associated with a decreased risk of MetS [AdjustedORs(95%CIs)=0.65(0.50-0.86)]. After inspected by five thousand times permutationtest, the frequency distribution of those sites genotype between the case group andcontrol group still existed statistical differences. No evidence suggested other SNPswere associated with MetS(P>0.05).3. Stratified analysis by gender of miRNA target SNPThe stratified analysis by gender show that the variant genotypes GA[AdjustedORs(95%CIs)=1.37(1.06-1.72)] and the combined genotype GA/AA[AdjustedORs(95%CIs)=1.33(1.03-1.71)] of rs5750146were still all associated with asignificant risk effect for MetS compared with the GG genotype in the female subjects. Similar changes in rs5999924were seen. On the contrary, the variantgenotypes AA of rs11724758was not associated with a decreased risk effect for MetSin the female subjects, association still existed in the male subjects [AdjustedORs(95%CIs)=0.52(0.34-0.80)]. For both males and females, other SNPs were notsignificantly associated with MetS.4. Haplotype analysis on APOL6variationsWe performed haplotype analysis on APOL6variations. There were nohaplotypes which may increase or decrease risk of MetS compared with the mostcommon haplotype GArs5750146-rs5999924, even after adjusting for age, gender, smoking,drinking and physical activity5. Conjoint analysis between SNPs rs5750146and rs11724758and MetS risk.Compared with the wild homozygote of rs5750146and rs11724758, subjectswith variant alleles of the two SNPs had increased risk for MetS susceptibility in adose-response manner (P trend=5.4×10-5).Part ⅡAssociation of polymorphisms in lipid metabolismrelated miRNA binding and the components of MetSIn this study, we based on the PartⅠof Association of Polymorphisms in lipidmetabolism related miRNA binding and the susceptibility of MetS, and selectedrs5750146and rs11724758which had shown the association with MetS in the logisticregression analysis. We further researched the association of these tow SNPs and thecomponents of MetS in group case and control. The main results were as follows:1. Comparison of the levels of triglycerides (TG) of SNPs in miRNA binding sites.In control group, people who carriers with rs5750146AA had higher levels ofTG (1.48±0.95mmol/L)than those with the genotype GG (1.23±0.77mmol/L) (P=0.034), after adjusting for age, gender, HDL-C, FPG, BP and WC, smoking,drinking, physical activity.2. Comparison of the levels of fasting glucose (FPG) of SNPs in miRNA bindingsites.Patients who carriers with rs11724758AA had lower levels of FPG(7.66±3.46mmol/L) than those with the genotype GG(8.99±3.93mmol/L),(P=0.002), after adjusting for age, gender, TG, HDL-C, WC, smoking, drinking andphysical activity.3. Conjoint analysis between SNPs rs5750146and rs11724758and levels of fastingglucose (FPG).Compared with the wild homozygote of rs5750146and rs11724758, Patientswith variant alleles of the two SNPs had increased levels of fasting glucose in adose-response manner (β=0.43295%CI=0.043-0.820, P=0.030).

脂代谢相关miRNA靶基因结合区域多态性与代谢综合征及其组分的分子流行病学研究

摘要5-10Abstract10-14前言15-19第一部分 脂代谢相关 miRNA 靶基因结合区域多态的筛选及与代谢综合征易感性的关系19-43    材料与方法19-26    结果26-39    讨论39-43第二部分 脂代谢相关 miRNA 靶基因结合区域单核苷酸多态性与代谢综合征组分的相关性43-61    对象与方法44    结果44-59    讨论59-61小结61-62参考文献62-69附录一、论文缩写词汇表69-70附录二、居民健康状况调查表70-74综述74-90    参考文献83-90已发表论文90-92致谢92

  本文关键词：脂代谢相关miRNA靶基因结合区域多态性与代谢综合征及其组分的分子流行病学研究，由笔耕文化传播整理发布。