本文關鍵詞：脂代謝相關miRNA靶基因結(jié)合區(qū)域多態(tài)性與代謝綜合征及其組分的分子流行病學研究，由筆耕文化傳播整理發(fā)布。

        代謝綜合征(metabolic syndrome, MetS)是一組以肥胖、高血糖(糖尿病(DM)或糖調(diào)節(jié)異常(IGR))、血脂異常(高甘油三酯(TG)和(或)低高密度脂蛋白膽固醇(HDL-C))以及高血壓等聚集發(fā)病，嚴重影響人類健康的臨床征候群。2005年國際糖尿病聯(lián)盟(International Diabetes Federation, IDF)估計全球約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險因素之一。近年來，隨著經(jīng)濟高速發(fā)展，人們的飲食、生活方式發(fā)生了巨大改變，MetS的發(fā)病率快速增長，已成為嚴重威脅人類健康的公共衛(wèi)生問題。因此，有關MetS的病因?qū)W機制及其防治研究成為研究熱點。MetS是一種環(huán)境因素與遺傳因素共同作用引起的疾病，由于它本身為多種因素的集合，遺傳特點復雜，其主要機制為機體熱量過剩導致脂肪在脂肪組織和非脂肪組織中過度蓄積及其相關的胰島素抵抗。其中脂肪代謝紊亂是MetS發(fā)病的重要環(huán)節(jié)。研究表明microRNA (miRNA)在多種生物進程中發(fā)揮著重要的作用，其中包括脂肪細胞的增殖分化、代謝整合、胰島素抵抗及食欲的調(diào)節(jié)。MiRNA在代謝綜合征發(fā)展的各個階段也起到不容忽視的調(diào)控作用，在與MetS發(fā)病機理有關的脂代謝和脂肪形成中, miRNA不但參與動物調(diào)控脂肪細胞的分化，還參與脂類代謝及與脂類代謝相關的內(nèi)分泌激素調(diào)節(jié)，有的甚至與肥胖、T2DM、MetS等脂類代謝病的基本病理相關。越來越多的證據(jù)表明，，miRNA靶基因3’UTRs(untranslated regions)或5’UTRs內(nèi)的單核苷酸多態(tài)性(single-nucleotide polymorphism, SNP)可以影響miRNA與靶區(qū)域的結(jié)合效率，造成miRNA調(diào)控作用發(fā)生改變，最終導致相應表型—疾病狀態(tài)。因此，探討脂代謝相關miRNA靶基因結(jié)合區(qū)域SNPs與MetS發(fā)病風險間的聯(lián)系，將進一步豐富MetS分子病因?qū)W內(nèi)容。此外，近年來，生物信息學方面提供了一系列基于miRNA序列及靶基因的數(shù)據(jù)庫()，同時可獲取多種miRNA靶基因預測的方法和生物信息學軟件，這為系統(tǒng)深入地研究miRNA的作用機制提供了便利條件。本研究擬以脂代謝相關miRNAs靶基因結(jié)合區(qū)域的單核苷酸多態(tài)性為切入點，采用病例-對照研究，選擇中國東南方漢族人群，利用全基因組和分子生物遺傳學的研究成果，結(jié)合MetS發(fā)生機制路徑，采用生物信息學技術，篩選候選脂代謝相關miRNAs靶基因結(jié)合區(qū)域的SNP位點，結(jié)合環(huán)境因素，探討其與MetS遺傳易感性的關聯(lián)，并進一步分析其與代謝綜合征組分的相關性。系統(tǒng)探討脂代謝相關miRNAs靶基因結(jié)合區(qū)域的多態(tài)性與中國東南方漢族人群MetS遺傳易感性的關系，為MetS的病因?qū)W一級預防、高危險人群篩查提供科學依據(jù)。第一部分脂代謝相關miRNA靶基因結(jié)合區(qū)域多態(tài)的篩選及與代謝綜合征易感性的關系本文采用病例-對照研究設計，利用國際人類基因組miRBase公共數(shù)據(jù)庫()提供的miRNA信息，結(jié)合PUBMED等數(shù)據(jù)庫收錄的分子生物遺傳學方面的文獻，基于miRNA靶基因預測軟件和NCBI( nih.gov/)的SNP數(shù)據(jù)庫，篩選出脂代謝相關的miRNA靶基因結(jié)合區(qū)域SNP位點，應用Taqman-MGB探針的技術，檢測全部研究對象(MetS患者1026例，正常對照1032例) SNP位點的多態(tài)性，分析其不同基因型及組成的單倍型在中國東南方漢族人群中頻率的分布及其與MetS的關聯(lián)。應用Logistic回歸模型計算不同基因型人群患MetS的風險(OR)及其95%可信區(qū)間(95%CI)。主要結(jié)果如下：1.脂代謝相關的miRNA靶基因結(jié)合區(qū)域SNP位點篩選利用生物信息學方法共篩選出7個脂代謝相關的miRNA，及其靶基因結(jié)合區(qū)域SNP位點11個，分別為rs1042094，rs741191，rs5750146，rs11724758，rs6710015，rs4597342，rs2292899，rs5999924。2.單個脂代謝相關的miRNA靶基因結(jié)合區(qū)域SNP位點與MetS的關系在對照組中，對11個SNPs位點三種基因型的頻率進行遺傳平衡檢驗，結(jié)果顯示實際頻數(shù)與理論頻數(shù)分布的差別均無統(tǒng)計學意義，說明該對照組人群11個位點基因型頻率分布均符合遺傳平衡法則，具有人群代表性。11個脂代謝相關的miRNA靶基因結(jié)合區(qū)域SNP位點與MetS易感性的關聯(lián)性分析顯示，調(diào)整年齡、性別、吸煙、飲酒和體力活動因素后，APOL6基因的rs5750146(G>A)和rs5999924(T>A)位點變異與MetS的發(fā)生呈正關聯(lián)，而FABP2基因的rs11724758(A>G)的位點變異與MetS的發(fā)生呈負關聯(lián)。與野生型rs5750146GG相比，雜合型rs5750146GA和rs5750146GA/AA可以增加MetS的發(fā)病風險[調(diào)整ORs(95%CIs)分別為1.31(1.08-1.58)和1.24(1.03-1.49)]。Rs5999924AT雜合型以及rs5999924AT/TT型的患病風險與rs5999924AA相比分別升高了27%、22%[調(diào)整ORs (95%CIs)分別為1.27(1.05-1.54)、1.22(1.01-1.46)]。此外，rs11724758位點的突變純合型AA與野生型GG相比可以顯著降低MetS的發(fā)病風險[調(diào)整ORs(95%CIs)為0.65(0.50-0.86)]。經(jīng)5000次置換檢驗(Permutation test)方法校正后，這3個位點的基因型頻率分布在病例組與對照組間仍存在統(tǒng)計學差異。未發(fā)現(xiàn)其他8個位點的多態(tài)性與MetS存在統(tǒng)計學關聯(lián)(P>0.05)。3.脂代謝相關的miRNA靶基因結(jié)合區(qū)域多態(tài)性與MetS的性別分層分析在女性中，與野生型rs5750146GG相比，攜帶雜合型rs5750146GA和GA/AA可增加患MetS的風險[調(diào)整OR及其95%CI為1.37(1.06-1.72)、1.33(1.03-1.71)]；Rs5999924AT雜合型以及rs5999924AT/TT型與野生型rs5999924AA相比也可增加患MetS的風險[調(diào)整OR及其95%CI為1.36(1.05-1.77)、1.33(1.04-1.72)]。在男性中，與野生型rs11724758GG相比，攜帶突變型rs11724758AA可降低患MetS的風險[調(diào)整OR及其95%CI為0.52(0.34-0.80)]。對其他位點多態(tài)性的性別分層分析顯示各層病例對照間的基因型頻率分布差異無統(tǒng)計學意義(P>0.05)。4.單倍型分析對APOL6基因2個SNP位點(rs5750146,rs5999924)構(gòu)成的4種單倍型分析中，未發(fā)現(xiàn)其單倍型在病例與對照組間的頻率分布差異具有統(tǒng)計學意義(P>0.05)。5. rs5750146和rs11724758與MetS易感性的聯(lián)合分析對rs5750146和rs11724758兩位點與MetS易感性的聯(lián)合分析顯示，在顯性模型下，隨著攜帶危險等位基因個數(shù)的增加，MetS的發(fā)病風險也增加，呈劑量—反應關系(趨勢性檢驗P值=5.4×10-5)。第二部分脂代謝相關miRNA靶基因結(jié)合區(qū)域單核苷酸多態(tài)性與代謝綜合征組分的相關性基于第一部分脂代謝相關miRNA靶基因結(jié)合區(qū)域單核苷酸多態(tài)性與MetS易感性的關聯(lián)研究，并且，考慮到多態(tài)性位點可能在病例組與對照組中對組分作用效應存在差異。本研究選取與MetS的發(fā)生有相關性的2個SNPs位點，進一步分別在病例組和對照組中分析兩位點與MetS各組分的關聯(lián)性，并對兩位點與MetS各組分的關系進行聯(lián)合分析。主要結(jié)果如下：1.兩候選基因多態(tài)性位點基因型與甘油三酯水平的相關性在對照組中，調(diào)整年齡、性別、高密度脂蛋白膽固醇、空腹血糖、血壓、腰圍、吸煙、飲酒和體力活動因素后，攜帶突變型rs5750146AA者的甘油三酯平均水平(1.48±0.95mmol/L)高于攜帶野生型GG者(1.23±0.77mmol/L)，差異具有統(tǒng)計學意義(P=0.034)。未發(fā)現(xiàn)rs11724758位點不同基因型與甘油三酯平均水平間的差別存在統(tǒng)計學關聯(lián)(P＞0.05)。2.兩候選基因多態(tài)性位點基因型與空腹血糖水平的相關性在病例組中，調(diào)整年齡、性別、甘油三酯、高密度脂蛋白膽固醇、腰圍、吸煙、飲酒和體力活動因素后，攜帶突變型rs11724758AA者的空腹血糖平均水平(7.66±3.46mmol/L)低于攜帶野生型GG者(8.99±3.93mmol/L)，差異具有統(tǒng)計學意義(P=0.002)。未發(fā)現(xiàn)位點rs5750146不同基因型的空腹血糖平均水平間的差別存在統(tǒng)計學意義(P＞0.05)。3. rs5750146和rs11724758兩位點與血糖水平的聯(lián)合分析調(diào)整年齡、性別、甘油三脂、高密度脂蛋白、血壓、腰圍、吸煙、飲酒和體力活動因素后。在病例組中，空腹血糖與危險等位基因的增加存在相關性，回歸系數(shù)為0.432(95%CI=0.043-0.820, P=0.030)，即顯性模型下，每增加一個危險等位基因的突變，病例組中個體空腹血糖水平將增加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).

脂代謝相關miRNA靶基因結(jié)合區(qū)域多態(tài)性與代謝綜合征及其組分的分子流行病學研究

摘要5-10Abstract10-14前言15-19第一部分 脂代謝相關 miRNA 靶基因結(jié)合區(qū)域多態(tài)的篩選及與代謝綜合征易感性的關系19-43    材料與方法19-26    結(jié)果26-39    討論39-43第二部分 脂代謝相關 miRNA 靶基因結(jié)合區(qū)域單核苷酸多態(tài)性與代謝綜合征組分的相關性43-61    對象與方法44    結(jié)果44-59    討論59-61小結(jié)61-62參考文獻62-69附錄一、論文縮寫詞匯表69-70附錄二、居民健康狀況調(diào)查表70-74綜述74-90    參考文獻83-90已發(fā)表論文90-92致謝92

  本文關鍵詞：脂代謝相關miRNA靶基因結(jié)合區(qū)域多態(tài)性與代謝綜合征及其組分的分子流行病學研究，由筆耕文化傳播整理發(fā)布。