位于microRNA结合位点的基因多态与心脑血管疾病的相关性研究

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作者：杨涛 论文级别：博士 学科专业名称：遗传学 中文关键词：Angiopoietin1 ; 脑卒中 ; 单核苷酸多态性 ; MicroRNA ; 血管紧张素Ⅱ受体 ; 多态性 ; 冠心病 ; MicroRNA 英文关键词：Angiopoietin 1 ; stroke ; single nucleotide polymorphism ; microRNA ; AngⅡtype 1 receptor ; Polymorphism ; Coronary artery disease ; MicroRNA 学位年度：2008 导师：惠汝太 学科代码：071007 学位授予单位：中国协和医科大学 论文提交日期：2008-05-01

摘要

第一部分血管生成素-1基因多态性与出血性脑卒中相关
     心脑血管病是当今威胁人类健康的严重疾病之一。据最新的统计资料显示:在我国,心脑血管病是仅次于癌症的第二大致死疾病。脑卒中是心脑血管疾病主要类型之一,存在着明显三高(发病率高、致残率高、死亡率高)现象。引起脑卒中的原因众多,如高龄、吸烟、饮酒、高血压、高血脂、高血糖、心脏病、凝血机制障碍等,此外许多证据表明遗传因素在脑卒中的发病中有显著作用。寻找脑卒中的致病基因,从分子水平揭示脑卒中的发病机理,可为及早发现脑卒中的危险人群、预防脑卒中的发生提供帮助。
     脑卒中是一种复杂疾病,发病过程涉及动脉粥样硬化病变。在动脉粥样硬化和动脉疾病的病理生理学过程中,血管发生足研究的热点。血管生成素-1(Angiopoietin1,ANGPT1)是一种特异作用于血管内皮细胞的生长因子,足酪氨酸激酶受体Tie-2的特异性配体。血管生成素-1在血管发生过程中起到了重要作用,它可以促进血管重塑、成熟、阻止内皮细胞凋亡,具有维持血管的完整性,促进血管损伤修复的功能。血管生成素-1的功能提示我们,任何影响其表达的基因多态性,都可能会与血管性疾病相关。通过对文献和数据库的查询,我们发现,血管生成素-1的3'-UTR区域存在SNP+1602 A/T,该位点被预测位于microRNA的结合识别区域,可能会影响microRNA与mRNA的结合。因此我们假设,+1602A/T可以影响血管生成素-1的表达,可能与脑卒中相关。
     为了证明我们的假设,我们设计了荧光素酶报告系统实验,通过检测荧光素酶的表达来验证SNP是否影响基因表达。首先我们构建了含有SNP的荧光素酶质粒,然后与micorRNA或者对照一起共转染培养细胞,最后通过双荧光素酶检测来确定基因表达的情况。
     实验结果证实:在microRNA存在的条件下,与SNP位点为T的荧光素酶质粒相比,SNP位点为A的荧光素酶质粒表达下降超过50%(P＜0.01)。而在对照或者空白条件下,无论携带哪一种SNP位点,荧光素酶的表达均不发生改变。这说明microRNA可以通过识别不同的SNP对表达进行调控。
     在验证了SNP的功能以后,我们进一步采用病例对照的研究方法,以两个独立的中国汉族样本为研究对象,探讨了血管生成素-1的SNP+1602 A/T在脑卒中患者各个亚型和正常对照中的分布,检测SNP是否与脑卒中相关。基因型鉴定采用聚合酶链反应-连接酶检测反应(PCR-LDR)。基因型频率的分布符合Hardy—Weinberg平衡。
     在第一个研究人群中,共计有脑梗患者803例,腔梗患者499例,出血性脑卒中患者489例,正常对照1843例进行了基因分型,各组间年龄、性别相匹配。结果显示:在正常对照中该基因分布为AA型31.9%、AT型48.5%、TT型19.6%;等位基因A和T的频率分别为0.561和0.439。在脑梗患者组中该基因分布为AA型34.9%、AT型46.9%、TT型18.2%;等位基因A和T的频率分别为0.583和0.417。与对照组相比,脑梗患者组中三种基因型分布没有显著性差异。在腔梗患者组中该基因分布为AA型39.5%、AT型48.3%、TT型12.2%:等位基因A和T的频率分别为0.636和0.364。与对照组相比,腔梗患者组中AA型和TT型分布存在显著性差异(P＜0.01)。在出血性脑卒中组中该基因分布为AA型41.1%、AT型49.1%、TT型9.8%;等位基因A和T的分布频率分别为0.656和0.344。与对照组相比,出血性脑卒中组中AA型和TT型分布存在显著性差异(P＜0.01)。Logistic回归分析结果证明:在校正了传统危险因素以后,血管生成素-1的+1602 AA基因型是引起出血性脑卒中的独立危险因素(P＜0.01)。
     在第二个研究人群中,共计有缺血性脑卒中患者238例,出血性脑卒中患者157例,正常对照790例进行了基因分型,各组间年龄、性别相匹配。结果显示:在正常对照中该基因分布为AA型32.9%、AT型47.8%、TT型19.3%;等位基因A和T的频率分别为0.568和0.432。在缺血性脑卒中组中该基因分布为AA型40.8%、AT型48.7%、TT型10.5%;等位基因A和T的频率分别为0.595和0.405。与对照组相比,AA型和TT型分布存在显著性差异(P＜0.01)。在出血性脑卒中组中该基因分布为AA型43.3%、AT型47.1%、TT型9.6%;等位基因A和T的频率分别为0.669和0.331。与对照组相比,AA型和TT型分布存在显著性差异(P＜0.01)。Logistic回归分析结果证明:在校正了传统危险因素以后,血管生成素-1的+1602 AA基因型是引起缺血性脑卒中的独立危险因素(P=0.047);同时血管,生成素-1的+1602 AA基因型也是引起出血性脑卒中的独立危险因素(P＜0.01)。
     通过分析两个独立样本的研究结果,确认血管生成素-1的+1602 AA基因型可以增加出血性脑卒中的发病风险,但对于缺血性脑卒中的发病影响尚存在不同的结果,需要进一步研究。
     本研究选择血管生成素-1的SNP+1602 A/T作为研究对象,研究SNP的功能以及与脑卒中的相关性。结果显示发现SNP+1602 A/T可以通过影响microRNA与mRNA的结合来影响血管生成素-1的表达。进一步的病例对照研究则显示+1602 AA基因型与出血性脑卒中明显相关,可以增加发病风险。这一发现可以用于开发新的检测方法,以便及早发现脑卒中的危险人群、预防脑卒中的发生。同时,这一发现也为治疗脑卒中提供了新的治疗靶点和思路。
     第二部分血管紧张素Ⅱ1型受体基因多态性与冠心病相关
     冠心病是一种严重威胁人类健康的疾病,是心血管疾病中最主要的死亡原因。肾素—血管紧张素系统(renin-angiotensin system,RAS)作为心血管系统中最重要的调控通路,在冠心病的病理生理学过程中起到了非常重要的作用。血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)是RAS系统中的一个重要的成员,而AngⅡ1型受体(AT1R)作为AngⅡ最主要的受体,对于AngⅡ的功能发挥有着重要的影响。
     最新的研究报告,位于AT1R的3'-UTR上的SNP rs5186(A1166C)可以影响miR-155与mRNA的结合。当1166位点为A时,miR-155可以结合AT1R的mRNA,下调AT1R的表达,而当1166位点为C时,miR-155不能结合。而AT1R的表达不一样,对冠心病的发病也可能会产生影响。因此我们采用病例对照研究,来研究SNP A1166C与冠心病的发病是否相关。
     通过冠状动脉造影检查,我们共收集了455例冠心病患者和465例对照。提取被调查者的基因组DNA,采用等位基因特异性PCR对AT1R SNP位点rs5186(A1166C)进行基因分型,最后进行统计分析。结果发现,在冠心病病人和对照之间,SNP rs5186基因型存在差异。冠心病病人中,AA基因型为229人(50.3%),AC基因型为188人(41.3%),CC基因型为38人(8.4%),而正常对照中,AA基因型为304人(65.4%),AC基因型为151人(32.5%),CC基因型为10人(2.2%)。AA基因型可以降低冠心病的发病风险(OR=0.214,0.084-0.548,P＜0.01)。实验结果表明,血管紧张素Ⅱ1型受体基因多态性与冠心病的发病相关,SNP 1166AA是冠心病发病的保护因素。
Cardio-cerebral vascular disease is the leading cause of morbidity and mortality. Stroke is a main type of the cardio-cerebral vascular diseases.Importantly,stroke is the No.1 causes of disability in china.Many factors could contribute stroke,such as aging,smoking,drinking,hypertension,hyperlipidemia,hyperglycemia,heart diseases,and blood coagulation mechanism obstacles.In addition,genetic factors have been shown to play an essential role in the pathogenesis of stroke.The genetic markers of stroke susceptibility are helpful for the prediction and prevention of stroke.
     Stroke evolves depending on the accumulation of risk factors which leads to atherosclerotic lesions.The role of angiogenesis within diseased blood vessels has emerged as a hotspot debate in the biology of atherosclerosis and arterial diseases.
     Angiopoietin 1(ANGPT1) is a vascular endothelial cell growth factors and the ligand for Tie2,a tyrosine kinase receptor.ANGPT1 plays a critical role in angiogenesis,it can promote blood vessel remodeling and maturing,prevent apoptosis of endothelial cells,and maintain the integrity of blood vessels and recovery function of vascular injury.We can speculate that the expression of ANGPT1 may be associated with vascular disease,including stroke.Based on the literature and database enquiries,SNP+1602 A/T in the 3'-UTR of ANGPT1 was found in the region for microRNA recognition for binding.Variations in this region could influence microRNA binding to the mRNA of ANGPT1 and change ANGPT1 expression. Therefore,we hypothesized that SNP+1602 A/T could influence ANGPT1 expression and are associated with stroke.
     To test our hypothesis,we first determined whether the SNP could influence ANGPT1 expression by Luciferase reporter assay.Luciferase reporter vectors containing the SNP were co-transfected into cultured cells with the micorRNA or control.The expression of gene was determined through the double-Luciferase test. Our results showed that expression level of Luciferase reporter vector with SNP A was significantly lower than that of the vector with SNP T(median,74.96 versus 180.82;P＜0.01) in the presence of microRNA.But no significantly change was found in the expression level of Luciferase reporter vector carrying the two SNPs in either the control or blank conditions.
     Next,we carried out a case-control study in two different Chinese Han populations.SNP+1602A/T was genotyped with polymerase chain reaction-Ligase detection reaction(PCR-LDR).Genotype frequencies fulfilled expectation of Hardy-Weinberg equilibrium.In the first population,1791 cases with stroke(803 cerebral thrombosis,499 lacunar infarctions and 489 intracerebral hemorrhages) and 1843 controls were included.Age and sex were matched between the cases and controls.Our results showed that the frequency of AA genotype was significantly higher in the intracerebral hemorrhage than in the control(41.1%versus 31.9%; P＜0.01).Aider adjustment for age,sex and other conventional risk factors with multiple logistic regression analysis,the AA genotype of ANGPT1+1602 remained independently associated with increased risk for hemorrhagic stroke(odds ratio,3.21; 95%CI,1.45 to 7.11;P＜0.01).In second population,395 cases with stroke(238 ischemic strokes and 157 hemorrhagic strokes) and 790 controls were included.Our results showed that the frequency of AA genotype was significantly higher in the hemorrhagic stroke than in the control(43.3%versus 32.9%;P＜0.01).After adjustment for age,sex and other conventional risk factors with multiple logistic regression analysis,the AA genotype of ANGPT1+1602 was independently associated with increased risk for hemorrhagic stroke(odds ratio,2.45;95%CI,1.34 to 4.50;P＜0.01).
     In conclusion,our results support that SNP+1602 A/T could influence the expression of ANGPTI by change in the binding of microRNA.Case-control study further showed that +1602 AA genotype conferred significant risk of hemorrhagic stroke.The polymorphism may be a novel genetic marker for the prediction and prevention of stroke,and a new target for the treatment of stroke.
     Coronary heart Disease is the leading cause of morbidity and mortality in the world.Renin-angiotensin system(RAS) is important in regulation of cardiovascular functions and plays a critical role in pathophysiology of coronary heart disease. AngiotensinⅡ[(AngⅡ) is an important member of RAS system,and performs its functions mainly by AngⅡtype 1 receptor(AT1R).
     The latest report showed that the SNP rs5186(A1166C) in the 3'-UTR of AT1R can affect mRNA and miR-155 combination.MiR-155 down-regulates the expression of AT1R by the binding with AT1R mRNA in the presence of rs5186 A allele,but not the C allele.The expression of AT1R may be associated with risk of coronary heart disease.Therefore a case-control study was performed to examine whether SNP A1166C correlates with risk of coronary heart disease.
     A total of 455 patients with coronary heart disease and 465 controls by coronary angiography were recruited.SNP rs5186(A1166C) was genotyped by using allele-specific PCR.Genotype frequencies fulfilled expectation of Hardy-Weinberg equilibrium.
     Our results showed that the frequency of 1166AA genotype in controls was significant higher than that in patients,(65.4%versus 50.3%;P＜0.01);and AA genotype could reduce the risk of coronary heart disease(odds ratio,0.214;95%CI, 0.084 to 0.548;P＜0.01).We can conclude that the polymorphism of AT1R gene was associated with coronary heart disease,the A allele is a protective factor of coronary heart disease.

引文

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