阿尔茨海默病基因关联性分析及APOE缺陷小鼠脑组织中异常表达miRNA的鉴定和初步功能研究

【作者】 毕秀华；

【导师】 张俊武；

【作者基本信息】 中国协和医科大学 ， 生化与分子生物学， 2008， 博士

【摘要】 大量流行病学研究显示叶酸缺乏和同型半胱氨酸升高与阿尔茨海默病（Alzheimer’s disease,AD）相关。体内叶酸和同型半胱氨酸水平可能受饮食因素影响,也可能受参与两者代谢途径的基因的多态性影响。我们采用干酪乳酸菌法和高压液相色谱方法测定了106例AD病人和120例正常人的血浆叶酸、同型半胱氨酸和半胱氨酸水平,并用限制性内切酶片段长度多态性方法（Restriction Fragment Length Polymorphism,RFLP）检测了368例AD病人和375例正常人中叶酸和同型半胱氨酸代谢途径相关的5个基因中的5个多态性位点,分别是5,10—亚甲基四氢叶酸还原酶677 C/T（5,10-methylenetetrahydrofolate reductase,MTHFR）、叶酸还原载体1 80 A/G（reduced folate carrier 1,RFC1）、胱硫醚β合成酶（cystathionine beta-synthase,CβS）844ins68、5,10—亚甲基四氢叶酸脱氢酶1958A/G（5,10-methylenetetrahydrofolate dehydrogenase,MTHFD1）以及磷脂酰乙醇胺甲基转移酶523 G/A（phosphatidylethanolamine N-methyltransferase,PEMT）。此外,也检测了载脂蛋白E（apolipoproteine E,APOE）的基因多态性。结果显示AD病人中同型半胱氨酸含量显著高于正常人群（p＜0.05）,叶酸含量显著低于正常人（p＜0.001）,同时还发现AD病人中半胱氨酸含量也显著低于正常人群（p＜0.001）。在基因多态性与AD关联研究方面,我们发现RFC1 80A/G多态性的GG基因型和G等位基因频率在AD病人中显著高于正常人（G等位基因:p=0.008,OR=1.312,95%CI:1.072-1.605;GG基因型:p=0.042,OR=1.383,95%CI:1.012-1.890）。按照APOEε4,年龄和性别分层后,发现在不携带APOEε4的人群,女性和≥65岁的人群中,G等位基因在AD病人中的分布显著高于正常人。这表明RFC1 80G等位基因可能是一个不依赖APOEε4的晚发性AD风险因子,在女性中的作用更为明显。在未分层样本中,MTHFR 677C/T多态性的基因型和等位基因分布在AD组和正常对照组间不存在显著性差异。根据年龄和性别分层后,也未发现显著性差异。但根据是否携带APOEε4等位基因分层后,发现在携带APOEε4等位基因的个体,AD病人中TT基因型和T等位基因的频率显著高于正常对照组（T vs.C:p=0.031,OR=1.586,95%CI:1.042-2.414;TT vs.CC plus CT:p=0.028,OR=2.250,95%CI:1.074-4.712）。我们没有发现CβS 844ins68,MTHFD1 1958G/A和PEMT 523G/A多态性与AD存在关联性。分析基因多态性对血浆叶酸和同型半胱氨酸含量的影响时发现,所有样本中携带RFC1 80GG基因型的人群中血浆叶酸水平低于AA和GA基因型人群血浆叶酸水平的平均值,而同型半胱氨酸水平高于AA和GA基因型人群,但是这种差异未达到显著性。在AD组和正常组也存在这样的趋势,尤其是正常人中的同型半胱氨酸含量,在GG基因型的人群中高于AA+GA的人群（10.25μmol/L vs.7.96μmol/L）,差异接近但未达到统计学显著性（p=0.086）。没有发现MTHFR 677多态性,CβS 844ins68和MTHFD1 1958A/G对血浆叶酸和同型半胱氨酸含量有影响。上述结果表明叶酸代谢途径中的RFC1 80G等位基因和GG基因型能够增加患AD的风险,但可能不是通过影响血浆叶酸和同型半胱氨酸含量而实现的,确切机理还需进一步研究。此外,MTHFR 677T等位基因可能与APOEε4协同作用增加患AD的风险。小RNA（miRNA）是一类长度约为21-23个核苷酸的重要调节分子,它可以通过剪切靶基因mRNA或抑制靶基因翻译的方式在转录后水平抑制蛋白编码基因的表达。近年来鉴别了大量的miRNAs,已证明它们通过在转录后水平调控基因表达参与许多重要的生理过程,并涉及多种疾病的发生和发展。哺乳动物神经系统表达丰富的miRNAs,已确定它们在神经系统发育中起重要调节作用,它们在成熟的神经细胞,脑老化及神经退行性疾病中也可能行使重要功能。阿尔茨海默氏病（Alzheimer’sdisease,AD）是老年痴呆中最普遍的一种,其典型的病理改变是β淀粉样肽在大脑皮层和海马神经细胞外沉积以及过度磷酸化的微管相关Tau蛋白在神经细胞内缠结。APOE基因多态性是目前唯一确定的散发性AD的危险因素,该基因缺陷的小鼠能够显示部分AD病理特征,可用作研究载脂蛋白E在阿尔茨海默氏病病理过程中的作用的动物模型。我们采用Multiplex qRT-PCR方法鉴定了41个神经系统丰富或特异表达和我们感兴趣的miRNAs在7月龄APOE基因缺陷小鼠(apoE^-/-)和正常小鼠大脑皮层和海马中的表达。发现mmu-miR-7f2和miR-29a在apoE^-/-小鼠大脑额叶皮层的表达显著低于正常小鼠;其中,mmu-miR-143在apoE^-/-小鼠额叶皮层和海马中的表达显著均低于正常小鼠,用单一qRT-PCR方法进一步验证也确证了mmu-miR-143的表达变化。随后我们检测了miR-143在2月龄和13月龄小鼠额叶皮层和海马的表达。发现正常小鼠中,随着年龄增加,miR-143的表达逐渐升高;在apoE^-/-小鼠中大脑额叶皮层中miR-143在2月龄时略高于正常小鼠,之后也随年龄增加,但增加趋势远不如正常小鼠明显,7月龄和13月龄时显著低于正常小鼠;apoE^-/-小鼠海马中,2月龄时miR-143的表达与正常小鼠无差异,到了7月龄时,表达较2月龄有所升高,但13月龄时表达较2月龄显著降低,同时也低于同时期的正常小鼠。miR-143表达的变化以及在正常小鼠和apoE^-/-小鼠中的差异提示它可能在载脂蛋白E相关的AD病理过程中起调节作用。为进一步了解miR-143的作用机制,我们对其可能的靶基因做了分析预测,用荧光素酶报告基因的方法检测了通过软件筛选到的4个基因:cAMP反应元件结合蛋白Zhangfei因子（cAMP response element binding protein Zhangfei factor,CREBZF）,cAMP反应元件结合蛋白5（cAMP response element binding protein 5,CREB5）,TAOK1（TAO kinase 1）,DYRK1B（dual-specificity tyrosine-（Y）-phosphorylation regulatedkinase 1b,DYRK1B）以及已知的细胞外激活激酶5（extracellular activated kinase 5,ERK5）基因3’-UTR区可能的miR-143作用位点。发现miR-143可以使携带ERK5,CREBZF和TAOK1基因3’—UTR区的萤光素酶报告基因表达量分别下降约30%,28%和25%,提示除了ERK5外,CREBZF和TAOK1基因也有可能是miR-143的靶基因。ERK5和TAOK1基因是MAPKK—MAPK—ERK信号通路中的成员,已有研究表明该信号通路中的ERK1/2,p38在AD病人中表达升高且被激活;CREBZF是ATF4的共同作用因子,在神经系统中与长时程记忆形成受限有关。提示miR-143可能通过影响MAPK-ERK信号通路并限制长时程记忆形成参与apoE^-/-小鼠的病理过程。更多还原

【Abstract】 Folate is a cofactor in one-carbon metabolism,during which it promotes the remethylation of homocysteine.Recent epidemiological and experimental studies have linked folate deficiency and resultant increased homocysteine levels with Alzheimer’s disease.Then,association between folate related genes polymorphisms and AD is a logic extension of the well-established connection between folate,homocysteine and AD.In this study,we tested single nucleotide polymorphisms in folate metabolism related genes for association with late onset AD（LOAD） by restriction fragment length polymorphism（RFLP） method.Polymorphisms in 5,10-methylenetetrahydrofolate reductase（MTHFR）,the reduced folate carrier 1（RFC1）,5,10-methylenetetrahydrofolate dehydrogenase（MTHFD1）,cystathionine beta-synthase（CβS） and phosphatidyl-ethanolamine N-methyltransferase（PEMT） were examined in 386AD cases and 375 matched controls.Plasma levels of homocysteine,folate and cysteine were measured in 106 cases and 120 controls by microbiological assay and high-performance liquid chromatography（HPLC）.Results showed that AD patients had significantly decreased plasma folate and cysteine concentrations,and increased plasma homocysteine levels.Significant associations of RFC1 80G allele and GG genotype with AD（p=0.008,OR=1.312,95%CI 1.072-1.605, and p=0.042,OR=1.383,95%CI 1.012-1.890） were found.Further stratification of total samples by APOEε4 status,age/age at onset and gender reveled that RFC1 80G allele was an APOEε4-independent risk factor for late-onset AD,and it might increase the risk of AD in females.No significant associations of MTHFR 677C/T allele and genotype with AD were observed in total samples,but significant associations of T allele and TT genotype with AD（p=0.031,OR=1.586,95%CI=1.042-2.414,and p=0.028,OR=2.250, 95%CI=1.074-4.712） were identified in APOEε4 carrier subgroup.No relationship between MTHFD1 1958G/A,CβS 844in68 and PEMT 523G/A polymorphisms with AD were found. For the total samples,the subjects with the RFC1 80 GG genotype exhibited lower plasma folate levels,and higher homocysteine levels than the subjects with GA plus AA genotype,but the difference was not statistically significant.The similar results were observed in the subgroups of AD and control samples.It seemed to suggest a trend of lower folate levels and higher homocysteine levels in the subjects with GG genotypes than that the subjects with the GA plus AA genotypes although the difference did not reach statistical significance.No significant differences for the plasma levels of folate and homocysteine between the genotypes of MTHFR 677C/T,CβS 844ins68 and MTHFD1 1958G/A were observed.Our findings suggest that genetic variant of RFC1 80A/G increases the risk of Alzheimer’s disease,,suggesting that MTHFR 677T allele and APOEε4 allele may synergistically act to increase AD risk.The mechanism need further studied. A class of small,non-coding transcripts called microRNAs（miRNAs） that provide a crucial and pervasive layer of post-transcriptional gene regulation has recently emerged and become the focus of intense research,miRNAs are abundant in the nervous system, where they have key roles in development and are likely to be important mediators of plasticity.There are several evidence that miRNAs may play an important role in aging and neurodegeneration disease.Alzheimer’s disease is one of the most prevalent dementia, and characterized with neuronal loss,extracellular senile plaques containing the peptideβamyloid,and neurofibrillary protein tau.Polymorphism in APOE is an risk factor of Alzheimer’s disease,apoE-deficient mouse offer an opportunity to study some of the possible functions of apoE in the Alzheimer’s disease.We examined the expression of 41 miRNAs in the frontal cortex and hippocampus of 7 months old apoE-deficient mouse and control mouse by multiplex qRT-PCR.The data showed that mmu-miR-7f2,mmu-miR-29a levels were decreased significantly in frontal cortex of apoE^-/- mouse,and mmu-miR-143 levels was decreased significantly both in the frontal cortex and hippocampus regions of apoE^-/- mouse.Then we analyzed the expression of miR-143 in 2 and 13 months old apoE^-/- and control mouse,found that miR-143 levels was increased with age both in frontal cortex and hippocampus in control mouse.In the frontal cortex of apoE^-/- mouse,miR-143 levels was a little higher than that in the control mouse at the second month,and it increased with age too,but failed to paralle to the control mouse when 7 and 13 months old.In the hippocampus of apoE^-/- mouse,the expression of miR-143 was not defferent with control mouse significantly at 2 months old, there was a increase at the 7th month,but in the 13months old mouse,the miR-143 level decreased to a lower levels than 2 months old,and lower than control mouse too.The 3’-untranslated region（UTR） of ERK5,CREBZF,CREB5,DYRK1B and TAOK1 were presumed to be targeted by miR-143 through computational analysis.The luciferase reporter assay suggested that the ERK5,CREBZF and TAOK1 were the target gene of miR-143.The function of the three genes suggest that miR-143 may change the MAPKK-MAPK-ERK pathway and long-term synaptic changes and memory formation, which need further confirmation.更多还原