MicroRNAs对帕金森病致病基因LRRK2表达调控机制研究
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摘要
第一部分LRRK2基因相关microRNAs的预测及验证
帕金森病(Parkinson Disease, PD)是一种常见的神经变性疾病。PD的发病机制十分复杂,目前认为其是由遗传因素与环境因素的共同作用所导致的。PD遗传致病机制的研究已取得明显进展,迄今已定位16个PD相关位点,克隆了包括富亮氨酸重复激酶2基因(Leucine-rich repeat kinase2, LRRK2)在内的11个致病基因,LRRK2不仅是常染色体显性遗传性PD重要致病基因,还与部分原发性PD有关。
目前LRRK2基因突变导致PD发生的机制并不十分清楚,但研究提示某些LRRK2致病突变(如G2019S)可能引起其激酶功能增强而具有细胞毒性,并且过表达野生型LRRK2也具有细胞毒性作用。此外LRRK2可能参与PD发生过程中异常蛋白沉积。过表达野生型LRRK2、LRRK2-G2019S突变体或LRRK2激酶结构域可加速a-Synuclein A53T的聚集。由此可见,对LRRK2的表达和功能的精确调控在PD发病机制中具有重要作用。
MicroRNA (miRNA)是一类由内源基因编码的长度约为21-23个核苷酸的非编码单链RNA分子,由具有发夹结构的约70-90个碱基大小的单链RNA前体经过Dicer酶加工后生成,有5’端磷酸基和3’羟基,定位于RNA前体的3’端或5’端。在不同组织、不同发育阶段中miRNA的水平有显著差异,这种miRNAs表达模式具有分化的位元相性和时序性(differential spatial and temporal expression patterns),因此1miRNAs作为参与调控基因表达的分子具有重要意义。本课题将探讨miRNA对LRRK2基因表达调控机制在PD发病机制中的作用。
方法：通过UCSC Genome Bioinformatics数据库(http://genome.ucsc. edu/)查询LRRK2基因的RefSeq序列,根据查询到的LRRK2基因3'UTR序列运用targetscan (http://www.targetscan.org/),miRanda(http://www.microrna.org/microrna/home.do)等根据LRRK2基因3’UTR序列预测能对其进行转录后调控的microRNAs,从]niRbase数据库获得相关microRNAs序列,比对生物信息学软件的预测结果,选择评分值高的microRNAs进行生物学验证。构建野生型及突变型LRRK2基因3'UTR序列荧光素酶报告基因载体,在N2a细胞中与miRNAs瞬时共转染,于转染后24小时及48小时进行荧光素酶活性分析,验证miRNAs对LRRK2基因mRNA的转录后抑制作用。瞬时转染miRNAs进入HEK293细胞系,于转染后24小时及48小时检测内源性LRRK2蛋白表达水平。根据共转microRNA后荧光素酶报告系统活性以及细胞系体内的内源性LRRK2蛋白表达水平判断,二者均具有统计学意义时判定该microRNA分子对LRRK2基因具有转路后抑制作用。
结果：本研究对LRRK2基因的RefSeq序列NM_198578的全长3'UTR序列共1517个碱基进行分析,以蛋白翻译终止位点"TAG"的G碱基为+1位进行描述,结合TargetScan预测结果显示hsa-miR-205与LRRK2基因3'UTR序列结合位点+116-122具有高保守性,107个人类miRNAs分子与人类LRRK2基因3’UTR序列存在保守性较低的潜在结合性；miRanda预测结果显示57个人类miRNAs分子与人类LRRK2基因3'UTR序列存在潜在结合性,选取23个潜在调控miRNAs进行生物学验证,结果显示hsa-miR-205mimics与携带野生型LRRK2基因3'UTR序列以及位点+116-122突变型LRRK2基因3'UTR序列的Luc报告系统质粒共转染N2a细胞株后,携带野生型组的Luc报告基因相对活性下降,t检验显示差异具有统计学意义(P<0.05,t=6.479,95%CI值为-1.614～-0.617)；hsa-miR-454mimics与构建的携带野生型LRRK2基因3'UTR序列以及位点+506-512突变型LRRK2基因3'UTR序列的Luc报告系统质粒共转染N2a细胞株后,野生型组的Luc报告基因相对活性明显下降,t检验显示差异具有统计学意义(P<0.001,t=6.479,95%CI值为-1.614--0.617)。瞬时转染hsa-miR-205、hsa-miR-301b及hsa-miR-454mimics进HEK293细胞于24小时及48小时检测内源性LRRK2表达,结果显示转染hsa-miR-205mimics后内源性LRRK2蛋白生成明显减少,转染hsa-miR-454mimics后内源性LRRK2蛋白生成减少,而转染hsa-miR-301b mimics后HEK293细胞的内源性LRRK2蛋白减少不明显。
结论：hsa-miR-205可与LRRK2基因3'UTR序列的5'-AUGAAGG-3'互补结合,抑制LRRK2基因的表达；hsa-miR-454可与LRRK2基因3'UTR序列的5'-UUGCACU-3'互补结合,抑制LRRK2基因的表达hsa-miR-301b可能与LRRK2基因3'UTR序列的5'-UUGCACU-3'互补结合,抑制LRRK2基因的表达,但仍需深入研究。
第二部分miRNAs及LRRK2mRNA在干细胞-神经元分化细胞系的表达水平研究
组织发育与生理过程中的稳态通过于细胞与组织干细胞的调控完成,是否存在一些细胞内机制介导干细胞间的信号转导、表观遗传,转录,翻译以及转录后调控水平的分化基因表达等细胞内机制。microRNA,作为基因表达、修饰、转录和翻译的调节者,在干细胞的自我复制、定向分化和组织再生中起着十分重要的作用。它是干细胞特性、维持、转化功能的一个关键调控者,参与细胞周期、细胞重建(Reprograming)、多能干细胞生成、信号传递、特异分化、聚集、修复、再生、变异、代谢等所有过程,是当前干细胞调控研究的一个重点。
LRRK2基因是PD发病的重要致病基因,而病理学研究表明LRRK2蛋白广泛表达于所有脑组织,但以多巴胺能神经支配区为主。对大鼠的胚胎发育中神经元与神经元附属组织的LRRK2mRNA表达研究表明LRRK2可能在控制神经元细胞的增殖、迁移以及分化过程中发挥作用。本章节将在验证miR-454及miR-205对LRRK2基因转录后调控的基础上初步探讨miR-454及miR-205与LRRK2在胚胎干细胞向神经元的分化过程中不同分化阶段胚胎干细胞及胎脑组织的表达之间的相关性,为进一步深入研究miRNAs与LRRK2在神经元增殖、迁移以及分化过程中的作用奠定基础。
方法：分化人源胚胎干细胞hESCs H9-P57细胞(ES),分别收集ES,拟胚体(EB),神经前体细胞以及人类胎脑组织共3个不同胚胎发育阶段的细胞及组织,提取总RNA,应用实时荧光定量PCR检测miRNAs, LRRK2基因mRNA的表达水平；提取胎脑组织蛋白,应用western blotting技术检测胎脑组织中LRRK2蛋白的表达水平。
结果：相对定量PCR结果显示LRRK2mRNA在胚胎分化的前期到神经前体细胞阶段表达均很低,在拟胚体阶段甚至检测不到LRRK2mRNA,而在发育到26周的有成熟神经元细胞存在的胎脑组织中LRRK2mRNA则呈现高表达,其中以包含大脑皮质及髓质结构的组织表达最高,仅有皮质结构的脑组织表达量较前者低,而在小脑中则表达更低；miR-454在胚胎干细胞,拟胚体阶段以及神经前体细胞阶段的表达均较低,而在发育到26周的胎脑组织中表达则升高,在胎大脑组织(包括大脑皮质及髓质)中的表达多于大脑皮层组织内的表达,大脑皮质及小脑组织中miR-454表达水平相当；miR-205在胚胎干细胞,拟胚体阶段存在表达,神经前体细胞阶段基本没有miR-205的表达,但在发育到26周的胎脑组织中均有表达,在胎大脑组织(包括大脑皮质及髓质)相对较低,而小脑中表达相对较高。胎小脑组织中LRRK2蛋白表达最高,胎大脑组织(包括大脑皮质及髓质)相对较低,仅包含皮质的胎脑组织中表达最低。
结论：miR-454及miR-205在中枢神经系统中存在表达；miR-205在胚胎干细胞分化过程中存在表达。
第三部分帕金森病患者血清miRNAs表达水平检测
miRNAs是一类内源性表达的短小的,非编码RNAs,可通过调控众多基因表达在不同生理功能中发挥作用。miRNAs主要与靶基因的mRNAs的3'UTR序列的结合位点相结合通过降解靶基因的mRNAs或者减少靶基因蛋白翻译而达到转录后抑制作用。目前研究显示miRNAs表达异常与众多疾病的发生相关,miRNAs的表达异常可能作为潜在的疾病诊断标志物或者与疾病相关的miRNAs可能成为潜在的疾病治疗靶点。多项研究显示人类血清或者血浆中存在由组织释放的游离：miRNAs,并且这些游离miRNAs表达具有稳定性,可再生性,在同一个体中表达水平一致,并不易被自身血液系统中的RNAse酶降解等特性,研究表明一些疾病如肺癌、前列腺癌等患者血清中游离miRNAs与正常人比较表达存在差异,因此目前认为血清中的1niRNAs可能作为诊断癌症等某些疾病的标志物。
多年来,众多研究组致力于寻找准确诊断PD的生物标志物与生理学检查,但是目前并没有找到确凿的诊断标志物,因此检测PD患者脑脊液,或者血液中的niRNAs水平看是否能作为包括PD在内的神经变性病的诊断标志物或者治疗靶点。本研究在前面章节的研究基础上,进一步检测PD患者与正常对照血清中是否存在游离miR-205及miR-454水平差异性作为一种寻找PD生物标志物的探索性研究。
方法：采集16例散发性PD患者以及20例正常对照者5ml静脉血,促凝管静置4-6小时,分离血清于-70℃保存,应用Ambion-mirVanaTM PARISTMkit进行游离小RNAs分离,测OD,应用Qiagen公司的microRNAs RT-kit逆转录成DNA,应用荧光定量PCR定量PD组及对照组血清中Hsa-miR-205, hsa-miR-454的表达水平。
结果：本章研究利用qReal-Time PCR对16例PD患者及20例健康对照者的血清中游离miR-205进行相对定量分析,PD患者血清中的1niR-205表达较正常对照组低,应用T检验进行统计学分析,两者差异的P值=0.0001,差异具有统计学意义；PD患者血清中的miR-454表达较正常对照组低,应用T检验进行统计学分析,两者差异的P值=0.04,差异具有统计学意义。
结论：PD患者血清中游离miR-205水平较正常对照明显减低,miR-205很可能为PD的生物标记物；PD患者血清中游离miR-454水平较正常对照组减少,miR-454可能为PD的生物标记物。
ABSTRACT OF SECTION1The Isolation of miRNAs to regulate the expression of the Parkinson Disease Causitive Gene LRRK2
Parkinson's disease (PD) is the second most globally prevalent neurodegenerative disorder. The cause of PD is complex and multifactorial, involving both hereditary and environmental factors. Recent progress in molecular genetic studies of familial PD has led to the identification of16susceptible loci and11genes responsible for PD. Mutations in LRRK2are thus far the most prevalent genetic cause associated with autosomal dominant and idiopathic PD. Although the mechanism of how PD-associated LRRK2mutations cause disease is yet unknown, several studies indicated that certain kinds of mutations including G2019S are likely associated with toxic gain of function. Moreover, to overexpress wildtype LRRK2was found to be toxic in cultured cells and transgenic fly model. Becides, LRRK2was found to be associated with abnormal protein deposition in the brains of patients with lowy body disease including PD. To overexpress wildtype LRRK2, G2019S mutant or kinase domain alone accelerate the formation of aggregates caused by α-synuclein A53T mutant. These results suggest that precise regulation of LRRK2expression and function is necessary for maintaining homeostasis of the organism, and the disruption of the regulation mechanism may cause disease. microRNAs (miRNAs) are a class of endogenous22base-long single-stranded, noncoding RNAs that regulate gene expression in a sequence-specific manner in plants and animals. miRNAs are derived from long precursor transcripts by the action of nucleases Drosha and Dicer, and the resulting mature functional miRNAs bind to their target sequence in the3'-untranslated region (UTR) of mRNA with imperfect complementarity and lead to repression of translation.Therefore, to elucidate the mechanism of LRRK2post-transcriptional regulation is helpful to understand PD aetiology. In this study, the promoter activity of the LRRK23'-untransation region (UTR) was analized, which is essenssial to understand the post-transcriptional regulation of LRRK2.
Methods:Several on-line softwares were involved in our study to predict the miRNAs to regulate the expression of LRRK2.23miRNAs was selected base on the predictation results, and further constructed the luciferase reporter plasmids. The plasmids were then transfected transiently into the N2a cell lines, and the luciferase relative activity were analysed to identified regulation of miRNAs to LRRK2. Finelly, miRNAs were transfected transiently into the Hek293cell lines, and the expression of endougenous LRRK2protein were analysed to identified the repression of miRNAs to LRRK2.
Results:The study Found243miRNAs bind potentially with the sites of LRRK23'UTR of RefSeq NM_198578using several software to predict the targets of miRNAs.23miRNAs was selected base on the predictation results, and further constructed the luciferase reporter plasmids. The luciferase relative activity decreased after miR205and miR454were transfected transiently into N2a cell line with wild type LRRK23'UTR Luc report system or mutation the binding site ones,the luciferase relative activity decreased lightly after miR-301b were transfected transiently into N2a cell line with wild type LRRK23'UTR Luc report system or mutation the binding site ones. The expression of endougenous LRRK2obviously decreased when miR-205and miR-454transfected transiently into the HEK293cell line. The expression of endoug-enous LRRK2decreased lightly when miR-301b transfected transiently into the HEK293cell line.
Conclution:In summary,miR-205can regulate the expression through to bind the site of5'-AUGAAGG-3'in LRRK23'UTR; miR-454can regulate the expression through to bind the site of5'-UUGCACU-3'in LRRK23'UTR; miR-301b probably regulate the expression through to bind the site of5'-UUGCACU-3'in LRRK23'UTR.
ABSTRACT OF SECTION2Identify expression of miRNAs and LRRK2mRNA for embryo stem-cell Neuron differentiated cell line and fetal brain tissue
The hallmark of a stem cell is its ability to self-renew and to produce numerous
differentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a new
dimension of gene regulation in controlling stem cell fate and behaviour. This is shown deregulated synthesis of E2F1/DP caused by the miRNA pathway impairment as a key event in LRRK2pathogenesis and suggest novel miRNA-based therapeutic strategies.
Mutations in the LRRK2gene cause autosomal dominant, late-onset parkinsonism, which presents with pleomorphic pathology including alpha-synucleopathy. The expression and localization of LRRK2to various neuronal populations in brain regions implicated inPD including the cerebral cortex, caudate-putamen and substantia nigra pars compacta. The localization of LRRK2to key neuronal populations throughout the nigrostriatal dopaminergic pathway is consistent with the involvement of LRRK2in the molecular pathogenesis of familial and sporadic parkinsonism.
Methods:Using quantitative Real-time PCR, we analyzed the relative copies of miR-454and miR-205for embryo stem cell-Neuron differentiated cell line and fetal brain tissue which from the fetus developed about26weeks. We analyzed the expression of LRRK2mRNA and protein in different fetal brain tissue by quantitative real-time PCR and western blotting.
Results:The expression profile of miR-454and LRRK2mRNA were rather low in the embryo stem cell-neuron differentiated cell line,however,miR-454and miR-205and LRRK2mRNA shows very high expression in fetal brain tissue,the most highest exression in cerebral tissue composed cerebral cortex,medulla,basal ganlia and striatum, high expression in cerebral cortex, and relatively lower expression in cerebellum. MiR-205expressed in the embryo stem cell and embryo body, but could not be detected in NPC.
Conclusion:MiR454and miR-205expressed in central neuronal system.MiR-205expressed in embryo stem cell-neuron differentiated cell line
ABSTRACT OF SECTION3Identify expression of serum miRNAs for PD and Normal Subjects
Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including neurodegenerative disorders. MiRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are in a remarkably stable form that is protected from endogenous RNase activity, reproducible, and consistent among individuals of the same species. the study identified specific expression patterns of serum miRNAs for prostate cancer,lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.
There has been tremendous effort by the field to find biomarkers and physiological tests that accurately diagnose PD. There is still no conclusive diagnostic test. The goal of this proposal is to test the utility of using a miRNA signature to selectively diagnose and track Parkinson's disease. By the completion of this study, we will know if blood contains a better diagnostic miRNA signature for PD.
Methods:Using quantitative Real-time PCR, we analyzed the relative copies of miR-205and miR-454in serum among16sopradic PD patients and20genetically unrelated control individuals.
Results:The expression profile of serum miR-205in PD was significantly different from that of normal subjects, P value of T test was0.0001. The expression profile of serum miR-454in PD was significantly different from that of normal subjects, P value of T test was0.040.
Conclusion:We conclude that miR-205is in all a probability as a biomarker to diagnosing Parkinson's disease, and miR454is probability as a biomarker to diagnosing Parkinson's disease.
帕金森病(Parkinson Disease, PD)是一种常见的神经变性疾病。PD的发病机制十分复杂,目前认为其是由遗传因素与环境因素的共同作用所导致的。PD遗传致病机制的研究已取得明显进展,迄今已定位16个PD相关位点,克隆了包括富亮氨酸重复激酶2基因(Leucine-rich repeat kinase2, LRRK2)在内的11个致病基因,LRRK2不仅是常染色体显性遗传性PD重要致病基因,还与部分原发性PD有关。
目前LRRK2基因突变导致PD发生的机制并不十分清楚,但研究提示某些LRRK2致病突变(如G2019S)可能引起其激酶功能增强而具有细胞毒性,并且过表达野生型LRRK2也具有细胞毒性作用。此外LRRK2可能参与PD发生过程中异常蛋白沉积。过表达野生型LRRK2、LRRK2-G2019S突变体或LRRK2激酶结构域可加速a-Synuclein A53T的聚集。由此可见,对LRRK2的表达和功能的精确调控在PD发病机制中具有重要作用。
MicroRNA (miRNA)是一类由内源基因编码的长度约为21-23个核苷酸的非编码单链RNA分子,由具有发夹结构的约70-90个碱基大小的单链RNA前体经过Dicer酶加工后生成,有5’端磷酸基和3’羟基,定位于RNA前体的3’端或5’端。在不同组织、不同发育阶段中miRNA的水平有显著差异,这种miRNAs表达模式具有分化的位元相性和时序性(differential spatial and temporal expression patterns),因此1miRNAs作为参与调控基因表达的分子具有重要意义。本课题将探讨miRNA对LRRK2基因表达调控机制在PD发病机制中的作用。
方法：通过UCSC Genome Bioinformatics数据库(http://genome.ucsc. edu/)查询LRRK2基因的RefSeq序列,根据查询到的LRRK2基因3'UTR序列运用targetscan (http://www.targetscan.org/),miRanda(http://www.microrna.org/microrna/home.do)等根据LRRK2基因3’UTR序列预测能对其进行转录后调控的microRNAs,从]niRbase数据库获得相关microRNAs序列,比对生物信息学软件的预测结果,选择评分值高的microRNAs进行生物学验证。构建野生型及突变型LRRK2基因3'UTR序列荧光素酶报告基因载体,在N2a细胞中与miRNAs瞬时共转染,于转染后24小时及48小时进行荧光素酶活性分析,验证miRNAs对LRRK2基因mRNA的转录后抑制作用。瞬时转染miRNAs进入HEK293细胞系,于转染后24小时及48小时检测内源性LRRK2蛋白表达水平。根据共转microRNA后荧光素酶报告系统活性以及细胞系体内的内源性LRRK2蛋白表达水平判断,二者均具有统计学意义时判定该microRNA分子对LRRK2基因具有转路后抑制作用。
结果：本研究对LRRK2基因的RefSeq序列NM_198578的全长3'UTR序列共1517个碱基进行分析,以蛋白翻译终止位点"TAG"的G碱基为+1位进行描述,结合TargetScan预测结果显示hsa-miR-205与LRRK2基因3'UTR序列结合位点+116-122具有高保守性,107个人类miRNAs分子与人类LRRK2基因3’UTR序列存在保守性较低的潜在结合性；miRanda预测结果显示57个人类miRNAs分子与人类LRRK2基因3'UTR序列存在潜在结合性,选取23个潜在调控miRNAs进行生物学验证,结果显示hsa-miR-205mimics与携带野生型LRRK2基因3'UTR序列以及位点+116-122突变型LRRK2基因3'UTR序列的Luc报告系统质粒共转染N2a细胞株后,携带野生型组的Luc报告基因相对活性下降,t检验显示差异具有统计学意义(P<0.05,t=6.479,95%CI值为-1.614～-0.617)；hsa-miR-454mimics与构建的携带野生型LRRK2基因3'UTR序列以及位点+506-512突变型LRRK2基因3'UTR序列的Luc报告系统质粒共转染N2a细胞株后,野生型组的Luc报告基因相对活性明显下降,t检验显示差异具有统计学意义(P<0.001,t=6.479,95%CI值为-1.614--0.617)。瞬时转染hsa-miR-205、hsa-miR-301b及hsa-miR-454mimics进HEK293细胞于24小时及48小时检测内源性LRRK2表达,结果显示转染hsa-miR-205mimics后内源性LRRK2蛋白生成明显减少,转染hsa-miR-454mimics后内源性LRRK2蛋白生成减少,而转染hsa-miR-301b mimics后HEK293细胞的内源性LRRK2蛋白减少不明显。
结论：hsa-miR-205可与LRRK2基因3'UTR序列的5'-AUGAAGG-3'互补结合,抑制LRRK2基因的表达；hsa-miR-454可与LRRK2基因3'UTR序列的5'-UUGCACU-3'互补结合,抑制LRRK2基因的表达hsa-miR-301b可能与LRRK2基因3'UTR序列的5'-UUGCACU-3'互补结合,抑制LRRK2基因的表达,但仍需深入研究。
第二部分miRNAs及LRRK2mRNA在干细胞-神经元分化细胞系的表达水平研究
组织发育与生理过程中的稳态通过于细胞与组织干细胞的调控完成,是否存在一些细胞内机制介导干细胞间的信号转导、表观遗传,转录,翻译以及转录后调控水平的分化基因表达等细胞内机制。microRNA,作为基因表达、修饰、转录和翻译的调节者,在干细胞的自我复制、定向分化和组织再生中起着十分重要的作用。它是干细胞特性、维持、转化功能的一个关键调控者,参与细胞周期、细胞重建(Reprograming)、多能干细胞生成、信号传递、特异分化、聚集、修复、再生、变异、代谢等所有过程,是当前干细胞调控研究的一个重点。
LRRK2基因是PD发病的重要致病基因,而病理学研究表明LRRK2蛋白广泛表达于所有脑组织,但以多巴胺能神经支配区为主。对大鼠的胚胎发育中神经元与神经元附属组织的LRRK2mRNA表达研究表明LRRK2可能在控制神经元细胞的增殖、迁移以及分化过程中发挥作用。本章节将在验证miR-454及miR-205对LRRK2基因转录后调控的基础上初步探讨miR-454及miR-205与LRRK2在胚胎干细胞向神经元的分化过程中不同分化阶段胚胎干细胞及胎脑组织的表达之间的相关性,为进一步深入研究miRNAs与LRRK2在神经元增殖、迁移以及分化过程中的作用奠定基础。
方法：分化人源胚胎干细胞hESCs H9-P57细胞(ES),分别收集ES,拟胚体(EB),神经前体细胞以及人类胎脑组织共3个不同胚胎发育阶段的细胞及组织,提取总RNA,应用实时荧光定量PCR检测miRNAs, LRRK2基因mRNA的表达水平；提取胎脑组织蛋白,应用western blotting技术检测胎脑组织中LRRK2蛋白的表达水平。
结果：相对定量PCR结果显示LRRK2mRNA在胚胎分化的前期到神经前体细胞阶段表达均很低,在拟胚体阶段甚至检测不到LRRK2mRNA,而在发育到26周的有成熟神经元细胞存在的胎脑组织中LRRK2mRNA则呈现高表达,其中以包含大脑皮质及髓质结构的组织表达最高,仅有皮质结构的脑组织表达量较前者低,而在小脑中则表达更低；miR-454在胚胎干细胞,拟胚体阶段以及神经前体细胞阶段的表达均较低,而在发育到26周的胎脑组织中表达则升高,在胎大脑组织(包括大脑皮质及髓质)中的表达多于大脑皮层组织内的表达,大脑皮质及小脑组织中miR-454表达水平相当；miR-205在胚胎干细胞,拟胚体阶段存在表达,神经前体细胞阶段基本没有miR-205的表达,但在发育到26周的胎脑组织中均有表达,在胎大脑组织(包括大脑皮质及髓质)相对较低,而小脑中表达相对较高。胎小脑组织中LRRK2蛋白表达最高,胎大脑组织(包括大脑皮质及髓质)相对较低,仅包含皮质的胎脑组织中表达最低。
结论：miR-454及miR-205在中枢神经系统中存在表达；miR-205在胚胎干细胞分化过程中存在表达。
第三部分帕金森病患者血清miRNAs表达水平检测
miRNAs是一类内源性表达的短小的,非编码RNAs,可通过调控众多基因表达在不同生理功能中发挥作用。miRNAs主要与靶基因的mRNAs的3'UTR序列的结合位点相结合通过降解靶基因的mRNAs或者减少靶基因蛋白翻译而达到转录后抑制作用。目前研究显示miRNAs表达异常与众多疾病的发生相关,miRNAs的表达异常可能作为潜在的疾病诊断标志物或者与疾病相关的miRNAs可能成为潜在的疾病治疗靶点。多项研究显示人类血清或者血浆中存在由组织释放的游离：miRNAs,并且这些游离miRNAs表达具有稳定性,可再生性,在同一个体中表达水平一致,并不易被自身血液系统中的RNAse酶降解等特性,研究表明一些疾病如肺癌、前列腺癌等患者血清中游离miRNAs与正常人比较表达存在差异,因此目前认为血清中的1niRNAs可能作为诊断癌症等某些疾病的标志物。
多年来,众多研究组致力于寻找准确诊断PD的生物标志物与生理学检查,但是目前并没有找到确凿的诊断标志物,因此检测PD患者脑脊液,或者血液中的niRNAs水平看是否能作为包括PD在内的神经变性病的诊断标志物或者治疗靶点。本研究在前面章节的研究基础上,进一步检测PD患者与正常对照血清中是否存在游离miR-205及miR-454水平差异性作为一种寻找PD生物标志物的探索性研究。
方法：采集16例散发性PD患者以及20例正常对照者5ml静脉血,促凝管静置4-6小时,分离血清于-70℃保存,应用Ambion-mirVanaTM PARISTMkit进行游离小RNAs分离,测OD,应用Qiagen公司的microRNAs RT-kit逆转录成DNA,应用荧光定量PCR定量PD组及对照组血清中Hsa-miR-205, hsa-miR-454的表达水平。
结果：本章研究利用qReal-Time PCR对16例PD患者及20例健康对照者的血清中游离miR-205进行相对定量分析,PD患者血清中的1niR-205表达较正常对照组低,应用T检验进行统计学分析,两者差异的P值=0.0001,差异具有统计学意义；PD患者血清中的miR-454表达较正常对照组低,应用T检验进行统计学分析,两者差异的P值=0.04,差异具有统计学意义。
结论：PD患者血清中游离miR-205水平较正常对照明显减低,miR-205很可能为PD的生物标记物；PD患者血清中游离miR-454水平较正常对照组减少,miR-454可能为PD的生物标记物。
ABSTRACT OF SECTION1The Isolation of miRNAs to regulate the expression of the Parkinson Disease Causitive Gene LRRK2
Parkinson's disease (PD) is the second most globally prevalent neurodegenerative disorder. The cause of PD is complex and multifactorial, involving both hereditary and environmental factors. Recent progress in molecular genetic studies of familial PD has led to the identification of16susceptible loci and11genes responsible for PD. Mutations in LRRK2are thus far the most prevalent genetic cause associated with autosomal dominant and idiopathic PD. Although the mechanism of how PD-associated LRRK2mutations cause disease is yet unknown, several studies indicated that certain kinds of mutations including G2019S are likely associated with toxic gain of function. Moreover, to overexpress wildtype LRRK2was found to be toxic in cultured cells and transgenic fly model. Becides, LRRK2was found to be associated with abnormal protein deposition in the brains of patients with lowy body disease including PD. To overexpress wildtype LRRK2, G2019S mutant or kinase domain alone accelerate the formation of aggregates caused by α-synuclein A53T mutant. These results suggest that precise regulation of LRRK2expression and function is necessary for maintaining homeostasis of the organism, and the disruption of the regulation mechanism may cause disease. microRNAs (miRNAs) are a class of endogenous22base-long single-stranded, noncoding RNAs that regulate gene expression in a sequence-specific manner in plants and animals. miRNAs are derived from long precursor transcripts by the action of nucleases Drosha and Dicer, and the resulting mature functional miRNAs bind to their target sequence in the3'-untranslated region (UTR) of mRNA with imperfect complementarity and lead to repression of translation.Therefore, to elucidate the mechanism of LRRK2post-transcriptional regulation is helpful to understand PD aetiology. In this study, the promoter activity of the LRRK23'-untransation region (UTR) was analized, which is essenssial to understand the post-transcriptional regulation of LRRK2.
Methods:Several on-line softwares were involved in our study to predict the miRNAs to regulate the expression of LRRK2.23miRNAs was selected base on the predictation results, and further constructed the luciferase reporter plasmids. The plasmids were then transfected transiently into the N2a cell lines, and the luciferase relative activity were analysed to identified regulation of miRNAs to LRRK2. Finelly, miRNAs were transfected transiently into the Hek293cell lines, and the expression of endougenous LRRK2protein were analysed to identified the repression of miRNAs to LRRK2.
Results:The study Found243miRNAs bind potentially with the sites of LRRK23'UTR of RefSeq NM_198578using several software to predict the targets of miRNAs.23miRNAs was selected base on the predictation results, and further constructed the luciferase reporter plasmids. The luciferase relative activity decreased after miR205and miR454were transfected transiently into N2a cell line with wild type LRRK23'UTR Luc report system or mutation the binding site ones,the luciferase relative activity decreased lightly after miR-301b were transfected transiently into N2a cell line with wild type LRRK23'UTR Luc report system or mutation the binding site ones. The expression of endougenous LRRK2obviously decreased when miR-205and miR-454transfected transiently into the HEK293cell line. The expression of endoug-enous LRRK2decreased lightly when miR-301b transfected transiently into the HEK293cell line.
Conclution:In summary,miR-205can regulate the expression through to bind the site of5'-AUGAAGG-3'in LRRK23'UTR; miR-454can regulate the expression through to bind the site of5'-UUGCACU-3'in LRRK23'UTR; miR-301b probably regulate the expression through to bind the site of5'-UUGCACU-3'in LRRK23'UTR.
ABSTRACT OF SECTION2Identify expression of miRNAs and LRRK2mRNA for embryo stem-cell Neuron differentiated cell line and fetal brain tissue
The hallmark of a stem cell is its ability to self-renew and to produce numerous
differentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a new
dimension of gene regulation in controlling stem cell fate and behaviour. This is shown deregulated synthesis of E2F1/DP caused by the miRNA pathway impairment as a key event in LRRK2pathogenesis and suggest novel miRNA-based therapeutic strategies.
Mutations in the LRRK2gene cause autosomal dominant, late-onset parkinsonism, which presents with pleomorphic pathology including alpha-synucleopathy. The expression and localization of LRRK2to various neuronal populations in brain regions implicated inPD including the cerebral cortex, caudate-putamen and substantia nigra pars compacta. The localization of LRRK2to key neuronal populations throughout the nigrostriatal dopaminergic pathway is consistent with the involvement of LRRK2in the molecular pathogenesis of familial and sporadic parkinsonism.
Methods:Using quantitative Real-time PCR, we analyzed the relative copies of miR-454and miR-205for embryo stem cell-Neuron differentiated cell line and fetal brain tissue which from the fetus developed about26weeks. We analyzed the expression of LRRK2mRNA and protein in different fetal brain tissue by quantitative real-time PCR and western blotting.
Results:The expression profile of miR-454and LRRK2mRNA were rather low in the embryo stem cell-neuron differentiated cell line,however,miR-454and miR-205and LRRK2mRNA shows very high expression in fetal brain tissue,the most highest exression in cerebral tissue composed cerebral cortex,medulla,basal ganlia and striatum, high expression in cerebral cortex, and relatively lower expression in cerebellum. MiR-205expressed in the embryo stem cell and embryo body, but could not be detected in NPC.
Conclusion:MiR454and miR-205expressed in central neuronal system.MiR-205expressed in embryo stem cell-neuron differentiated cell line
ABSTRACT OF SECTION3Identify expression of serum miRNAs for PD and Normal Subjects
Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including neurodegenerative disorders. MiRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are in a remarkably stable form that is protected from endogenous RNase activity, reproducible, and consistent among individuals of the same species. the study identified specific expression patterns of serum miRNAs for prostate cancer,lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.
There has been tremendous effort by the field to find biomarkers and physiological tests that accurately diagnose PD. There is still no conclusive diagnostic test. The goal of this proposal is to test the utility of using a miRNA signature to selectively diagnose and track Parkinson's disease. By the completion of this study, we will know if blood contains a better diagnostic miRNA signature for PD.
Methods:Using quantitative Real-time PCR, we analyzed the relative copies of miR-205and miR-454in serum among16sopradic PD patients and20genetically unrelated control individuals.
Results:The expression profile of serum miR-205in PD was significantly different from that of normal subjects, P value of T test was0.0001. The expression profile of serum miR-454in PD was significantly different from that of normal subjects, P value of T test was0.040.
Conclusion:We conclude that miR-205is in all a probability as a biomarker to diagnosing Parkinson's disease, and miR454is probability as a biomarker to diagnosing Parkinson's disease.
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