【作者】 戚艳婷；

【导师】 袁崇刚； 赖立辉；

【作者基本信息】 华东师范大学 ， 生理学， 2008， 博士

【摘要】 小分子RNA在脑内含量丰富。现有研究表明这些非编码小RNA与脑的发生、发育及脑疾患相关,然而他们在神经元老化和神经退行性疾病中的作用机制有待揭示。本论文从研究恒河猴脑microRNA时空表达特征入手,发现mir-34a呈年龄相关性表达。在此基础上,我们验证了多个mir-34a作用靶标,并发现过表达mir-34a引起神经元凋亡的现象。尤为重要的是,本研究进一步探讨了mir-34a在AD的发病中的可能机制以及mir-34a在脑肿瘤发生过程中的网络调控机制。第一部分,mir-34a转录本在脑内的表达特性。我们证明,mir-34a在恒河猴脑内随着年龄老化而表达增加,这一表达特性在芯片和Northern Blotting中具有一致性。相同的表达趋势也存在于大鼠胚胎脑皮层原代培养神经元中,且mir-34a高表达主要集中在皮层颗粒细胞和锥体神经元(Ⅱ/Ⅲ层)。联合免疫荧光检测发现,mir-34a主要在神经元胞体表达。mir-34a在脑内的时空表达提示其与脑功能休戚相关。第二部分,mir-34a靶标的预测。利用我们自主开发的软件KeyTar进行mir-34a保守作用位点的预测。KeyTar中所采用的新算法大幅度地提高了miRNA靶标预测的准确性(结果未发表)。结合分析mir-34a在生理和病理情况下的表达特性,BCL-6,FoxP1,Notch1,R-Ras和CCND1被预测为是mir-34a的靶标。第三部分,过表达mir-34a诱导神经元凋亡的可能机制。基于上述研究,我们进一步用原代培养神经元来研究mir-34a的功能。MTT实验表明,过表达mir-34a可以影响神经元的存活。采用检测caspase-3活性体的方法进一步验证,过表达mir-34a可以引发神经元凋亡。通过mir-34a靶标鉴定,我们发现其过表达诱导神经元凋亡的机制可能与其抑制BCL-6的翻译有关。业已表明,BCL-6系一转录抑制因子,其可抑制p53的生成。我们的研究结果提示mir-34a可能在神经元发育和老化中具有重要作用。第四部分,mir-34a参与AD病理过程的可能机制。在该项研究中,我们发现mir-34a在阿尔茨海默氏病模型小鼠脑中和人阿尔茨海默氏病脑中表达失调。同时,我们验证了FoxP1和Notch1系mir-34a作用靶标。进一步研究表明,AD神经元中mir-34a表达呈下调趋势,而FoxP1和Notch1则表达升高。已有的研究表明,Notch1,CCND1及CDC25a在神经元中的表达失调与阿尔茨海默氏病的发生有关,我们的研究结果表明FoxP1也与阿尔茨海默氏病的发生相关,mir-34a因调控这些细胞周期相关因子而参与了阿尔茨海默氏病的病理过程。因此,我们的研究为揭示miRNA转录与神经退行性病症之间的关系提供了有价值的线索。第五部分mir-34a抑制R-Ras引起脑肿瘤细胞凋亡的研究。新近研究表明,p53通过调控mir-34a表达对多种肿瘤具有抑制作用。在该部分研究中,我们首先对mir-34a在不同物种体内的时空表达情况进行了检定。其次,我们采用RNA印迹和ISH发现mir-34a在脑胶质瘤及神经母细胞瘤细胞中表达下调。进一步的研究表明,mir-34a可以通过对R-Ras的转录后翻译起抑制作用,并且mir-34a导致的脑肿瘤细胞凋亡可能与其作用于R-Ras有关。我们的这一研究结果首先发现了mir-34a具有调控R-Ras通路的作用,为mir-34a成为治疗脑肿瘤的靶标提供可能性。综上所述,我们的工作在于探讨mir-34a参与脑老化过程与脑肿瘤形成的分子机制。本研究论文已在三个方面得到了初步结论,即呈年龄梯度变化的mir-34a可能参与控制神经元衰老过程;mir-34a可能参与阿尔茨海默氏病发病机制;mir-34a具有调控R-Ras通路的作用,有望成为脑肿瘤治疗的靶标之一。更多还原

【Abstract】 MicroRNAs (miRNAs) are abundant in the brain and have been implicated in neurogenesis and brain disorders. However, the essential rules of miRNAs in regulation of neuronal aging and progressive neurodegeneration have not been established. In this study, the spatial and temporal pattern of mir-34a expression in the brain was determined. In addition, several novel targets of mir-34a were validated and induction of mir-34a mediates neuronal apoptosis. Importantly, the deregulation of mir-34a expression in neurons was found to correlate with Alzheimer’s pathogenesis. Moreover, the correlations between the changes of mir-34a expression in brain tumors and its targets demonstrated that miR-34a might play critical roles in brain cancer development.Part I. Characterization of miR-34a transcripts in the brain. In this part of study, we demonstrated that miR-34a expresses with aging in the brain of rhesus monkey using microarray, northern blotting and ISH. Correspondingly, the same trend was found in the primary cultures of rat cortical neurons. Furthermore, high level expression of miR-34a was found in the external granular layer and outer pyramidal layer (II/III layers). Co-localization of miRNA positive cells with a specific cellular marker Map-2 showed that mir-34a signals were exclusively detected in cell bodies of neurons. The determination of the spatial and temporal patterns of mir-34a expression in the brain provided insight into its biological functions.Part II. Prediction of mir-34a targets. The putative conserved binding sites were predicted by KeyTar, which is a sequence and structure based algorithm developed by our team. This algorithm would largely improve the accuracy of miRNA target prediction when the real status of both miRNAs and their targets in the biological systems are evaluated (unpublished data). Considering the possible rules of mir-34a and its expression patterns in the physiological and pathological conditions, the genes Bcl-6, FoxP1, Notch1, R-Ras and CCND1, are selected as the putative targets of mir-34a.Part III. Induction of mir-34a mediates neuronal apoptosis. In this part, we extended our study to test the function of this miRNA in the primary culture of rat cortical neurons. We found induction of mir-34a led to neuronal apoptosis. The data was further confirmed by the analysis of cleaved activated caspase-3. Moreover, we found that induction of miR-34a might be associated with the putative targets BCL-6. These results indicate that miR-34a mediates neuronal death versus suppressing a transcriptional repressor bcl6 that is known to direct a negative feedback loop to p53. Thus, we hypothesize that it is possible mir-34a has a critical role in neuronal development and aging.Part IV. Deregulation mir-34a is correlated with Alzheimer’s pathogenesis though modulating multiple cell cycle associated factors. In this part of the study, we showed mir-34a was deregulated in the brain of a mouse AD model and in the brain of human AD. Moreover, we found FoxP1 and Notch1 are the targets of mir-34a. Correspondingly, the decreased level of mir-34a in AD neurons was detected to correlate with the increased levels of its targets Foxp1 and notch1. Of noted is that deregulations of Notch1, CCND1 and CDC25a have been known to associate with Alzheimer’s pathogenesis in human. Our data demonstrate that FoxP1 is involved in Alzheimer’s pathogenesis and the deregulation mir-34a is correlated with Alzheimer’s pathogenesis versus modulating multiple anti-apoptotic factors. Thus, this study provides valuable clues to unravel the complex interrelationships between miRNA transcripts and neurodegenerative pathogenesis.Part V. mir-34a could promote apoptosis in brain tumor cells through translational repression of genes including R-Ras. It has been reported that mir-34a modulates the gene expression program initiated by p53 and serves as a potential tumor suppressor in a variety of tumor cells recently. In this part of the study, the spatial and temporal patterns of mir-34a expression in different species were determined. Northern Blotting and ISH analyses showed miR-34a was poorly expressed in glioma and neuroblastoma cells. Moreover, induction of mir-34a was found to promote tumor cell apoptosis. We demonstrated here firstly that miR-34a regulates R-ras pathway in brain tumors. The molecule, mir-34a, therefore, might be a possible target that could be employed in anti-brain cancer therapy.In summary, this doctoral thesis is focused on understanding molecular mechanisms how mir-34a is involved in aging and in tumor in the brain. The dissertation provides three clear contributions in this area: age-related mir-34a regulates neuron senescence, deregulation mir-34a is correlated with Alzheimer’s pathogenesis and miR-34a might suppress the R-Ras pathway, thus might be a possible target that could be employed in anti-brain cancer therapy.更多还原