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题名: 基于microRNA和mRNA表达谱的二恶英体外神经毒理学研究
作者: 徐团1
学位类别: 博士
答辩日期: 2017-05
授予单位: 中国科学院大学
授予地点: 北京
导师: 赵斌 ; 谢群慧
关键词: 二恶英,系统生物学,microRNA,芳香烃受体,乙酰胆碱酯酶 ; dioxins, systems biology, microRNA, aryl hydrocarbon receptor, acetylcholinesterase
其他题名: In vitro toxicology study of dioxin based on microRNA and mRNA expression profiles
学位专业: 环境科学
中文摘要: 二恶英暴露能够引起实验动物和人类的神经系统发育障碍和高级脑功能损 伤(如运动协调、情绪、认知与心理障碍等)。二恶英诱导基因表达的变化是二 恶英毒理效应的重要分子基础,其中基因表达的转录水平调控主要通过芳香烃受 体(Aryl hydrocarbon receptor, AhR)信号通路调控一系列下游基因的表达实现的。 随着表观遗传学的兴起,越来越多的证据提示二恶英与表观遗传调控机制关系密 切,如二恶英可造成 microRNAs(miRs)的表达异常;反之,miRs也可以参与 AhR信号通路的调控等。因此基于上述二恶英发挥作用的分子基础的研究是目 前二恶英神经毒理机制研究的一个热点领域。但是目前的相关机制研究多集中于 对单个基因或单一毒理效应的研究,而在系统性阐述二恶英对神经源性基因表达 的干扰作用方面的研究十分有限。另外,miRs作为基因表达的负调控因子在二 恶英神经毒理中的作用尚未见报道。鉴于系统生物学的方法和手段在揭示基因整 体性改变及生物学意义方面的优势,本研究以人源神经母细胞瘤细胞系 SK-N-SH 细胞为研究模型,采用芯片技术,围绕二恶英基因干扰的两个方面,开展 miR 和 mRNA表达谱的研究,结合生物信息学分析及细胞分子生物学研究手段,揭 示低浓度二恶英处理后 miR和 mRNA表达谱的整体变化情况,并从中发现具有 神经学意义并参与二恶英神经干扰作用的新的 miRs、基因及神经毒理学效应。 围绕这一目标获得了如下实验结果: (1)低浓度二恶英处理(10-10 mol/L TCDD)能够导致 SK-N-SH细胞内 miR 和 mRNA表达谱发生显著变化。二恶英处理组共有 277个 miRs(包括 148个下 调和 129个上调的 miRs)及 4378个基因(1838个下调和 2540个上调的基因) 的表达发生显著性变化。通过生物学功能聚类分析发现,一些差异表达的 mRNA 参与神经系统功能和细胞凋亡等信号通路,这些信号通路大部分与文献报道的二 恶英的毒理效应密切相关。据文献报道,在 53个表达变化超过 40%的 miRs中 有 13种 miRs参与神经退行性疾病或脑肿瘤等神经系统疾病发生发展过程。 (2)结合 miR表达谱数据及实验验证,我们首次证明二恶英能够显著上调 miR-146b-5p的表达,其调控机制可能是 AhR信号通路介导的。MiR-146b-5p能 够与胆碱能神经传递的重要酶类-乙酰胆碱酯酶(acetylcholinesterase,AChE)T 亚型 mRNA的 3′端非编码区作用从而抑制 AChE的酶活性。通过对 miR-146b-5p 的靶基因进行功能聚类分析发现,miR-146b-5p可以参与已知的二恶英神经毒性 相关的信号通路及生物进程,如脑肿瘤、突触传导、细胞凋亡等。而 AChE在部 分生物进程中的作用提示二恶英对 AChE表达的干扰可能最终导致与这些生物 进程相关的有害结局。 (3)我们还首次证明了二恶英能够显著上调 SK-N-SH细胞中灵长类特异性 miR-608的转录表达,其功能可能涉及神经元分化和肌动蛋白细胞骨架组装等。 而在细胞骨架组装相关基因中,CDC42被证明是神经源性 miR-608的靶基因, 且二恶英能够显著上调 SK-N-SH细胞中 CDC42的表达。作用机制研究显示转录 上调机制和 miR-608介导的转录后抑制机制共同参与二恶英对 CDC42的表达调 控,且 AhR是两种调控机制共同的上游信号分子。 (4)通过整合 miR和 mRNA表达谱数据,在生物信息学分析及细胞分子 生物学实验验证的基础上,发现二恶英能够促进神经母细胞瘤细胞的迁移能力, 且具有时间-浓度效应,其机理可能是通过 AhR介导的细胞迁移相关基因的表达 上调造成的。 综上所述,系统生物学方法在二恶英神经毒性研究中具有重要应用价值,并 首次获得了低浓度二恶英处理对神经细胞中基因表达影响的系统性数据。本论文 不仅弥补了二恶英对神经源性 miRs干扰作用方面研究的不足,也为今后二恶英 神经毒理机制及效应的研究提供铺垫。
英文摘要: Exposure to dioxins has been associated with impaired development of the nervous system and losses of brain function (e.g. motor coordination, emotion, cognition and psychological disorders) in experimental animals and humans. Alterations of various genes caused by dioxins are considered as a critical action mechanism for dioxins to exert the biological and toxicological effects. Aryl hydrocarbon receptor (AhR) is known as a key mediator of transcriptional regulation of the altered genes caused by dioxin eposures. With the rise of epigenetics, emerging evidence suggested possible involvement of dioxins in epigenetic regulation, such as in dysregulation of of microRNAs (miRs), which may be involved in the regulation of the gene expression mediated by the AhR signaling pathway. However most of the present neurotoxicity studies of dioxins are focused on single gene or single toxicological effect, systematic studies on gene expression interferences caused by dioxin are lack. Moreover, as a down-regulator of gene expression, the role of miRs in the neuro-toxicology of dioxins has not been reported. Due to the advantages of systems biology in revealing the overall genetic changes and the related functional significance, microarray technology was employed in this study for revealing miR and mRNA expression profiles in dioxin (10-10 mol/L TCDD) -treated human neuroblastoma cell line SK-N-SH. With the aids of bioinformatics analysis, overall changes in coding gene and non-coding gene expressions were revealed based on miRs and mRNA expression profiling study. Novel dioxin responsive miRs, mRNA and neurotoxic effect were identified in dioxin treated SK-N-SH cells.The major findings are as follows: (1) MiR and mRNA expression profiles were significantly changed in dioxin-treated SK-N-SH cells. Some differentially expressed mRNA were involved in the nervous system function related and apoptosis signal pathways by functional clustering analysis, most of which have been reported as toxicological effects of dioxins. There were 13 differentially expressed miRs involving in the development of nervous system diseases, such as neurodegenerative diseases or brain tumors. (2) Consistent with miR expression profile, the expression of miR-146b-5p can be significantly increased by dioxin. MiR-146b-5p can suppress acetylcholinesterase (AChE) enzymatic activity through targeting 3’-non encoding region of AChE T subunit mRNA. The target genes of miR-146b-5p were involved in brain functions or cytotoxicitiy related to dioxin effects, including synapse transmission, in which AChE may serve as a responsive gene for the effect. (3) The expression of a primate-specific miR, miR-608, was significantly increased by dioxin via AhR, which may be involved in neuron differentiation and actin cytoskeleton oragnization. Among genes related to actin cytoskeleton oragnization, CDC42 has been shown to be a target gene for neuronal miR-608. Dioxin significantly increased the expression of CDC42 via AhR, suggesting that AhR not only mediates transcriptional induction of CDC42, but also miR-608-induced post-transcriptional suppression of CDC42 in dioxin treated neuroblastoma cells. (4) Based on integration of miR and mRNA expression profiles, we proposed and demonstrated that dioxin could promote the migration ability of neuroblastoma cells in time- and concentration- dependent manners, which may be mediated by the up-regulation of migration promoting genes. In conclusion, with the aids of systems biology method, a systematic assessment of dioxin effects on human neuronal gene expression was firstly obtained in the present study. The outcome of the study not only makes up the deficiency in understanding the dioxin effect on neuronal miRs, but also provides the basic data for neuro-toxicological study on dioxins.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38716
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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