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学科主题: 神经生物学
题名:
MiR-211-5p在阿尔茨海默病的作用及发病机制研究
作者: 樊春英
答辩日期: 2016-05-11
导师: 万峻
专业: 神经生物学
授予单位: 北京大学
授予地点: 北京大学深圳医院
学位: 硕士
关键词: 阿尔茨海默病 ; microRNA-211-5p ; NUAK1 ; 神经发生 ; β淀粉样肽
其他题名: The role of miR-211-5p in the pathogenesis of Alzheimer’s disease
分类号: R749.1
摘要:

阿尔茨海默病(Alzheimer’s disease,AD)是一种进行性不可逆性恶化的中枢神经系统退行性疾病,是老年期痴呆最常见的类型,严重威胁着老年人的健康,但其发生机制尚未阐明。近年来的研究显示,AD进展中不仅发生了神经元的变性,而且神经发生也受到了损害。MicroRNAs(miRNAs)是一类主要通过改变靶基因表达水平来参与调节多种细胞生物过程的非编码小RNA。多项研究表明,miRNA可通过调节神经发生参与AD的进展。研究发现,miR-211具有调节细胞的增殖、分化、生存等功能,但目前miR-211在神经元分化、AD中的作用及作用机制还没有相关报道。

有研究证实NUAK1调节了神经元的轴突生长和分支,且是miR-211的靶点之一。我们通过转染验证了miR-211-5p对NUAK1的调控作用,荧光定量PCR技术及免疫印迹结果显示,与对照组比较,无论在Neuro-2a 细胞系还是在体外原代培养的皮层神经元中转染miR-211-5p mimic后NUAK1的mRNA或蛋白表达水平显著下降,转染miR-211-5p inhibitor后NUAK1的mRNA或蛋白表达水平没有显著变化。进而我们应用荧光定量PCR技术及免疫印迹在ICR小鼠脑皮层神经元发育期检测miR-211-5p及NUAK1的表达谱和在皮层神经元中通过转染和免疫荧光技术检测miR-211-5p是否通过靶向NUAK1也调控了神经元的轴突生长和分支,结果表明,在神经元分化期间miR-211-5p呈现低表达而NUAK1 mRNA和蛋白表达上调;过表达miR-211-5p的神经元与对照组比较,轴突的长度和分支数目都明显降低,而抑制miR-211-5p表达的神经元轴突的长度没有明显变化但分支数目显著增加;miR-211-5p mimic和NUAK1共转染的神经元与miR-211-5p mimic 和PT-GFP共转组比较,轴突的长度和分支数目都明显升高。接下来我们用荧光定量PCR技术及免疫印迹在2月-18月龄APPswe/PS1ΔE9双转基因小鼠模型和体外细胞模型中检测miR-211-5p和NUAK1是否参与了AD的进展,在9个月到18个月年龄段APPswe/PS1ΔE9双转基因小鼠脑皮层组织中miR-211-5p表达水平高于野生型小鼠而NUAK1的mRNA和蛋白表达水平低于野生型小鼠;体外原代培养胚胎E18.5天脑皮层神经元经Aβ1-42处理后与溶剂对照组比较miR-211-5p表达水平升高而NUAK1的蛋白表达水平下调,其变化趋势与小鼠脑皮层组织的变化趋势一致。为了检测miR-211-5p对Aβ1-42处理的神经元分化的影响,我们用miR-211-5p mimic 和PT-GFP或者miR-211-5p inhibitor 和PT-GFP共转染E15.5天的皮层神经元后经Aβ1-42处理,结果显示,与对照组比较,过表达miR-211-5p显著降低了经Aβ1-42处理的神经元轴突的长度和分支数目;miR-211-5p inhibitor显著增加了Aβ1-42处理的神经元轴突的长度和分支数目,减轻了Aβ1-42的损害。我们用MTT实验检测了miR-211-5p mimic及inhibitor对有无Aβ1-42处理的E18.5天皮层神经元活性的影响,结果表明,与对照比较,无论Aβ存在与否过表达miR-211-5p降低了神经元的活性;抑制miR-211-5p表达则对神经元的活性没有明显影响。

综上所述,我们的实验表明miR-211-5p降低了皮层神经元的活性及通过调节NUAK1水平抑制了皮层神经元轴突的分化,可能参与了阿尔茨海默病的发生。

 

英文摘要:

Alzheimer’s disease (AD) is the most common irreversible neurodegenerative disease, which is serious to old people. However, the underlying mechanism is not fully known. Research in recent years indicates that in addition to neuronal degeneration, adult neurogenesis in AD is impaired. MicroRNAs (miRNAs), small non-coding RNAs, modulate cellular regulatory processes via their ability to alter gene expression. Some studies have identified miRNAs that are involved in AD by regulating neurogenesis. Studies have shown that miR-211 regulates cell proliferation, differentiation, survival, invasion and metastasis. However, there are no relative reports about the roles of miR-211 in neuronal differentiation and Alzheimer’s disease at present.

It has been reported that NUAK1, a known target of miR-211, regulates both axon growth and branching. To determine whether NUAK1 is regulated by miR-211-5p in Neuro-2a cells and cortical neurons, we transfected miR-211-5p mimic and inhibitor and examined their effects on NUAK1 mRNA and protein levels using qRT-PCR and Western Blot assays. Over-expression of miR-211-5p mimic resulted in a significant decrease of NUAK1 mRNA and protein levels while miR-211-5P inhibitor didn’t show a significant effect on NUAK1 level. Then to gain insight into the role of miR-211-5p in neurogenesis, qPCR and western-blot were performed to assess the expression levels of miR-211-5p and NUAK1 during mouse embryonic and postnatal cortex development. MiR-211-5p expression is down-regulated while NUAK1 mRNA and protein is highly expressed during the differentiation of neurons. According to above changes, we cotransfected cortical neurons from E15.5 with miR-211-5p mimic and PT-GFP or miR-211-5p inhibitor and PT-GFP and observed that neurons transfected miR-211-5p mimic displayed significantly decreased axon growth and branching while miR-211-5p inhibitor induced markedly increased axon branching without a significant effect on axon length. Cotransfection of miR-211-5p mimic and NUAK1 significantly increased axon length and branching compared to the group cotransfected with miR-211-5p mimic and PT-GFP. To explore the dynamic changes of miR-211-5p and NUAK1 expression in the cortexes of APPswe/PS1ΔE9 double transgenic mice and wild type controls with ages spanning from 2 to 18 months, we examined their levels using qRT-PCR and Western Blot assays. MiR-211-5p expression was markedly increased and NUAK1 mRNA and protein levels were dramatically reduced accordingly since 9 months of age compared to the control group. To corroborate these changes, cortical neurons from E18.5 were exposed to Aβ1-42 after DIV7, and dynamic changes of miR-211-5p and NUAK1 were consistent with that from mice tissues. We determined the effect of miR-211-5p on the differentiation of neuron treated with Aβ1-42 and results showed that over-expression of miR-211-5p dramatically decreased axon length and branching in Aβ1-42-treated cortical neurons compared to the control group. MiR-211-5p inhibitor induced markedly increased axon length and branching in Aβ1-42-treated cortical neurons compared to the control group exposed to Aβ1-42, alleviating the insult of Aβ1-42 on axon length and branching. MTT was used to examine the effect of miR-211-5p on cell viability. Whether Aβ1-42 existed or not the viability of primary neurons was dramatically decreased following over-expression of miR-211-5p. When miR-211-5p was inhibited, cell viability didn’t get affected.

In conclusion, our results suggest that miR-211 inhibits axon differentiation of cortical neuron by regulating the level of NUAK1, decreases neuronal viability and may be involved in AD.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125376
Appears in Collections:北京大学深圳医院_学位论文

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作者单位: 北京大学深圳医院

Recommended Citation:
樊春英. MiR-211-5p在阿尔茨海默病的作用及发病机制研究[D]. 北京大学深圳医院. 北京大学. 2016.
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