MicroRNA调控BACE1在AD发病中的作用与机制研究

The role and mechanism of epigenetic regulation in AD pathogenesis have received increasing attention recently. Our previous work showed that, the expression of miR-338-5p in the hippocampal brain tissue was significantly decreased, while the protein expression level of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) was markedly increased, in both transgenic 5XFAD mice and AD patients. By target gene analysis and fluorescence report vector expression, we confirmed the specific binding of miR-338-5p and 3' untranslated region of BACE1 mRNA. Cellular experiments preliminary confirmed that miR-338-5p upregulation could inhibit the protein expression levels of BACE1. BACE1 is a key rate-limiting enzyme in the procedure of amyloid precursor protein (APP) cleaving to produce Aβ polypeptide and starting the cascade of AD pathological injury. Thus, we assume the decreased expression of miR-338-5p palyed an important role in the pathogenesis of AD. Meanwhile, gene sequence analysis suggests there are two bindig motifs of nuclear transcription factor-B (NF-κB) in the promoter of miR-338-5p gene. Therefore, we suppose the neuroinflammation in the AD process could induce the NF-κB activation and then cause the inhibition of miR-338-5p transcription, and thus lead to the increased BACE1 protein and Aβ production, also the aggravation of AD pathology. To confirm this hypothesis, we intend to use 5XFAD transgenic mice and primary cultured neurons as the study model, aslo by using technical means such as molecular biology, tissue pathology, electrophysiological and behavioral tests, to explore the molecular mechanism of NF-κB regulation on miR-338-5p transcriptional expression, and to further clarify the post-transcriptional regulation of miR-338-5p on BACE1 and its role in the pathogenesis of AD. This study will provide a new theoretical basis on the AD pathogenesis, and also molecular targets for AD prevention and treatment.

表观遗传调控在阿尔茨海默病（AD）发病中的作用与机制受到重视。我们的前期工作发现：miR-338-5p在转基因AD小鼠及AD患者脑组织中显著低表达，其能与β位淀粉样前体蛋白裂解酶1（BACE1）的3'非编码区特异性结合；细胞水平初步证实miR-338-5p抑制BACE1的蛋白表达。BACE1是产生Aβ多肽并启动AD病理级联的关键限速酶，由此提示miR-338-5p的下调在AD发病中起重要作用。基因分析发现miR-338-5p基因的启动子区含有炎症转录因子NF-κB的结合位点。因此，本课题拟采用5XFAD转基因小鼠和原代培养神经元模型，通过表观遗传学、分子生物学、组织病理学、电生理及行为学等技术，探索NF-κB调控miR-338-5p转录表达的分子机制，并进一步阐明miR-338-5p对BACE1表达的调控效应及其在AD发病中的作用。本课题将为AD发病机制及防治靶标的研究提供新的理论基础。

本项目研究旨在阐明miR-338-5p分子调控环路在AD病理过程中的作用。我们提取不同月龄5×FAD转基因小鼠和AD患者海马组织总RNA，通过荧光实时定量PCR检测miR-338-5p的表达。将数据库预测的结合位点进行突变，体外通过双荧光素酶报告基因载体验证BACE1的3’UTR与miR-338-5p的结合。构建表达miR-338-5p的慢病毒质粒，体外转染SH-SYSY细胞系和原代神经元，在体将该慢病毒注射至4月龄5×FAD转基因小鼠海马区，通过水迷宫实验检测该AD小鼠（6月龄）的空间学习和记忆能力，通过Western blot检测APP及其剪切片段、BACE1的表达情况，通过免疫荧光染色检测海马区淀粉样斑块沉积情况、氧化应激、Aβ42水平以及星形胶质细胞、小胶质细胞的激活情况，通过电生理技术记录该小鼠DG区LTP来反映其神经突触功能。此外，采用免疫共沉淀-PCR技术寻找miR-338-5p与NF-κB的结合位点序列，并通过原代神经元验证NF-κB对miR-338-5p表达水平的影响。结果我们发现AD患者和5×FAD小鼠海马组织中的miR-338-5p表达水平较正常对照均明显降低，miR-338-5p与BACE1的3’UTR区有特异性结合位点，且能抑制BACE1的表达。SH-SYSY细胞以及原代神经元转染miR-338-5p慢病毒后，细胞中的BACE1表达均有明显减少。5×FAD转基因小鼠海马区注射miR-338-5p慢病毒2月后，该AD小鼠的空间学习和记忆能力均较病毒载体对照组有更明显恢复，其海马区的BACE1、APP剪切片段β-CTF表达降低，且AD小鼠海马区淀粉样斑块沉积及Aβ42水平均有显著减少，星形胶质细胞、小胶质细胞的炎性激活也有明显降低。同时，该AD小鼠海马DG区记录到的LTP衰减现象得到部分恢复。此外，miR-338-5p基因上游启动子区域存在两个与NF-κB结合的位点，NF-κB活化后核转移并抑制miR-338-5p的表达而增加BACE1的表达水平。因此，miR-338-5p负调控BACE1的表达且其自身受到NF-κB的负性转录调控，miRNA-338-5p过表达可明显改善5×FAD小鼠海马的病理损害、学习记忆能力和突触功能。