阿尔茨海默病炎症中circRNA介导的竞争性内源RNA（ceRNA）研究

Alzheimer's disease (AD) is neuronal degeneration with irreversible process. The role of neuroinflammation in AD dementia has gained attention recently, implicating an early involvement of inflammation in the disease onset and progression. The expression of inflammation related genes and its intrinsic regulation are cell-specific. In addition to mRNA, non-coding RNA (ncRNA) plays an important role in the level of gene transcription, post-transcriptional and translation. In recent years, a new mode of gene regulation: competitive endogenous RNA (ceRNA), has attracted great attention. As a special kind of non-coding RNA, Circular RNA (circRNA) is not affected by RNA exonuclease, but easy to change in the disease. Therefore, we hypothesized that circRNA associated ceRNA play a key role in the regulation of inflammation related genes in AD. In this study, we selected 3-month-old and 12-month-old APP / PS1 miceas the model. Then, the neuron, microglia and astrocyte were separated from cerebral cortex. RNA-Seq (mRNA-seq, miRNA-seq and circRNA-seq) were used to identify differential gene expression pattern at the various stages and to complete the ceRNA map, which participate in the regulating of inflammation related genes in AD. At the same time, Northern-blot, in situ hybridization, RNA interference and overexpression were used to verify the results. It helped us gain new insight into neuroinflammation of AD, and providing theoretical basis for AD treatment.

阿尔茨海默病(AD)是慢性进行性神经退行性疾病，发病早期即有炎症反应。AD炎症相关基因的表达变化及其调控具有细胞特异性。除了mRNA外，非编码RNA在基因转录水平、转录后水平及翻译水平起了重要调控作用。近年竞争性内源RNA（ceRNA）逐渐被认可。环状RNA（circRNA）是一类特殊的非编码RNA分子，稳定性强，推测AD炎症中circRNA介导 ceRNA模式是参与AD炎症相关基因调控的重要部分。本课题选取APP/PS1小鼠，于3月龄、2月龄分离神经元、小胶质细胞和星形胶质细胞，进行RNA-Seq分析，比较不同阶段AD炎症相关基因表达的差异，并以 circRNA-miRNA-mRNA为主线绘制完整参与AD炎症相关基因调控的竞争性内源RNA (ceRNA) 图谱；利用Northern-blot、原位杂交、RNA干扰、过表达等技术进行验证，从基因表达与调控角度理解AD炎症的发生、发展与防治。

阿尔茨海默病（Alzheimer’s disease，AD）发病早期即有炎症反应。Aβ 在脑内积聚形成老年斑为AD主要病理特征，与炎症密切相关。Aβ作用下，神经元、小胶质细胞、星形胶质细胞中炎症相关基因表达变化及其调控存在差异；此外，非编码RNA也在基因转录水平、转录后水平及翻译水平也起到重要调控作用，竞争性内源RNA（ceRNA）为非编码RNA调控的主要机制。本课题从单细胞分辨率和全转录组水平，全面、系统的解析了Aβ刺激对神经细胞的影响，包括mRNA和非编码RNA的表达变化。存在内源Aβ下，12月龄APP/PS1小鼠单细胞测序结果显示三种神经组织细胞可通过NF-κB信号通路调节炎症，其中小胶质细胞可分化为高表达Apoe、Lilrb4标记基因且具有吞噬Aβ作用的DAM型细胞，并发现了一个高表达干扰素基因Ifi204以及趋化因子Ccl12的促炎型小胶质细胞亚群；在外源Aβ刺激下，全转录组测序结果提示三种神经组织细胞的应激基因各有不同，但是均存在可靶向NF-κB信号通路的炎症相关基因。我们还根据对三种神经组织细胞差异Lnc-RNA、circRNA、miRNA的筛选分析，分别绘制了三种细胞的ceRNA网络图谱，包括lncRNA-miRNA-mRNA 以及circRNA-miRNA-mRN图谱。并筛选出了可靶向NF-κB炎症通路的ceRNA通路，包括mmu_circ_0010413/mmu-miR-694/Cux1、mmu_circ_0010413/mmu-miR-6946-3p/Rufy3、mmu_circ_0010941/mmu-miR-446n-3p/Rbm39、mmu_circ_0006650/mmu-miR-6903-3p/Lpp、mmu_circ_0005599/mmu-miR-350-3p/Xiap等。综上，本研究揭示了神经元、小胶质细胞、星形胶质细胞中炎症相关基因表达变化的异同，并筛选到多条炎症相关ceRNA通路，为抑制Aβ病理以及神经炎症提供新靶点。