Notch3基因突变引起CADASIL病的分子病理学新机制：miR-181a及其靶基因所扮演的角色

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in Notch3 gene encoding a transmembrane receptor Notch3. However, the functional nature of the Notch 3 mutations causing CADASIL and their mechanistic connection to small-vessel disease remain enigmatic. Given the importance of Notch3 signaling in regulating gene expression, the downstream expression profile of mRNA, protein and miRNA of Notch3 signaling will provide insight into the molecular pathogenic mechanism of CADASIL. However, so far, the influence of Notch 3 mutations on the miRNA expression profile in the brain has not been investigated. We recently established that, in CADASIL patients biopsies and in R90C mutation knock-in mice (TgNotch3R90C), expression level of miR-181a is markedly upregulated while two targets of Notch3 (Kcnmb1、Lrrfip1) were significantly down-regulated. Additional evidence suggested a negative regulatory role of Notch3-RBPJ signaling on miR-181a expression. Based on these results, in this proposal, we are going to further investigate the molecular mechanism of Notch3-RBPJ signaling on miR-181a expression. Moreover, functional role of miR-181a and its targets on vascular function in mice (in vivo) will be dissected. Finally, miR-181a antagomir（anti-miR-181a）will be injected into R90C mutation knock-in mice (TgNotch3R90C) to assess if blocking miR-181a would alleviate CADASIL phenotype. Our project will provide a new therapeutic tool for the control of CADASIL.

伴有皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病（CADASIL）是由于Notch3基因发生突变所致。作为一个调控基因转录表达的受体，Notch3对下游基因、蛋白、miRNA的表达调控无疑会对CADASIL的分子病理过程提供重要线索。目前为止，Notch3下游miRNA表达变化未见报道。在前期实验中，我们在CADASIL病例和Notch3-突变鼠里发现miR-181a表达升高及其靶基因（Kcnmb1、Lrrfip1）表达下降的现象；而且有证据提示Notch3-RBPJ通路对miR-181a可能有负调控作用。在此基础上，我们一方面将深入研究Notch3-RBPJ通路调控miR-181a的分子机制；另一方面将在活体动物体内解析miR-181a及其靶基因对血管功能的调节作用；最后观察miR-181a阻断剂能否减轻CADASIL模型小鼠的疾病表型。本项目将为CADASIL提供新的分子治疗策略。

我们采用差异表达RNA的策略，初步筛选得到了Cadasil相关的差异表达7639个mRNA与49个miRNA。针对任意差异表达的RNA进行组合，基于斯皮尔曼等级相关，计算得到3284个差异表达mRNA与48个差异表达miRNA之间的5009对miRNA-mRNA关系对，其中度排名前十位的mRNA功能主要富集在三个层面，即信号通路的调控、细胞质，以及剪接。更进一步的，筛选同时具有靶向关系的miRNA-mRNA关系对，构成了包含17个miRNA与31个mRNA之间的31对关系对的，基于表达与靶向关系的Cadasil相关miRNA-mRNA生物标记物网络。mRNA的功能主要富集于神经发生、转录因子活性调控、信号转导、蛋白质转运、染色体定位、亚细胞质定位以及选择性剪接。我们发现miR-181a表达的升高可以增加大鼠MACO 后的脑梗死面积以及神经功能缺损。我们的研究结果表明，基于miRNA与mRNA的表达相关性以及靶向关系分析，可能从新的角度诠释了Cadasil的发病机制，具有重要的理论意义。