MiR-132介导巯基亚硝基化修饰在先天性巨结肠发生中的作用及其调控机制研究

Hischsprung’s disease (HSCR) is a complex genetic disorder caused by congenital defect of the enteric nervous system (ENS), which are derived from neural crest cells (NCCs). MicroRNAs (miRNAs) have been suggested to be involved in the pathophysiology of HSCR. Recent studies have shown that miR-132 modulates radial migration of cortical neurons in embryonic mouse neocortex, and thus may be required for proper neural development. We have recently observed a significant down-regulation of miR-132 in HSCR cases by employing miRNA expression profiling, and have demonstrated that miR-132 regulates NO production and S-nitrosylation (SNO) of proteins in SH-SY5Y cells.Moreover, it has been demonstrated that nNOS-derived NO can S-nitrosylate specific proteins, including ECM (extracellular matrix) proteins, and abnormal S-nitrosylation (SNO) may play a key role in the pathophysiology of the ENS.We will therefore explore the miR-132-mediated regulation of NO-SNO signaling pathway in enteric neural crest cells (ENCCs). Using the CRISPR-Cas9 system, we will generate a miR-132 knockdown mouse model, and ENCCs will thus be isolated from the mouse embryonic guts. We will then examine the SNO levels of ECM (extracellular matrix) proteins in the miR-132 knockdown ENCCs.The colonization and migration of ENCCs upon miR-132 knockdown will be assessed taking advantage of whole-mount immunofluorescence staining and ex vivo time-lapse imaging respectively. Collectively, we will for the first time uncover the miR-132-mediated modulation of S-nitrosylation in the pathogenesis of HSCR.

先天性巨结肠（HSCR）为复杂遗传疾病，源自神经嵴细胞发育异常导致的远端肠段肠神经系统（ENS）缺失。MiR-132可调节神经元径向迁移并影响神经系统发育过程，我们前期以microRNA表达谱首次证实miR-132在HSCR中显著下调，并在SY5Y细胞模型中发现miR-132参与调控NO生成并影响巯基亚硝基化（SNO）修饰水平。已有研究表明NO驱动的SNO修饰可作用于胞外基质（ECM）蛋白并在ENS相关病理过程中起关键作用。故本项目拟借助肠神经嵴细胞（ENCCs），研究miR-132对NO-SNO信号通路的调控机制；并以CRISPR-Cas9系统构建miR-132敲减小鼠模型，分析miR-132对ECM蛋白SNO修饰的调节作用，同时结合离体延时成像等技术探索miR-132敲减对ENCCs定植及迁移过程的影响。本研究将首次从翻译后修饰水平以深度揭示miR-132在HSCR发生中的调控作用。

先天性巨结肠（Hirschsprung disease, HSCR）是由神经嵴细胞发育异常而导致的一类致命性出生缺陷，属于复杂遗传疾病。研究表明包括miR-132在内的多种microRNAs（miRNAs）可通过调控神经元迁移进而影响神经系统发育过程。本课题以HSCR miRNA差异表达谱为基础，剖析相关先天性巨结肠发生的miRNA转录后调控网络，并揭示miR-132等差异表达miRNAs通过调控NO水平及ECM蛋白巯基亚硝基化修饰状态从而改变先天性巨结肠发病进程。我们借助1015例样本（包括502例HSCR散发病例和513例正常对照），首次证实miR-4516及其靶基因MAPK10对HSCR发病风险的影响，并阐释了转录后调控与顺式调控之间的互作在HSCR发病机制中的重要作用。此外，我们在已有研究基础上深度解析HSCR潜在易感基因以及miRNA相关靶基因，并首次在中国汉族人群中揭示RET、ARHGEF3、CTNNAL1、GAL、及GAP43等基因及其互作遗传网络对于先天性巨结肠遗传易感性的影响。同时我们利用HSCR家系样本鉴别出包括编码区DNVs（de novo variants）和亲本嵌合突变在内的多种RET基因常见及罕见遗传突变，并首次揭示HSCR中RET常见突变可影响其罕见无效突变外显率。本课题首次系统地从转录后调控、顺式调控、翻译后修饰、以及基因互作网络等多个层面剖析HSCR发生的分子遗传机制，期望借此可以为先天性巨结肠的早期诊断及临床防治提供新的线索。