lncRNA-uc.385影响神经嵴细胞迁移致胚胎心脏发育畸形的机制研究

Based on the technology of lncRNA microarray, we identified a novel lncRNA-uc.385 which is highly conversed among different species and pathologically upregulated in myocardial tissue of fetuses with heart malformation. We previously found that uc.385 is highly expressed in the neural tube during embryogenesis in mice. We also found that the overexpression of uc.385 could cause cardiac malformation in zebrafish embryos together with affecting the migration of neural crest cells. Bioinformatics and preliminary data support the candidate downstream target of uc.385 could be meis2, which is related to neural crest cell development and congenital heart disease (CHD) according to the published data. We found that meis2a is downregulated in zebrafish embryos overexpressed with uc.385. Since neural crest cell migration is one of the key steps during vertebrate heart formation, our previous data indicates uc.385 could transcriptionally regulate meis2, and this regulation plays an important role in neural crest cell migration and cardiac development. Our study will focus on further clarifying the role of uc.385 in neural crest cell migration and cardiac development. Also, we will put effort on uncovering the detailed relationship between uc.385 and meis2, and the underlying mechanism controling cardiac development and CHD. Taken together, our study will uncover the mechanism how the novel lncRNA uc.385 is involved in the cardiac development and CHD, which will provide us a new bio-target in the prevention and treatment of fetal cardiac malformations.

lncRNA-uc.385是我们基于芯片技术筛选获得的、一条差异高表达于人发育异常胚胎心脏组织中、多物种间高度保守、功能未知的lncRNA。前期发现, uc.385主要在小鼠胚胎神经管中高表达；过表达uc.385不但可致斑马鱼胚胎心脏发育畸形，而且也显著减弱神经嵴细胞的迁移；软件预测及初步实验提示与神经嵴细胞发育相关的meis2为其潜在靶基因；斑马鱼胚胎中过表达uc.385显著降低meis2a的表达；而神经嵴细胞迁移是心脏发育的重要事件，提示uc.385可能通过转录调控meis2的机制、影响神经嵴细胞迁移而致心脏发育畸形。本研究拟进一步深入分析uc.385在神经嵴细胞迁移和心脏发育中的作用；并以meis2为线索，论证uc.385与meis2间的相互作用及影响心脏发育的机制。揭示uc.385这一功能未知lncRNA的作用与机制，无疑将为胚胎心脏发育畸形的防治提供新靶标。

先天性心脏病 （先心病）是最常见的一类先天性畸形，但其发病机制，特别是lncRNA在其中的作用，目前尚不明确。因此，本课题对前期工作中通过基因芯片筛选所得的uc.385以及后续通过RNASeq筛选所得的NONDRET002342.2（lncRNA1）和NONDRET003632.2（lncRNA2）进行研究。我们发现lncRNA1和lncRNA2在先心病胎儿心脏组织中的差异表达稳定性优于uc.385。我们采用了Crispr-dCas9的方法，在斑马鱼胚胎上成功实现了内源性lncRNA1和lncRNA2的敲降，敲降后胚胎发育在50hpf时出现畸形和部分发育停滞现象。通过对敲降的不同时期胚胎进行转录组测序，我们发现了lncRNA1及lncRNA2敲降所影响的下游基因以及相关信号通路。这些结果初步揭示了lncRNA1和lncRNA2在心脏发育中所起的作用及相应的机制。为了扩展对先心病机制的理解，我们尝试了通过液相色谱联合串联质谱技术探索先心病特异性的内源性多肽并做了初步的分析。此外，为了更好的研究先心病，我们建立了基于先心病患者来源iPS的疾病模型体系，以及基于靶向捕获测序的先心病产前诊断体系。这些结果使得可以更好的认识先心病的机制，研究潜在的防治方法。