LncRNA GATA6-AS1调控GATA6参与心脏发育的分子机制研究

Congenital heart disease (CHD) is the major non-infectious cause of infant mortality. Its pathogenesis is complex and the etiology for CHD in an overwhelming majority of patients remains unclear. During our preliminary study by bioinformatics analysis and in vitro cell experiments, we identified GATA6-AS1 as a novel heart-associated lncRNA. GATA6-AS1 has a highly correlated expression pattern with several CHD related genes and its knockout can reduce the expression of GATA6. As a clear CHD pathogenic gene, the dose changes of GATA6 contribute to several kinds of severe CHD in human and mice. Based on their sequence characteristics, we speculate that GATA6-AS1 may regulate the expression of GATA6 through the epigenetic modification or competition with miRNA. This project aims to reveal the role of GATA6-AS1 in the heart development by using cardiomyocyte differentiation model combined with gene knockout mice model. By means of transcriptome sequencing, dual-luciferase reporter assay system and RNA pull down, we intend to investigate the regulation mechanism of GATA6-AS1 in heart development. The achievements of this project will elucidate the molecular regulatory network of GATA6-AS1 and also provide new mechanism for lncRNA roles in CHD pathogenesis.

先天性心脏病（CHD）是新生儿死亡最常见的原因之一，主要由心脏发育调控网络异常所致，但其致病机理远未明了。我们前期研究发现lncRNA GATA6-AS1与众多心脏发育关键基因的表达具有高度相关性，其敲除可降低邻近GATA6基因的表达。作为已明确的CHD致病基因，GATA6的剂量改变可引起人及小鼠胚胎心脏发育畸形。根据两者的序列特征，我们推测GATA6-AS1可能通过表观遗传修饰或与miRNA竞争结合调控GATA6的表达，进而参与心脏发育过程。本项目拟通过人心肌细胞诱导分化模型和小鼠基因敲除模型，以GATA6-AS1为切入点，利用转录组测序、双荧光素酶报告系统及RNA pull-down结合质谱检测，探讨GATA6-AS1调控GATA6的作用机制，阐明GATA6-AS1对心肌细胞分化及心脏发育的影响。本项目的研究将揭示GATA6-AS1的分子调控网络，为CHD致病机制研究提供新的理论依据。

基因组非编码区尤其是长链非编码RNA（lncRNA）已被证明在胚胎发育、器官形成等过程中发挥重要的调控作用。我们前期研究发现lncRNA GATA6-AS1与众多心脏发育关键基因的表达具有高度相关性，本项目进一步利用人诱导多能干细胞向心肌细胞分化模型和基因敲除小鼠模型，系统地研究了GATA6-AS1在心肌分化和心脏发育中的作用。本项目主要研究成果如下：（1）在HEK293细胞中进行了GATA6-AS1序列的敲除，结果显示GATA6-AS1敲除可降低邻近GATA6基因的表达，RNA-seq结果显示，GATA6-AS1序列敲除后的差异基因涉及到心血管系统发育及循环系统调节中的多条通路。（2）利用慢病毒感染，我们进一步构建了GATA6-AS1基因敲减iPS细胞系，在iPSC向心肌细胞诱导分化模型分析中，我们发现基因敲减组iPSC向心肌细胞的分化效率明显低于对照组，GATA6-AS1敲减影响了iPSC向心肌细胞的分化过程。（3）我们利用CRISPR-CAS9的方法在小鼠Gata6os第二外显子前段插入polyA片段，以期在不影响Gata6编码序列的基础上实现该lncRNA的敲除，结果显示，片段插入小鼠Gata6os表达明显减低，基因敲除小鼠构建成功，同时繁育结果显示，部分Gata6os完全敲除小鼠生长发育受限。.本项目已发表标注SCI论文3篇，正在撰写的SCI论文1篇，并培养了1名博士研究生。本项目的研究，进一步阐明了lncRNA GATA6-AS1的作用机制，为心脏发育的机制研究提供参考。