LincRNA-Hrt在胚胎干细胞向心肌细胞分化过程中的分子机制研究

The mechanism research of cardiac defect plays important roles in the area of cardiac development. Our recent study found that the expression of lincRNA-Hrt, high-expressed in the tissue of fetal heart, was up-regulated during the ES differentiation. Further study found that downregulation of lincRNA-Hrt could significantly inhibit the differentiation ability of ES cells to cardiomyocytes. The preliminary data showed that lincRNA-Hrt might be involved in the transcriptional regulation of key molecules in the early stage of cardiomyocyte differentiation. Therefore, this project tried to systematically research on the molecular and epigenetic mechanisms of lincRNA-Hrt involved in the cardiomyocyte differentiation with molecular techniques such as RNA pull-down, RIP and ChIP. Furthermore, we would establish cardiac-specific conditional knockout mouse to determine whether lincRNA-Hrt participated in the regulation of heart development. According to the preliminary data and characteristic of lincRNA, we speculated that lincRNA-Hrt might regulate the cardiac differentiation through forming the RNA-transcription factors-epigenetic enzymes complex. The research of regulatory complex is not only conducive to develop a better understanding of gene regulatory network during the cardiac differentiation and provide more molecular cues and theoretical basis for the research of heart development, but also is beneficial for the study of cardiac development defect.

心脏发育缺陷的机理研究已成为当今研究热点。我们最近发现lincRNA-Hrt在ES细胞分化中表达上调且在胚胎心脏中高表达，进一步研究发现干扰ES细胞中的lincRNA-Hrt使ES细胞分化成心肌细胞的能力受到明显抑制。已有结果还显示lincRNA-Hrt可能参与了心肌分化早期关键转录因子的转录调控。为此，本课题拟利用RNA pull-down、RIP、ChIP等技术，系统研究lincRNA-Hrt调节ES细胞心肌分化的分子和表观遗传机制，并构建心肌特异性敲除小鼠，进一步阐明lincRNA-Hrt在心肌发育过程中的功能。根据已有结果和lincRNA的作用特点，我们推测lincRNA-Hrt主要通过形成RNA-转录因子-表观修饰酶等蛋白复合物参与心肌分化的调控。这些复合物的揭示不仅有助于理解心肌分化的分子调控网络，为研究心脏发育过程提供更多线索和依据，对干预心肌发育异常相关疾病也具有潜在价值。

心脏发育缺陷的机理研究已成为当今研究重点，而ES细胞体外培养的成功极大促成了体外研究心脏器官的发生发育过程，这将更有助于理解心肌细胞分化发育的机制、认识心脏疾病的发生机理等。目前，对于心肌细胞分化过程中是否还存在其他重要因素参与调控心肌细胞分化的核心网络，并且如何从分子水平影响心肌分化过程还有待更多的研究。LincRNA已被公认在调控ES细胞的命运中具有关键作用。但是到目前为止，绝大多数lincRNA的功能和作用机理仍然是未知的，有待进一步探究。在本项目执行期间，不仅顺利完成了项目任务书既定计划和任务，还在多个方面取得了重要的研究工作进展，具体成果总结如下：1）首次发现了lincRNA-Hrt联合转录因子和表观修饰子调控心肌分化和心脏发育的重要表观遗传机制；2）首次发现了linc1281上的m6A修饰具有维持多能干细胞特性和合适心肌分化潜能的关键作用，拓展了对“RNA表观遗传”领域的认知；3）发现了小鼠ES细胞中linc1614通过联合转录因子以及表观修饰复合物PRC2精确调控基因转录和ES细胞早期分化起始的重要机制；4）揭示了lncRNA LincU通过介导ERK信号活性调控方式，维持ES细胞原始态的重要机制；5）首次揭示了辅助因子Sin3a可通过联合DNA羟甲基化酶Tet1形成转录激活机器，调控ES细胞的分化全能性，提出了Sin3a结合特定表观修饰分子促进基因转录表达的崭新模式。