长链非编码RNA Fendrr对心肌肥厚的作用及机制研究

Cardiac hypertrophy is the common pathophysiological process of a wide variety of cardiovascular diseases that ultimately leading to heart failure. However, no effective treatments exist for the process. Recently, it has been confirmed that long noncoding RNA (LncRNA) play an important role in the biological process of development, differentiation and growth. Fendrr, one member of the LncRNA family, was involved in cardiac development and differentiation. Our preliminary data indicated that the expression of lncRNA Fendrr in myocardial tissue is down-regulated significantly after 4 weeks' aortic banding in C57 mice, but its functions and mechanisms in this process remains unclear. We aim to observe phenotype changes in vivo through pressure overload, AngII and ISO induced cardiac hypertrophy in wild type, cardiac specific knock-out and transgenic mice, and explore the effect of LncRNA Fendrr on signal transduction, transcriptional activation and embryonic gene re-expression. In addition, we will construct vectors for silence or over-expression of genes regulated by LncRNA Fendrr to transfect the primary cardiomyocytes isolated from cardiac specific knock-out and transgenic mice , to further search for the role and mechanisms of LncRNA Fendrr on cardiomyocyte hypertrophy induced by AngII and ISO. These studies are designed to find out new target for the prevention and treatment of cardiac hypertrophy and heart failure.

心肌肥厚是多种心血管疾病发展至心衰的共同病理生理过程，目前尚无根本有效防治措施。近年来研究显示，长链非编码RNA在生物发育、分化和生长中发挥重要作用，其家族成员Fendrr参与了心脏发育和分化调控过程。我们预实验发现：胸主动脉缩窄术后4周，野生型小鼠肥厚心肌组织Fendrr表达明显下降，提示其参与了心肌肥厚过程。但作用及机制未明。本项目组拟用野生型、Fendrr心脏特异性敲除和转基因小鼠构建压力负荷、血管紧张素II和异丙肾上腺素诱导的心肌肥厚在体模型，观察心肌肥厚表型变化，并探讨Fendrr对信号转导、转录调控和胚胎基因重新表达的影响。同时制作沉默或过表达受Fendrr调控的下游靶基因载体，并转染至心脏特异性Fendrr敲除和转基因小鼠乳鼠原代心肌细胞，从离体水平进一步探讨Fendrr在血管紧张素II和异丙肾上腺素诱导心肌细胞肥大中的作用及机制。旨在为心肌肥厚和心力衰竭防治提供新靶点。

长链非编码RNA Fendrr（LncRNA Fendrr，Fendrr）在心脏发育过程中发挥关键调控作用，Fendrr缺失后小鼠心脏发育不全并在出生前死亡。Fendrr是否在心肌重构的发生发展过程中起关键调控作用尚无研究报道。为了避免Fendrr缺失的致死作用，本研究方案中借助Crisp/Cas9系统构建成年小鼠心肌细胞特异Fendrr缺失小鼠，同时通过转基因技术借助心肌细胞特异性启动子在成年小鼠心肌细胞中特异性高表达Fendrr。在主动脉缩窄诱导的压力负荷模型中，Fendrr特异性缺失保护压力负荷诱导的心肌重构，相反Fendrr特异性高表达则加重压力负荷诱导的心肌重构；超声检测小鼠心功能发现Fendrr缺失可显著改善压力负荷诱导小鼠心功能障碍， Fendrr过表显著加重压力负荷诱导的小鼠心功能障碍；组织病理染色发现Fendrr缺失后显著减轻压力负荷诱导的心肌细胞肥大和心脏间质纤维化，Fendrr高表达显著促进压力负荷诱导的心肌细胞肥大和心脏纤维化；进一步RT-PCR和western-blot检测心脏中Gata6, Nkx2.5, Foxof1, Foxo1 和Foxo3a等一系列转录因子发现， Fendrr缺失后显著促进Gata6的表达， Fendrr高表达则显著抑制Gata6的表达；利用表观遗传学研究分析Gata6启动子的甲基化发现， Fendrr缺失促进Gata6启动子区域H3K4甲基化显著增强且抑制H3K27甲基化，相反Fendrr过表达则促进Gata6启动子区域H3K27甲基化并抑制H3K4甲基化。在分离的原代大鼠心肌细胞中，腺病素转染Fendrr干扰RNA沉默其表达后显著抑制苯紧上腺素（PE）诱导的心肌细胞肥大，而腺病毒过表达Fendrr后显著加重PE诱导的心肌细胞肥大；进一步在LncRNA Fendrr沉默的心肌细胞中转染siRNA沉默Gata6的表达发现，Gatat6沉默后Fendrr的缺失则不抑制PE诱导的心肌细胞肥大。本项目研究发现抑制Fendrr可促进H3K4甲基化并抑制H3K27甲基化从而促进Gata6的表达以改善病理性心肌重构和延缓心力衰竭的发生发展。本研究在体内外阐明了LncRNA Fendrr在心肌重构过程中的作用及作用机制，并指出特异性抑制心肌细胞中Fendrr的抑制是改善病理性心肌重构的潜在策略。