LncSync调控心血管系统发育的机制研究

Non-coding RNAs have diverse biological functions. In our previous research, we identified a long non-coding RNA, LncSync, which was initially highly expressed in E8.5 and 9.5 mouse embryonic hearts, then became enriched in the heart and placenta. This suggests that it may play important functions in the coordination of cardiovascular tissue growth and differentiation in the heart and placenta. Interestingly, LncSync is the host gene of several microRNAs that potentially regulate cell proliferation and migration. Moreover, it also contains the target sequence of several microRNAs enriched in cardiovascular tissues. We hypothesize that LncSync may form a feedback loop with microRNAs to coordinate the growth of the circulation system in mammalian embryos. In this study, we will dissect the relationship between LncSync and its related microRNAs in the cell culture system. We have generated LncSync knock-out mouse and embryonic stem cells to find out its function during development and cardiomyocyte differentiation. We also established a novel in vitro system to culture E8.0 embryos within the yolk sac, where they can develop further and maintain blood circulation and heart function. We will transfect LncSync and its related microRNAs in in vitro cultured wild type and LncSync knock-out embryos and investigate how this may affect the growth of heart, yolk sac and placenta blood vessels. This research may reveal a novel mechanism where LncRNA and microRNAs form feedback regulatory network to ensure the synchronized growth of the cardiovascular system throughout the embryo. Moreover, LncSync may also be used to promote cardiomyocyte in vitro differentiation which represents a novel method to produce useful cell types for regenerative medicine.

非编码RNA在心血管系统的发育过程中起重要作用。在前期研究中，我们发现一个长链非编码RNA， LncSync，在小鼠胚胎发育第9.5天在心脏中高表达, 之后在胚胎心脏和胎盘特异高表达。 LncSync包含多个可调控细胞增殖和迁移的前体microRNA的序列，同时也含有miR-181等心血管系统高表达的microRNA的靶序列，因此LncSync可能与microRNA形成一个反馈调控系统，精确控制哺乳动物胚胎、卵黄囊和胎盘循环系统的谐调发育。在本课题中，我们将利用LncSync敲除小鼠和胚胎干细胞，结合过表达、体外胚胎培养和心血管细胞培养、功能分析等手段，揭示LncSync调控心血管系统发育的机制以及在多能干细胞向心肌细胞分化中的作用。

本研究发现了一个位于X染色体上，在胎盘动物中保守的长非编码RNA：LncSync。LncSync在小鼠胚胎心脏组织高表达。 LncSync基因敲除后，小鼠E13.5天胚胎心肌和出生后8周的心肌细胞转录组均出现了肥厚型心肌病和铁死亡相关基因的显著富集。成年小鼠到6月后，发生左心室肥厚、心肌细胞体积增大、心脏纤维化等典型病理性心脏肥大表征。LncSync转录本可以产生包括miR-351，miR-503和miR-322的miR-351簇microRNA。我们发现LncSync通过miR-351簇促进小鼠胚胎干细胞体外的心肌分化。LncSync缺失则引起miR-351的靶基因Hddc3的上调。已有报道提出Hddc3能促进铁死亡的发生。我们检测到LncSync敲除小鼠心脏内铁死亡终产物丙二醛及相关标记基因Ptgs2显著升高，标志着心脏中确实发生了铁死亡，并继而引起心脏损伤。LncSync还包含miR-181靶序列。我们证明miR-181可可以降解LncSync从而调控miR-351簇的表达。基于以上研究结果，我们提出一种新颖的长非编码RNA-microRNA 调控模型。长非编码RNA作为重要代谢酶的“变阻器”, 而这一变阻器又收到上游microRNA的调控。这一调控体系对维持心肌细胞代谢平衡和稳态起重要作用。敲除LncSync后，会导致心肌细胞稳态失调，继而发展成病理性心脏肥大。这一研究为理解造成心脏肥大的分子机制提供了新的思路，同时解释了导致心肌铁焦亡的新机制， 并对如何更好的维持心脏稳态平衡，减轻心肌铁焦亡的产生提供了新的思路和实验依据。