长链非编码RNA uc.457致胚胎心脏发育畸形的机制研究

Uc.457 is a novel long noncoding RNA (lncRNA) of unknown function that is conserved in multiple species,and is highly expressed in myocardial tissue of fetuses with heart malformation identified by lncRNA microarray. Our previous studies found that: ①Overexpression of uc.457 inhibited cardiac differentiation of P19 cells;② Overexpression of uc.457 in zebrafish embryos induced cardiac malformation; ③Bioinformatic analyses and experiments suggested that HIRA was a putative uc.457 target gene. These results suggest that uc.457 may induce embryonic heart malformations by regulating HIRA gene. Based on these data,we propose to analyze the temporal and spatial expression pattern of uc.457 during cardiac differentiation and development, and investigate the function of uc.457 using gene overexpression and gene silencing technology in P19 cells and zebrafish. In addition, we take HIRA as the clue and analyze the interaction between uc.457 and HIRA,which will explore the molecular mechanism of uc.457-induced cardiac malformations in embryos. construct heart-special uc.457 overexpression transgenic mouse and analyze their phenotype.The effect and mechanism ofuc.457 is clarified in embryonic heart development,which will potentially provide a new target for the prevention and treatment of congenital heart disease.

人uc.457是我们采用芯片发现的、在不同物间高度保守、高表达于人发育异常胚胎心脏中、功能未知的lncRNA。前期发现：①过表达uc.457显著抑制P19细胞向心肌细胞分化；②过表达uc.457可致斑马鱼出现心脏畸形表型；③生物信息学与实验提示HIRA可能为uc.457调控靶标。本研究拟分析:uc.457在心脏发育中的时空表达规律；采用基因过表达/沉默技术，在P19细胞、斑马鱼中探讨uc.457对心肌细胞功能和心脏发育的影响；以HIRA为线索，分析uc.457与HIRA的相互作用，论证uc.457通过调控HIRA致胚胎心脏发育异常的机制；最后通过构建心脏特异性过表达uc.457转基因鼠，并进行表型分析。本研究具有源头创新性，揭示人uc.457这一未知功能lncRNA在胚胎心脏发育中的作用与机制，可为先天性心脏病早期防治提供新的干预靶标。

人uc.457是我们通过lncRNA芯片技术筛选到的、在脊椎动物中高度保守的、在发育异常胚胎心脏组织中高表达的lncRNA。本课题在前期预实验研究基础上，提出uc.457异常高表达通过调控HIRA致心脏发育异常的新机制。主要研究结果如下：.1）uc.457在斑马鱼胚胎发育过程中（4hpf开始至心脏发育基本成熟48hpf），随着时间的推移，表达量呈现逐渐升高的趋势；在P19细胞，心肌细胞诱导分化过程中其表达量逐渐减低，细胞内uc.457表达定位于细胞质中。.2）①过表达uc.457导致斑马鱼出现心包水肿，心房、心室位置异常，影响心房、心室的分化，且利用胚胎整体原位杂交的方法表明uc.457过表达、HIRA的表达减弱。注射最低剂量的12.5 ng/μl uc.457后的胚胎，在48与72 hpf发育时期与对照组相比都存在差异，提示过表达uc.457对心脏功能产生显著影响，②uc.457沉默对斑马鱼胚胎心脏发育没有影响。.3）①uc.457过表达可抑制细胞增殖，促进细胞凋亡，抑制心肌细胞分化进程。②uc.457表达沉默具有促进细胞增殖，抑制细胞凋亡，促进心肌细胞分化进程作用。③uc.457与HIRA相互作用的机制结果显示HIRA可以作为靶基因反向调控uc.457。.4）EF1A1、PKP1有可能在uc.457致胚胎心脏发育畸形过程中发挥作用，值得进一步研究探讨。.5）心脏特异性过表达uc.457小鼠心脏的形态检测结果显示野生型胎鼠心脏结构完整，纯合子胎鼠心结构完整性相对较差，与野生组相比，纯合组的ANP、cTnT、Mef2c、HIRA表达均下调。提示过表达uc.457对小鼠的心脏发育产生了负面影响。