一条与心脏发育畸形相关lncRNA-uc.4的功能与机制研究

Uc.4 is a novel long noncoding RNA (lncRNA) of unknown function that is conserved in multiple species (e.g.,human,rat,mouse,zebrafish),and is highly expressed in myocardial tissue of fetuses with heart malformation identified by lncRNA microarray. We previously found that overexpression of uc.4 inhibited cardiac differentiation of P19 cells,and overexpression of uc.4 in zebrafish embryos induced cardiac malformation. Furthermore, bioinformatic analyses and experiments suggested that CASZ1 was a putative uc.4 target gene. CASZ1 gene showed lower levels in myocardial tissue of fetuses with heart malformation, CASZ1 knock-out mice were reported to display cardiac malformations. These results suggest that uc.4 may induce embryonic heart malformations by regulating CASZ1 gene. Based on these data,we propose to analyze the temporal and spatial expression pattern of uc.4 during cardiac differentiation and development, and investigate the function of uc.4 using gene overexpression and gene silencing technology in P19 cells and zebrafish. In addition, we take CASZ1 as the clue and analyze the interaction between uc.4 and CASZ1,which will explore the molecular mechanism of uc.4-induced cardiac malformations in embryos. The effect and mechanism of uc.4 is clarified in embryonic heart development,which will potentially provide a new target for the prevention and treatment of fetal congenital heart disease.

uc.4是我们通过芯片筛选得到的一条在不同物种间高度保守、且高表达于人发育异常胚胎心脏中、功能未知的lncRNA。前期发现：uc.4过表达可致斑马鱼胚胎心脏发育畸形；uc.4过表达显著抑制P19细胞向心肌细胞分化；生物信息学分析与初步实验提示CASZ1可能为uc.4的调控靶标；CASZ1低表达于人发育异常胚胎心肌组织中，而文献报道CASZ1是维持心脏发育的关键基因之一，提示uc.4可能通过调控CASZ1致胚胎心脏发育畸形。本研究拟探索uc.4在心脏发育中的时空表达规律；以基因过表达/沉默技术，分别在P19细胞、斑马鱼中探讨uc.4对心肌细胞功能和心脏发育的影响；以CASZ1为机制主线，分析uc.4与CASZ1的相互调控作用，论证uc.4通过调控CASZ1致胚胎心脏发育异常的机制。阐明uc.4在胚胎心脏发育中的作用与机制，为先天性心脏病的基因防治提供新的靶标。

uc.4是我们通过芯片筛选得到的一条在不同物种间高度保守、且高表达于人发育异常胚胎心脏中、功能未知的lncRNA。前期发现：uc.4过表达可致斑马鱼胚胎心脏发育畸形；uc.4过表达显著抑制P19细胞向心肌细胞分化；生物信息学分析提示CASZ1可能为uc.4的调控靶标。通过本课题的研究，我们发现：①过表达lncRNA uc.4可显著抑制P19细胞向心肌细胞的分化，而对细胞的增殖、凋亡、周期无影响；②过表达lncRNA uc.4可显著抑制斑马鱼心脏的发育，出现心率减慢、心包水肿、室壁变薄等；③在uc.4稳定表达株诱导分化的第0天，第4天，第8天，第10天分别验证CASZ1在分化过程的变化，结果显示CASZ1在分化过程中无明显差异；④转录组芯片结果提示lncRNA uc.4可能通过TGF-β通路影响心脏发育。综上，通过本课题的研究，我们系统阐明了uc.4在心脏发育中的功能与机制，为先天性心脏病的防治提供潜在的干预靶标。