印记基因lncRNA-MEG8在胎盘早期发育和自然流产中的作用和机制研究

Unexplained spontaneous abortion is the commonest gynaecological emergency and is difficult to prevent and control. The early placental development is very important to the maintenance of pregnancy. The key processes of placenta implantation and formation are associated with the invasion of extravillous trophoblast cells. But the key cytokines and signaling pathways regulating the proliferation and invasion of trophoblasts are still poorly undertood.It has been found that the impairment of the methylation of the imprinting gene affects the function of the placental trophoblast cells. Long noncoding RNA especially imprinted lncRNA participates in the development of placenta through epigenetic regulation. In our previous studies, we have investigated the key role and mechanism of the imprinting gene lncRNA-H19 in the early development of placenta.Then we compared the gene expression profile of mouse placenta during the early, middle and late stages. We found that the expression of imprinted and lncRNA-Rian increased with the development of placenta. The homologous MEG8 are highly expressed in the human villous tissues of unexplained spontaneous abortion. We also found that the high expression of MEG8 can decrease the proliferation and invasion of human placental trophoblast cells. There is abnormal methylation in the promoter region of lncRNA-MEG8 in the human villous tissues of unexplained spontaneous abortion. Therefore, we conclude that lncRNA-MEG8 is involved in regulating the function of early trophoblast cells, and the aberrant methylation of imprinted gene promoter region may be one of the mechanisms of early placental dysplasia. In this proposal, we will further investigate the potential role and mechanism of lncRNA in the early placental development. We will show the multiple epigenetic network of genetic imprinting, DNA, RNA and methylation, and provide new ideas for studying the mechanism of placental development.

早期自然流产是常见不良妊娠结局之一，严重威胁母婴健康。胎盘滋养细胞的发育和功能是维持正常妊娠的关键。研究发现，印记基因甲基化异常影响胎盘滋养层细胞的功能，而长链非编码RNA特别是印记性lncRNA通过表观遗传调控胎盘发育的作用逐渐受到关注。我们的前期研究通过对比分析小鼠孕早、中、晚期胎盘的lncRNA基因表达谱及甲基化状态，发现印记基因lncRNA-Rian随胎盘发育表达量逐渐升高，而启动子甲基化水平降低。其人的同源基因MEG8在人自然流产绒毛组织中异常高表达。过表达MEG8可以降低胎盘滋养层细胞的增殖和侵袭能力。因此我们推测lncRNA-MEG8参与调控胎盘早期发育和自然流产的发生。本课题将在前期工作的基础上，以lncRNA-MEG8为切入点，对遗传印记、DNA甲基化等表观遗传学网络进行系统研究，探索胎盘早期发育和自然流产的机制，为自然流产的防治提供新的思路和干预靶点。

长链非编码RNA（lncRNA）是一类转录本长度大于200nt的非编码RNA。既往的研究表明它们在发育过程和生物学途径中发挥重要的调节作用，但是lncRNA对胎盘发育的影响尚不明确。本研究旨在通过lncRNA芯片筛选技术对胎盘发育过程中的lncRNA表达特征进行分析，找寻关键性lncRNA。项目组前期对不同妊娠时间的小鼠胎盘进行lncRNA芯片筛选，发现了一种可能在胎盘发育过程中发挥重要作用的lncRNA，即lncRNA-Rian，随后项目组发现它的同源序列lncRNA-MEG8在人类自发性流产绒毛组织中异常高表达，且调节lncRNA-MEG8的表达会影响人滋养层细胞的增殖和侵袭能力，DNA甲基化检测的结果显示lncRNA-MEG8启动子区域的甲基化在人类自然流产绒毛组织中增加，这表明lncRNA-MEG8参与早期滋养层细胞功能调控，其启动子甲基化异常可导致滋养细胞功能缺陷，这可能是是早期不明原因的自然流产的因素之一。随后项目组对人类子痫前期胎盘组织和正常妊娠胎盘组织进行lncRNA芯片筛选分析，发现了一种表达异常升高且与缺氧诱导因子（HIF-1α）通路密切相关的lncRNA，即lncRNA—HEIPP。缺氧诱导时，人滋养层细胞lncRNA-HEIPP表达量升高，染色质免疫共沉淀检测发现缺氧环境下lncRNA-HEIPP启动子区域的H3K4me3丰度明显增高，组蛋白修饰可能是缺氧时子痫前期胎盘lncRNA-HEIPP表达量增加的表观遗传学调控方式。而在人滋养层细胞中下调lncRNA-HEIPP表达，细胞侵袭能力明显增强。H3K4me3介导的缺氧环境下滋养层细胞lncRNA-HEIPP表达异常可能是子痫前期病理性胎盘发育的因素之一。