miRNA-200a靶向Tfam调控线粒体代谢及影响牛胚胎滋养层细胞命运的研究

During embryo implantation, the proliferation, infiltration, migration, autophagy or apoptosis and so on biological behaviors of trophoblast cells are closely related to the regulation of miRNA. Our previous studies showed that up-regulation of miRNA-200a in bovine embryo derived trophoblast cells resulted in a significant decrease in mitochondrial transcription factor Tfam levels, mitochondrial metabolic abnormalities, arrest of proliferation and migration, and the number of apoptotic cells increased. It was further predicted that there were miRNA-200a binding sites at the mRNA-3'UTR end of Tfam. Preliminarily inferred that during embryo implantation, miRNA-200a targets Tfam and regulates mitochondrial function, and then affects the invasion, migration and functional placenta formation of trophoblast cells. In order to confirm the direct action and key binding sites of miRNA-200a targeting Tfam gene, we established the implantation model between trophoblast cells and endometrial cells in vitro to clarify the role of miRNA-200a in the expression and regulation of Tfam gene. The mechanism of mitochondrial metabolism regulated by miRNA-200a/Tfam was studied on the premise of changing the level of miRNA-200a expression in mitochondria, cytoplasm and whole cells of trophoblast cells, respectively. Based on the changes of mitochondrial metabolism regulated by miRNA-200a/Tfam, the molecular mechanism of its effects on the proliferation, infiltration, migration, autophagy and apoptosis of trophoblast cells will be studied. This project can not only further clarify the mechanism of embryo implantation, but also improve the success rate of assisted reproduction.

胚胎着床早期滋养层细胞浸润、迁移或自噬、凋亡等命运的决定与miRNA调控紧密相关。我们前期研究发现上调牛胚胎滋养层细胞miRNA-200a导致线粒体转录因子Tfam水平显著下降，代谢异常，细胞迁移停止，凋亡增多；并预测到Tfam的mRNA-3ˊUTR端有miRNA-200a结合位点。推测胚胎着床过程中miRNA-200a靶向Tfam调控线粒体代谢，继而影响滋养层细胞的命运。本项目拟利用滋养层细胞体外黏附模型，验证miRNA-200a直接靶向Tfam基因及调控作用；分别改变滋养层细胞线粒体、胞质及全细胞miRNA-200a水平的前提下，研究miRNA-200a/Tfam调控线粒体代谢的机制，明确miRNA-200a/Tfam介导的线粒体代谢对滋养层细胞命运的影响。研究结果将有助于完善胚胎着床的机理，对提高辅助生殖的成功率有重要意义。

着床窗口期的胚胎着床失败是大多数牛被动淘汰的主要原因。在奶牛胚胎着床早期，着床失败或异常着床时的滋养层细胞凋亡率比成功着床的要高70%以上。我们早期采用Illumina Solexa测序技术对牛体内受精胚胎（IVF）和体细胞克隆胚胎（SCNT）来源的滋养层细胞线粒体miRNA分别进行了分析，发现miR-200a可能介导线粒体基因TFAM调控着胚胎着床。因此，本项目的主要目的是对miR-200a靶向TFMM进行验证，解析其调控机制，并阐明miR-200a/TFAM调控下的线粒体代谢影响胚胎滋养层细胞的生物学行为。我们首先分离纯化了奶牛胚胎滋养层细胞和子宫内膜细胞，构建了奶牛胚胎体外着床模型，利用双荧光素酶报告系统验证TFAM为miR-200a的靶基因，同时对miR-200a进行了加强和抑制调节，解析了其调控TFAM基因的机理，并且阐明了miR-200a调控下的线粒体功能变化对胚胎滋养层细胞生物学特性的影响。研究过程中，发现线粒体不但介导胚胎滋养层细胞发生不利于胚胎着床的变化，而且还介导子宫内膜细胞发生一系列的变化，同样不利于胚胎着床，由此，我们筛选到miR-200b是调控子宫内膜细胞炎症反应的关键因子，随后，采用脂多糖（LPS）进行模拟炎症反应，对线粒体基因和胚胎着床相关基因进行检测，发现奶牛子宫内膜细胞发生炎症反应时，miR-200b通过调控线粒体从而不利于子宫内膜细胞的容受性，导致胚胎着床失败。而LPS诱导的炎症反应在生产上常见于高精料饲喂导致的瘤胃酸中毒，由此，我们对规模化奶牛场进行了血液LPS跟踪检测，结果验证了我们的推断，即高精料饲喂导致的LPS浓度升高恰好发生于胚胎着床窗口期，本项目的实施找到了我国奶牛繁殖率低下、利用年限由过去5年缩短到如今2.5年的关键原因，研究结果对提高奶牛情期受胎率具有十分重要的参考意义。