NEDD8介导的neddylation在胚胎植入和蜕膜化中的作用和机制研究

Recurrent implantation failure is a major obstacle in assisted reproductive technology. Improving embryo implantation is an effective way to increase the pregnancy rate. Embryo implantation and decidualization, accompanying with massive stromal cell proliferation and differentiation, are two critical events occurred in the uterus, which directly determine the outcome of pregnancy. NEDD8 is an ubiquitin-like protein, which mediated neddylation to regulate cell proliferation and signal transduction during embryogenesis. Therefore, it would be interesting to explore the function of NEDD8-mediated neddylation in embryo implantation. Our preliminary research indicates that NEDD8 is down-regulated in recurrent implantation failure during embryo implantation. Employing MLN4924, a selective inhibitor of NEDD8-activating enzyme, which can efficiently block neddylation, leading to attenuate stromal cells proliferation and impair decidualization. Animal model shows implantation defect after using MLN4924. Herein, we make a hypothesis that NEDD8 mediated neddylation may affect embryo implantation through stromal cell decidualization. By using in vitro cell culture model and animal model, we are planning to confirm the function of NEDD8 in embryo implantation and to explore the key molecule by RNA sequencing. Understanding the mechanisms of NEDD8 mediated neddylation in embryo implantation is hopeful to provide new diagnosis strategy for the recurrent implantation failures.

胚胎反复种植失败是当前辅助生殖技术中一个亟待解决的难题，提高胚胎着床率是提升妊娠率的有效手段。胚胎着床和蜕膜化是成功妊娠的关键环节，它们直接决定了妊娠结局。类泛素蛋白NEDD8可以介导neddylation参与胚胎发育中的细胞增殖和信号转导等过程，但它在胚胎着床中的具体作用机制尚不明确。我们前期研究发现NEDD8在反复种植失败患者围着床期的子宫内膜中表达降低，使用特异性抑制剂降低NEDD8介导的neddylation后，基质细胞增殖能力降低和蜕膜化阻滞，小鼠胚胎着床数量明显减少。据此我们提出假设：NEDD8通过介导neddylation作用于基质细胞蜕膜化的过程，是胚胎着床所必须的。本项目拟利用人体子宫内膜组织、离体细胞培养和动物模型，进一步明确NEDD8对胚胎着床的影响，进而用转录组测序等手段发现其作用于胚胎着床的关键分子，揭示具体作用机制，有望为反复种植失败的机理提供新的理论依据。

NEDD8介导的neddylation对子宫内膜基质细胞的增殖和蜕膜化是必须的。我们前期研究发现NEDD8在反复种植失败患者围着床期的子宫内膜中表达降低，使用特异性抑制剂MLN4924降低NEDD8介导的neddylation后，我们观察到：基质细胞增殖能力降低和蜕膜化阻滞，细胞骨架结构的改变，细胞周期的异常，p21的异常积聚，细胞衰老的增加。进一步研究，我们发现p21敲降可以部分挽救细胞增殖的异常，有可能是关键分子之一。细胞实验和动物模型证实，抑制NEDD8介导的neddylation导致子宫内膜基质细胞增殖能力降低和蜕膜化的异常，这个过程也可能与Wnt4相关。我们构建了CRISPR-cas9敲除的细胞系，在细胞水平验证具体分子机制，同时建立了NEDD8基因特异性敲除的小鼠模型，拟进一步验证细胞实验的结果。课题研究仍在继续进行，以期为反复种植失败的机理提供新的理论依据。