YAP/TAZ在小鼠胚胎着床过程中的作用及其机制

Embryo implantation is crucial for the establishment of successful pregnancy in mammals and its mechanism is still unknown. YAP and TAZ are the major effectors of Hippo and Wnt signaling pathways and involved in several physiological processes, such as cell proliferation, differentiation and apoptosis, etc. Based on our previous microarray analysis, the expression levels of YAP and TAZ were significantly up-regulated in mouse uterus at implantation sites than that at inter-implantation sites (our unpublished data), indicating that YAP and TAZ might be closely associated with the process of embryo implantation. However, the function and regulation of YAP and TAZ during embryo implantation have not yet been reported. The project intends to study the expression of YAP and TAZ in mouse uterus during the peri-implantation, and the effects of YAP and TAZ on the proliferation and apoptosis of mouse uterine cells by taking advantage of early pregnancy, pseudopregnancy, artificial induced decidualization, delayed implantation and other animal models, and using immunohistochemistry, gene overexpression, RNAi technology, etc. to explore the relationships between YAP and TAZ genes and KLF5, BMP2, LIF, VEGF-A genes, etc. associated with embryo implantation so as to reveal the function and mechanism of YAP and TAZ in the process of embryo implantation in mice and provide theoretical basis for giving further clarification to the molecular mechanism of embryo implantation, the improvement of animal breeding rate and the treatment of infertility.

胚胎着床是哺乳动物妊娠建立过程中的一个关键环节，其机理十分复杂。YAP和TAZ是Hippo和Wnt信号通路中的主要效应因子，可调节细胞的增殖、分化以及凋亡等生理过程。我们前期基因芯片分析发现，与非着床位点相比，YAP和TAZ在小鼠子宫胚胎着床位点的表达显著上调，提示YAP和TAZ可能与小鼠胚胎着床过程密切相关。关于YAP和TAZ在胚胎着床过程中的功能与调节目前尚未见报道。本项目拟利用早期妊娠、假孕、人工诱导蜕膜化、延迟着床等动物模型，采用免疫组织化学、基因过表达、RNAi等技术，研究YAP和TAZ在小鼠胚胎着床期子宫中的表达规律及其对小鼠子宫内膜细胞增殖、凋亡等的影响，探讨YAP和TAZ与胚胎着床相关基因KLF5、BMP2、LIF、VEGF-A等的关系，揭示YAP和TAZ在小鼠胚胎着床过程中的作用及其机制，为进一步阐明胚胎着床的分子机理、动物繁殖率的提高及不孕症的治疗等提供理论依据。

本研究利用原位杂交、RNA干扰、基因过表达、流式细胞术等技术，研究YAP/TAZ在小鼠围着床期子宫中的表达及其对子宫基质细胞增殖、分化与凋亡的影响，探讨YAP/TAZ与HB-EGF、Bmp2、Wnt4、Rrm2和Foxm1间的相互关系，阐明TAZ在过氧化氢诱导的子宫蜕膜化氧化损伤过程中的保护作用。结果发现，TAZ高表达于子宫蜕膜化细胞中，可促进子宫基质细胞的增殖活性，诱使S期细胞增多，增加Ccnd3和Cdk4的表达；TAZ可增强子宫蜕膜化标志分子Prl8a2和Prl3c1的表达及ALP的活性，提示TAZ在子宫蜕膜化过程中起重要作用。沉默TAZ可减弱HB-EGF对子宫基质细胞增殖与分化的调节。在子宫基质细胞中转染TAZ siRNA后，细胞凋亡率并不发生明显改变。在子宫基质细胞氧化损伤模型中，HB-EGF可减少ROS、超氧阴离子和MDA含量，使mtDNA拷贝数、ATP和线粒体膜电位水平得到恢复，并促进TAZ的表达。进一步研究发现，过表达TAZ可增强SOD、CAT和GPX抗氧化酶及线粒体复合物I和III的活性，提高GSH在子宫基质细胞中的含量及GSH/GSSG比值，使mtDNA拷贝数和ATP水平得到恢复，进而减弱子宫基质细胞的氧化损伤，添加GPX、线粒体复合物I和III抑制剂可阻止TAZ过表达对子宫基质细胞氧化损伤的保护作用；沉默TAZ则呈现出相反的效应，并增强NOX酶活性，添加NOX抑制剂Apocynin后，子宫蜕膜化损伤可得到缓解。同时，YAP高表达于子宫蜕膜化细胞中，抑制YAP可减弱子宫基质细胞的增殖活性，导致S期细胞减少，但增加子宫基质细胞的分化。Bmp2和Wnt4可诱导YAP在子宫基质细胞中的表达，而添加YAP抑制剂Verteporfin或转染YAP siRNA后，Rrm2和Foxm1的表达受到了明显的抑制。研究结果可为进一步探明子宫蜕膜化的调控机理提供依据。