MARVELD1调控干细胞分化与体细胞重编程EMT/MET的机制研究

The transition of epithelial cells into motile mesenchymal cells, a process known as epithelial–mesenchymal transition (EMT), plays important roles in individual development and cancer progression. As the key stage of stem cell differentiation and somatic cell reprogramming, the better understanding of its molecular mechanisms is important. Our previous studies found that MARVELD1 regulated the migration and invasion of mouse trophoblast cells. In MARVELD1 knockout mice, we observed the abnormal EMT of mouse trophoblast cells, and indeed, the pregnant mice had a partial placenta accreta phenotype. More importantly, in mouse fibroblasts, MARVELD1 expression level was much higher than that of mouse embryonic stem cells. The knockout of MARVELD1 enhanced the efficiency of mesenchymal-epithelial transition during somatic cell reprogramming. Here, we attempt to clarify the roles of MARVELD1 in EMT/MET during stem cell differentiation and somatic cell reprogramming by using MARVELD1 knockout mice, as well as the in vitro mice embryonic stem cell differentiation and pluripotent stem cells induction through molecular cell biology and bioinformatics approaches. This project will help us to understand the molecular mechanisms of MARVELD1 mediated EMT/MET during stem cell differentiation and somatic cell reprogramming. Furthermore, it will provide more experimental basis to understanding MARVELD1 gene function in cell fate determination.

上皮间质转化（EMT）在个体发育及肿瘤发生发展过程中发挥重要作用，是细胞干性转化的重要环节，其机制研究具有重大意义。申请人前期研究发现MARVELD调控细胞的迁移运动能力，其敲除引发小鼠胎盘滋养层细胞的过度EMT，继而造成敲除孕鼠胎盘植入的难产表型。进一步研究发现，该基因在小鼠胎儿成纤维细胞中的表达显著高于其在小鼠胚胎干细胞中的表达，且敲除后促进体细胞重编程的MET过程。为此，本项目拟以小鼠胚胎干细胞的分化及胎儿成纤维细胞的重编程过程为切入点，基于MARVELD1敲除鼠模型、ES细胞体外分化系统及多潜能干细胞诱导体系，采用分子细胞生物学结合生物信息学的研究方法，探究MARVELD1调控EMT/MET的分子机制，揭示该基因在细胞干性转化中的调控作用。为深入认识EMT/MET的调控机制，解析体细胞重编程的分子机理奠定基础，进而为探讨MARVELD1在细胞命运决定中的生物学意义提供新线索。

在个体发育过程中，构成生命体的细胞经历着“命运决定”的事件。命运决定事件贯穿植入前胚胎发育的编程与重编程、干细胞的分化、细胞上皮态与间质态间的转化、细胞的衰老与恶性转化等过程。染色质组装、组蛋白乙酰化及甲基化修饰、DNA甲基化等影响着转录因子对基因的时空特异性调控，进而制约细胞的命运抉择，最终影响个体发育、衰老与恶性转化。为此，基于上述调控机制的基因功能研究具有重大意义。.MARVELD1为课题组发现的新核因子，前期研究提示该基因在细胞命运决定过程发挥作用，为此，本项目利用基因敲除鼠、体外上皮间质转化模型，基于干细胞分化和体细胞重编程体系，采用分子细胞生物、遗传学、生物信息学、动物生理学的研究手段，从组蛋白甲基化修饰、通路调控、细胞基本生物学特性角度，探究MARVELD1对细胞转化过程中的编程与重编程的调控机制。.本项目通过构建MARVELD1全身敲除小鼠，发现敲除鼠产仔数量显著低于对照野生小鼠，而数量减少发生在出生后；进一步发现MARVELD1的敲除导致胎儿难产、胎盘植入的表型；通过对机制的研究，发现MARVEDL1正向调控ITGB4，进而影响胎盘滋养层细胞的侵袭能力，致使胎盘植入，最终导致难产。在敲除鼠表型的基础上，进一步通过建立体外TGB-β1诱导的细胞侵袭模型，发现MARVELD1调控细胞H3K27me3修饰，进而抑制上皮间质转化（EMT）进程；同时发现MARVELD1的敲除显著提升MEF细胞的重编程效率，MARVELD1通过调控H3K4me3/H3K27me3修饰，进而影响Klf4因子与其靶序列的结合，实现其对于MEF重编程过程中间质上皮转化（MET）进程的调控。.本项目为MARVELD1基因在细胞命运决定过程中的功能研究奠定了基础，同时MARVELD1-H3K4me3/H3K27me3-Klf4-MET调控主线的建立，为EMT/MET在细胞命运决定中的生物学意义提供了新线索。