PLAC8在滋养层细胞侵润和迁移中的作用及其分子机制

The dysregulation of differentiation and function of extravillous trophoblast (EVT) may lead the malignant pregnancy outcomes, such as the preeclampsia. Finding specific markers of EVT is essential to understand its appropriate differentiation and the typical invasion characteristic. In the preliminary study, we found that Placenta Specific 8 (PLAC8) was exclusively expressed in EVT at human fetomaternal interface during pregnancy by immunofluorescent assay, and the expression of PLAC8 was significantly elevated along with the reinforcement of EVT invasion phenotype. PLAC8 was co-localization with actin filaments of EVT. Furthermore, the phosphorylation level of Cofilin 1, a key regulator of actin dynamic, was significantly increased by PLAC8 over-expression. These data suggest PLAC8 plays an important role in the regulation of EVT invasion and migration..In the present study, the in vitro trophoblast cell models and tissue culture models with PLAC8 overexpressed, silenced and Plac8 knockout in mouse trophoblast stem cell (TS) will be employed to verify the biological function of PLAC8 in regulation of EVT invasion and migration. Methods of Co-IP, Western blot and immunofluorescent assay (IF) will be applied to clarify the molecular mechanisms of PLAC8 in inducing the phosphorylation of Cofilin 1. Furthermore, he relationship between PLAC8 and preeclampsia will be investigated through primary EVT isolation, Western blot and immunofluorescent assay. This project will shed new light on understanding the molecular mechanisms of EVT invasion and migration and provide a new clue for the prediction and clinical treatment of preeclampsia.

绒毛外滋养层细胞（EVT）分化和功能异常将导致子痫前期等不良妊娠结局，发现新的EVT特异性分子标记对于理解其分化和侵润特性具有重要意义。我们前期研究结果显示，胎盘特异性基因8（PLAC8）在EVT特异性表达，并随着EVT侵润表型增强而表达升高；PLAC8在EVT与肌动蛋白纤维共定位，并促进肌动蛋白纤维关键调控蛋白Cofilin 1的磷酸化。本项目将采用多种细胞和组织模型，通过过表达、下调和完全敲除Plac8基因，确定PLAC8在EVT侵润和迁移中的生物学功能；结合免疫共沉淀和免疫荧光等技术，研究PLAC8促进Cofilin 1磷酸化的分子机制；并进一步以子痫前期患者胎盘为研究对象，采用原代EVT分离、Western blot和免疫荧光等技术，探讨PLAC8与子痫前期的相关性。该项目的实施对于进一步阐明EVT侵润和迁移的分子机制具有重要的学术价值，也将为子痫前期的预测和临床干预提供新的思路。

人胎盘中滋养细胞的适当分化是成功妊娠的先决条件，而这一过程的失调可能导致不良妊娠结局，如子痫前期等。寻找不同类型滋养细胞的特异性标记物是了解滋养细胞分化的基础。我们报道胎盘特异性蛋白8 (PLAC8)在人母胎交界面的间质外滋养细胞(iEVTs)中特异性表达。利用胎盘微绒毛-蜕膜共培养、原代滋养细胞或早期妊娠锚定绒毛外植体诱导iEVTs等模型系统，我们发现PLAC8促进了iEVTs的侵袭和迁移。经活细胞工作站平台延时拍摄成像、GTPase活性测定、co-IP和RNA-seq研究表明，PLAC8增加了Cdc42和Rac1的活性，并进一步诱导了迁移中的滋养细胞前缘丝状足的形成。更有趣的是，在缺氧条件下，PLAC8明显上调，子痫前期胎盘与正常对照胎盘相比，PLAC8在iEVTs中的表达更高。总之，PLAC8是一个我们在经典的HLA-G这一EVT分子标记之外新发现的另一个重要的分子标记，也是滋养层细胞研究领域目前报道发现的唯一的iEVTs分子标记，并在促进滋养层入侵和迁移方面、维系母胎界面iEVT功能稳态方面发挥重要作用。PLAC8的发现对于后续滋养层细胞分化研究领域的细胞谱系追溯，及进一步深入了解母胎界面滋养层细胞的功能，具有重要学术价值。