Huang Bo, Zhao Yating, Zhou Lin, Gong Tingyu, Feng Jiawen, Han Peilin, Qian Jianhua
Department of Gynecology and Obstetrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou City, 310003, Zhejiang Province, People's Republic of China.
J Inflamm Res. 2021 Jul 22;14:3489-3500. doi: 10.2147/JIR.S313422. eCollection 2021.
Peptidyl arginine deiminase, type VI (PADI6), a member of the subcortical maternal complex, plays an important role in oocyte growth and the development of fertilized oocytes. Human patients with PADI6 mutations can suffer from multiple reproductive deficiencies including hydatidiform moles and miscarriages. Recent studies have demonstrated that the Hippo signaling pathway plays a central role in the specification of the first cell fates and the maintenance of the human placental trophoblast epithelium. The present study aimed to verify the hypothesis that PADI6 regulates the biological functions of trophoblast cells by targeting YAP1 and to explore the mechanism by which PADI6 accomplishes this in trophoblast cells.
Villi from HMs and human trophoblast cell lines were used to identify the localization of PADI6 and YAP1 by immunohistochemistry and immunocytochemistry. PADI6 overexpression and knockdown were induced in human trophoblast cells. Co-immunoprecipitation was used to explore the interaction between PADI6 and YAP1. Wound healing, Transwell and EdU staining assays were used to detect migration, invasion and proliferation. Flow cytometric analysis was used to analyze the cell cycle and apoptosis. β-Tubulin and F-actin levels were determined by Western blot, quantitative real-time PCR and phalloidin staining.
The results showed that PADI6 and YAP1 had the same expression pattern in villi and colocalized in the cytotrophoblast. An interaction between PADI6 and YAP1 was also confirmed in human trophoblast cell lines. We found that PADI6 positively regulated the expression of YAP1. Functionally, overexpression of PADI6 promoted cell cycle progression and enhanced migration, invasion, proliferation and apoptosis, whereas downregulation of PADI6 showed the opposite effects.
This study demonstrates that YAP1 is a novel target of PADI6 that serves as an important regulator of trophoblast dysfunction. The crosstalk between the Hippo/YAP1 pathway and the SCMC might be a new topic to explore to uncover the pathological mechanisms of HMs.
肽基精氨酸脱亚氨酶6型（PADI6）是皮质下母体复合体的成员之一，在卵母细胞生长及受精卵发育过程中发挥重要作用。携带PADI6突变的人类患者会出现多种生殖缺陷，包括葡萄胎和流产。近期研究表明，Hippo信号通路在人类胎盘滋养层上皮细胞的最初细胞命运决定和维持过程中起核心作用。本研究旨在验证PADI6通过靶向Yes相关蛋白1（YAP1）调节滋养层细胞生物学功能这一假说，并探索PADI6在滋养层细胞中实现此功能的机制。
利用葡萄胎绒毛和人滋养层细胞系，通过免疫组织化学和免疫细胞化学鉴定PADI6和YAP1的定位。在人滋养层细胞中诱导PADI6过表达和敲低。采用免疫共沉淀法探究PADI6与YAP1之间的相互作用。使用伤口愈合实验、Transwell实验和EdU染色实验检测迁移、侵袭和增殖情况。通过流式细胞术分析细胞周期和凋亡。采用蛋白质免疫印迹法、定量实时聚合酶链反应和鬼笔环肽染色法测定β-微管蛋白和F-肌动蛋白水平。
结果显示，PADI6和YAP1在绒毛中具有相同的表达模式，且在细胞滋养层中共定位。在人滋养层细胞系中也证实了PADI6与YAP1之间存在相互作用。我们发现PADI6正向调节YAP1的表达。在功能上，PADI6过表达促进细胞周期进程，增强迁移、侵袭、增殖和凋亡，而PADI6下调则产生相反的效果。
本研究表明，YAP1是PADI6的一个新靶点，是滋养层功能障碍的重要调节因子。Hippo/YAP1信号通路与皮质下母体复合体之间的相互作用可能是探索葡萄胎病理机制的一个新课题。
