Chen Zhenlie, Zhong Wen, Zhang Ruiqing, Li Guigui, Zhang Yuanzhen, Zhang Ming
Reproductive Medicine Center, Zhongnan Hospital, Wuhan University, No. 169, East Lake Rd., Wuhan 430071, Hubei Province, P. R. China.
Shenshan Medical Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, No. 1, Henger Rd. Shanwei, 516621, P. R. China.
Biol Reprod. 2024 Nov 11;111(5):1142-1155. doi: 10.1093/biolre/ioae122.
Impaired extravillous trophoblast (EVT) invasion and resulted poor placentation play a vital role in the development of preeclampsia (PE). However, the underlying mechanisms of dysregulated EVTs remain unclear. This study aimed to explore the role of poly (C)-binding protein 2 (PCBP2), a multifunctional RNA-binding protein, in the pathogenesis of PE and to investigate the detailed signaling pathway. Using qRT-PCR, western blot, and immunohistochemistry, we confirmed that the expression of PCBP2 significantly decreased in placentas from 18 early-onset PE and 30 late-onset PE in comparison to those from 30 normotensive pregnancies. Besides, more significant suppression of PCBP2 was observed in the early-onset type. After transfection of HTR-8/SVneo with small-interfering RNA specific to PCBP2, the cellular biological behaviors including vitality, immigration, invasiveness, and apoptosis were evaluated by CCK-8 assay, wound-healing assay, transwell assay, and flow cytometry respectively. RNA-seq was applied to screen differentially expressed genes in HTR-8/SVneo upon PCBP2 silencing. GO and KEGG analysis indicated that WNT signaling pathway and the related processes such as extracellular matrix remodeling and cell adhesion were among the most enriched pathways or processes. Meanwhile, the alternative splicing of WNT5A regulated by PCBP2 was also identified by RIP-seq. Based on HTR-8/SVneo and villous explant, the regulatory roles of PCBP2 on trophoblast were confirmed to be mediated by WNT5A. Besides, it revealed that ROR2/JNK/MMP2/9 pathway was a vital pathway downstream WNT5A in trophoblast cells. In conclusion, this study suggests that down-regulated PCBP2 impaired the functions of EVTs via suppression of WNT5A-mediating ROR2/JNK/MMPs pathway, which may eventually contribute to the development of PE.
绒毛外滋养层细胞（EVT）侵袭受损及由此导致的胎盘形成不良在子痫前期（PE）的发生发展中起着至关重要的作用。然而，EVT功能失调的潜在机制仍不清楚。本研究旨在探讨多功能RNA结合蛋白多聚（C）结合蛋白2（PCBP2）在PE发病机制中的作用，并研究其详细的信号通路。通过qRT-PCR、蛋白质免疫印迹和免疫组织化学，我们证实与30例血压正常孕妇的胎盘相比，18例早发型PE和30例晚发型PE胎盘组织中PCBP2的表达显著降低。此外，早发型PE中PCBP2的抑制更为明显。用PCBP2特异性小干扰RNA转染HTR-8/SVneo细胞后，分别通过CCK-8检测、伤口愈合实验、Transwell实验和流式细胞术评估细胞的生物学行为，包括活力、迁移、侵袭和凋亡。应用RNA测序筛选PCBP2沉默后HTR-8/SVneo细胞中差异表达的基因。基因本体（GO）和京都基因与基因组百科全书（KEGG）分析表明，WNT信号通路以及细胞外基质重塑和细胞黏附等相关过程是最富集的通路或过程。同时，通过RNA免疫沉淀测序（RIP-seq）也确定了PCBP2对WNT5A可变剪接的调控作用。基于HTR-8/SVneo细胞和绒毛外植体，证实PCBP2对滋养层细胞的调控作用是由WNT5A介导的。此外，研究还发现ROR2/JNK/MMP2/9通路是滋养层细胞中WNT5A下游的一条重要信号通路。总之，本研究表明PCBP2表达下调通过抑制WNT5A介导的ROR2/JNK/MMPs信号通路损害了EVT的功能，这可能最终导致PE的发生发展。
