Abstract：
Recurrent miscarriage (RM) is related to the dysfunction of extravillous trophoblast cells (EVTs), but the comprehensive mechanisms remain largely unexplored. We analyzed single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing and microarray datasets obtained from Gene Expression Omnibus (GEO) database to explore the hub genes in the mechanisms of RM. We identified 1724 differentially expressed genes (DEGs) in EVTs from the RM, and they were all expressed along the trajectory of EVTs. These DEGs were associated with hypoxia and glucose metabolism. Single-cell Regulatory Network Inference and Clustering (SCENIC) analysis revealed that E2F transcription factor (E2F) 8 (E2F8) was a key transcription factor for these DEGs. And the expression of ENO1 can be positively regulated by E2F8 via RNA sequencing analysis. Subsequently, we performed immunofluorescence assay (IF), plasmid transfection, western blotting, chromatin immunoprecipitation (ChIP), real-time quantitative polymerase chain reaction (qRT-PCR), and transwell assays for validation experiments. We found that the expression of alpha-Enolase 1 (ENO1) was lower in the placentas of RM. Importantly, E2F8 can transcriptionally regulate the expression of ENO1 to promote the invasion of trophoblast cells by inhibiting secreted frizzled-related protein 1/4 (SFRP1/4) to activate Wnt signaling pathway. Our results suggest that ENO1 can promote trophoblast invasion via an E2F8-dependent manner, highlighting a potential novel target for the physiological mechanisms of RM.
摘要：
复发性流产（RM）与绒毛外滋养层细胞（EVT）的功能障碍有关，但是全面的机制在很大程度上仍未得到探索。我们分析了单细胞RNA测序（scRNA-seq），从基因表达Omnibus（GEO）数据库中获得的批量RNA测序和微阵列数据集，以探索RM机制中的hub基因。我们从RM的EVT中鉴定出1724个差异表达基因(DEG)，它们都是沿着EVT的轨迹表达的。这些DEGs与缺氧和葡萄糖代谢有关。单细胞调控网络推断和聚类（SCENIC）分析显示，E2F转录因子（E2F）8（E2F8）是这些DEGs的关键转录因子。而且经由过程RNA测序剖析，ENO1的表达可被E2F8正向调控。随后，我们进行了免疫荧光测定（IF），质粒转染，西方印迹,染色质免疫沉淀（ChIP），实时定量聚合酶链反应（qRT-PCR），和用于验证实验的transwell测定。我们发现RM胎盘中α-烯醇化酶1(ENO1)的表达较低。重要的是,E2F8可通过抑制分泌型卷曲相关蛋白1/4(SFRP1/4)激活Wnt信号通路,通过转录调控ENO1的表达促进滋养细胞的侵袭。我们的结果表明，ENO1可以通过E2F8依赖性方式促进滋养细胞的侵袭，突出了RM生理机制的潜在新目标。
