Li Chunyan, Ming Pingpo, Fan Cuifang, Chen Jiao, Yang Jing
Department of Obstetrics, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Department of Oncology, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Biomedicines. 2025 May 22;13(6):1268. doi: 10.3390/biomedicines13061268.
Preeclampsia (PE) is a pregnancy-specific disorder and a leading cause of maternal and fetal morbidity and mortality. Impaired trophoblast invasion is a hallmark of PE, and alternative splicing (AS) is crucial for trophoblast differentiation and placental development. However, the exact mechanisms of AS in PE remain poorly understood. To elucidate AS-mediated regulatory pathways in PE, a total of 38 fresh-frozen placental samples, including 13 pre-eclampsia samples and 25 normal control samples, were collected from Renmin Hospital of Wuhan University between 1 February and 30 July 2022. We performed transcriptome sequencing of seven PE and seven normal placentas to identify differentially spliced events. After quality control and adapter trimming, raw sequencing reads were aligned to the human reference genome using STAR. Differential exon usage was analyzed using DEXSeq (version 1.36.0), and exons with an adjusted -value < 0.05 and a fold change greater than 2 or less than 0.5 were considered significantly differentially spliced. Functional assays, including CCK8, colony formation, and cell cycle analyses, were conducted to assess trophoblast proliferation, whereas wound healing and Transwell assays were used to evaluate trophoblast migration and invasion using the HTR-8/SVneo cell line. RNA immunoprecipitation sequencing (RIP-seq) and RNA stability assays were employed to investigate mRNA interactions and stability. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) emerged as a key RNA-binding protein associated with alternative splicing regulation, intersecting both AS-related candidate genes and known splicing factors, although it is not a classical splicing factor itself. IGF2BP3 overexpression markedly enhanced HTR-8/SVneo trophoblast proliferation, migration, and invasion while suppressing ROS activation. RNA-seq, RIP-seq, and RNA stability assays revealed that IGF2BP3 directly interacts with and enhances the stability of PDE3A mRNA. Functional rescue experiments confirmed that PDE3A knockdown partially abrogated IGF2BP3-mediated trophoblast progression. Furthermore, miR-196a-5p was identified as a negative regulator of IGF2BP3 via miRNA inhibitor/mimic transfection, qRT-PCR, and functional assays, confirming that miR-196a-5p overexpression downregulates IGF2BP3, thereby impairing trophoblast migration and proliferation. Notably, restoring IGF2BP3 expression reversed these inhibitory effects. Our findings reveal a previously unrecognized regulatory axis in PE in which miR-196a-5p suppresses IGF2BP3 expression, leading to PDE3A mRNA destabilization and impaired trophoblast function. This study offers mechanistic insights into PE pathogenesis and identifies IGF2BP3 as a potential therapeutic target.
子痫前期（PE）是一种妊娠特有的疾病，是孕产妇和胎儿发病及死亡的主要原因。滋养层细胞侵袭受损是子痫前期的一个标志，而可变剪接（AS）对于滋养层细胞分化和胎盘发育至关重要。然而，子痫前期中可变剪接的确切机制仍知之甚少。为了阐明子痫前期中可变剪接介导的调控途径，2022年2月1日至7月30日期间，从武汉大学人民医院收集了38份新鲜冷冻的胎盘样本，其中包括13份子痫前期样本和25份正常对照样本。我们对7份子痫前期胎盘和7份正常胎盘进行了转录组测序，以鉴定差异剪接事件。经过质量控制和接头修剪后，使用STAR将原始测序读数与人类参考基因组进行比对。使用DEXSeq（版本1.36.0）分析差异外显子使用情况，调整后P值<0.05且折叠变化大于2或小于0.5的外显子被认为是显著差异剪接的。进行了包括CCK8、集落形成和细胞周期分析在内的功能测定，以评估滋养层细胞增殖，而伤口愈合和Transwell测定则用于使用HTR-8/SVneo细胞系评估滋养层细胞迁移和侵袭。采用RNA免疫沉淀测序（RIP-seq）和RNA稳定性测定来研究mRNA相互作用和稳定性。胰岛素样生长因子2 mRNA结合蛋白3（IGF2BP3）作为一种与可变剪接调控相关的关键RNA结合蛋白出现，它与可变剪接相关候选基因和已知剪接因子都有交集，尽管它本身不是经典的剪接因子。IGF2BP3过表达显著增强了HTR-8/SVneo滋养层细胞的增殖、迁移和侵袭能力，同时抑制了ROS激活。RNA测序、RIP-seq和RNA稳定性测定表明，IGF2BP3直接与PDE3A mRNA相互作用并增强其稳定性。功能挽救实验证实，敲低PDE3A可部分消除IGF2BP3介导的滋养层细胞进展。此外，通过miRNA抑制剂/模拟物转染、qRT-PCR和功能测定，鉴定出miR-196a-5p是IGF2BP3的负调节因子，证实miR-196a-5p过表达下调IGF2BP3，从而损害滋养层细胞迁移和增殖。值得注意的是，恢复IGF2BP3表达可逆转这些抑制作用。我们的研究结果揭示了子痫前期中一个以前未被认识的调控轴，其中miR-196a-5p抑制IGF2BP3表达，导致PDE3A mRNA不稳定和滋养层细胞功能受损。这项研究为子痫前期发病机制提供了机制性见解，并将IGF2BP3确定为潜在的治疗靶点。
