羊水外泌体miR-146a调控羊膜上皮细胞间质转化影响胎膜早破的分子机制

Premature rupture of membranes (PROM) is closely related with maternal and fetal complications, which is easy to diagnose in clinical medical,however, the unknown pathogenesis and lacked predictable diagnosis index are difficulties for clinical intervention and therapy in early stage of PROM. Amniotic epithelial cells (AECs) show an increase in the expression level of mesenchymal cell markers relative to epithelial cells in amnions of vaginal delivery including natural labor and preterm birth, however, the function of exosomal bioactivators derived from amniotic fluid in triggering epithelial-mesenchymal translation of AECs is poorly understood.In our previously research, we found that amniotic fluid derived exosome played a critical role in epithelial-mesenchymal translation of AECs for influencing PROM in LPS-treated mouse model. Meanwhile, exosomal miRNA-146a revealed significant change in expression level occurred between normal and LPS-treated mouse model, which could regulate target genes (Numb and Notch1) to increase progression of epithelial-mesenchymal translation of AECs for raising risk of PROM. Nevertheless, how exosomal miRNA-146a regulate intracellular Numb /NICD/β-catenin complex is unclear. To elucidate the mechanism of transcriptional regulation during the epithelial-mesenchymal translation of AECs and to better understand the role of exosmal miRNA-146a in PROM progression, in the current study, we will reveal: ① miRNA-146a target genes (Numb and Notch1) regulate activation of β-catenin to influence epithelial-mesenchymal translation of AECs, ② the influences in activation of β-catenin when Numb /NICD/β-catenin complex degraded in AECs, ③ miR-146a targeted Numb and Notch 1 to stabilize β-catenin that entered the nucleus and interacted with Lef/Tcf7, forming the β-catenin/ Lef/Tcf7 complex to enhance transcription of Snail and miR-146a. In conclusion, our studies focusing on exosomal-miRNAs involved in the epithelial-mesenchymal translation of AECs may advance the development of effective prevention and therapies for PROM.

胎膜早破可引母婴感染、早产等不良后果，胎膜破裂前羊膜上皮细胞（AECs）产生间质转化，以降低胎膜的承受能力增加破裂风险，但其调控机制尚未清晰。本项目前期在小鼠炎性早产模型中发现羊水外泌体miR-146a调控靶基因Numb和Notch1促进AECs间质转化并影响胎膜早破，但其在细胞内如何降解Numb/NICD/β-catenin复合物影响β-catenin活化，促进AECs间质转化的分子机制尚未明确。因此，本项目重点研究：①Numb和Notch1影响β-catenin活化调控AECs间质转化；②miR-146a解离Numb/NICD/β-catenin复合物促进β-catenin活化；③miR-146a激活β-catenin促进EMT基因Snail转录，同时增强miR-146a的转录表达形成正反馈调节，阐明炎性反应后羊水外泌体miR-146a促进AECs间质转化增加胎膜早破风险的分子机制。

羊膜上皮细胞（Amniotic Epithelial Cells，AECs）具有典型的顶面-底面极性的上皮细胞结构，细胞间的紧密连接增加了羊膜的承受压力能力，当AECs的上皮状态发生间质转化，覆盖在羊膜表层的AECs发生间质转化后使羊膜的完整性降低、承受压力的能力下降，导致羊膜的破裂，本研究绘制了AECs在炎性条件下上皮间质转化及胎膜早破发生的关系的分子生物学机制。应用LPS制备孕鼠流产模型，收集炎性羊水外泌体后发现，经LPS处理后的孕鼠羊水外泌体可加速AECs的上皮向间质转化进程，同时，炎症相关的miR-146a在外泌体中表达量显著升高。通过对子宫、蜕膜免疫组化实验及羊水流式分析发现，LPS处理后F4/80阳性巨噬细胞显著增加；体外处理巨噬细胞发现，LPS处理的M1型巨噬细胞外泌体中miR-146a表达丰度显著升高，将不同浓度外泌体子宫内原位注射，活体成像验证注射成功后发现，不同浓度炎性外泌体可引起AECs上皮标志物E-cadherin表达下调，间质标志物N-cadherin表达上调而引发生早产，并且引发早产的时间随浓度增加而缩短。进一步分析miR-146a在巨噬细胞内运送至外泌体的分子机制，研究携带miR-146a的蛋白，利用Pull down及产物质谱分析发现RNA结合蛋白Ptbp1可包裹进入外泌体进行运输，当干扰Ptbp1表达后，外泌体miR-146a的表达可显著下调。阐明外泌体miR-146a进入AECs调控其间质转化分子机制时发现，miR-146a通过下调靶基因Numb和Notch1进而激活Wnt/β-catenin 信号通路，增强EMT 重要调节因子Snail 和降解基质的金属蛋白酶MMP-9的转录，从而促进AECs发生上皮间质转化，促使羊膜的韧性降低而发生破裂，通过原子力显微镜分析羊膜的杨氏模量也证实炎性外泌体及外泌体miRNAs降低羊膜的承受能力。