Zhang Wan-Yu, Wang Han-Bi, Deng Cheng-Yan
Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China.
Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Beijing 100730, China.
World J Stem Cells. 2024 Nov 26;16(11):906-925. doi: 10.4252/wjsc.v16.i11.906.
Thin endometrium seriously affects endometrial receptivity, resulting in a significant reduction in embryo implantation, and clinical pregnancy and live birth rates, and there is no gold standard for treatment. The main pathophysiological characteristics of thin endometrium are increased uterine arterial blood flow resistance, angiodysplasia, slow growth of the glandular epithelium, and low expression of vascular endothelial growth factor, resulting in endometrial epithelial cell (EEC) hypoxia and endometrial tissue aplasia. Human umbilical cord mesenchymal stem cells (HucMSCs) promote repair and regeneration of damaged endometrium by secreting microRNA (miRNA)-carrying exosomes. However, the initiation mechanism of HucMSCs to repair thin endometrium has not yet been clarified.
To determine the role of hypoxic-EEC-derived exosomes in function of HucMSCs and explore the potential mechanism.
Exosomes were isolated from normal EECs (EEC-exs) and hypoxia-damaged EECs (EECD-exs), before characterization using Western blotting, nanoparticle-tracking analysis, and transmission electron microscopy. HucMSCs were cocultured with EEC-exs or EECD-exs and differentially expressed miRNAs were determined using sequencing. MiR-21-5p or miR-214-5p inhibitors or miR-21-3p or miR-214-5p mimics were transfected into HucMSCs and treated with a signal transducer and activator of transcription 3 (STAT3) activator or STAT3 inhibitor. HucMSC migration was assessed by Transwell and wound healing assays. Differentiation of HucMSCs into EECs was assessed by detecting markers of stromal lineage (Vimentin and CD13) and epithelial cell lineage (CK19 and CD9) using Western blotting and immunofluorescence. The binding of the miRNAs to potential targets was validated by dual-luciferase reporter assay.
MiR-21-5p and miR-214-5p were lowly expressed in EECD-ex-pretreated HucMSCs. MiR-214-5p and miR-21-5p inhibitors facilitated the migratory and differentiative potentials of HucMSCs. MiR-21-5p and miR-214-5p targeted STAT3 and protein inhibitor of activated STAT3, respectively, and negatively regulated phospho-STAT3. MiR-21-5p- and miR-214-5p-inhibitor-induced promotive effects on HucMSC function were reversed by STAT3 inhibition. MiR-21-5p and miR-214-5p overexpression repressed HucMSC migration and differentiation, while STAT3 activation reversed these effects.
Low expression of miR-21-5p/miR-214-5p in hypoxic-EEC-derived exosomes promotes migration and differentiation of HucMSCs into EECs STAT3 signaling. Exosomal miR-214-5p/miR-21-5p may function as valuable targets for thin endometrium.
薄型子宫内膜严重影响子宫内膜容受性，导致胚胎着床、临床妊娠及活产率显著降低，且治疗尚无金标准。薄型子宫内膜的主要病理生理特征为子宫动脉血流阻力增加、血管发育异常、腺上皮生长缓慢以及血管内皮生长因子表达降低，导致子宫内膜上皮细胞（EEC）缺氧和子宫内膜组织发育不全。人脐带间充质干细胞（HucMSCs）通过分泌携带微小RNA（miRNA）的外泌体促进受损子宫内膜的修复和再生。然而，HucMSCs修复薄型子宫内膜的起始机制尚未阐明。
确定缺氧EEC来源的外泌体在HucMSCs功能中的作用，并探索其潜在机制。
从正常EEC（EEC-exs）和缺氧损伤的EEC（EECD-exs）中分离外泌体，然后用蛋白质印迹法、纳米颗粒跟踪分析和透射电子显微镜进行表征。将HucMSCs与EEC-exs或EECD-exs共培养，并用测序法确定差异表达的miRNAs。将miR-21-5p或miR-214-5p抑制剂或miR-21-3p或miR-214-5p模拟物转染到HucMSCs中，并用信号转导和转录激活因子3（STAT3）激活剂或STAT3抑制剂处理。通过Transwell和伤口愈合试验评估HucMSC迁移。通过蛋白质印迹法和免疫荧光检测基质谱系（波形蛋白和CD13）和上皮细胞谱系（CK19和CD9）的标志物，评估HucMSCs向EECs的分化。通过双荧光素酶报告基因试验验证miRNAs与潜在靶点的结合。
miR-21-5p和miR-214-5p在EECD-ex预处理的HucMSCs中低表达。miR-214-5p和miR-21-5p抑制剂促进了HucMSCs的迁移和分化潜能。miR-21-5p和miR-214-5p分别靶向STAT3和活化STAT3的蛋白抑制剂，并负向调节磷酸化STAT3。STAT3抑制逆转了miR-21-5p和miR-214-5p抑制剂诱导的对HucMSC功能的促进作用。miR-21-5p和miR-214-5p过表达抑制了HucMSC迁移和分化，而STAT3激活逆转了这些作用。
缺氧EEC来源的外泌体中miR-21-5p/miR-214-5p低表达通过STAT3信号促进HucMSCs迁移和向EECs分化。外泌体miR-214-5p/miR-2l-5p可能成为薄型子宫内膜有价值的治疗靶点。
