Reproductive Medical Center, Renmin Hospital of Wuhan University & Hubei Clinic Research Center for Assisted Reproductive Technology and Embryonic Development, Wuhan 430060, China.
Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Theranostics. 2021 Mar 31;11(12):5813-5830. doi: 10.7150/thno.58731. eCollection 2021.
Emerging evidence demonstrates that insufficient migration and invasion of trophoblasts play critical roles in the pathogenesis of recurrent spontaneous abortion (RSA). Cell-to-cell communication at the maternal-fetal interface is essential to maintain the invasion and migration of trophoblasts. M1 macrophages, important immune cellular components at the maternal-fetal interface, have been reported to be elevated in decidua tissues from patients with RSA. Recent studies indicate that M1 macrophages modulate trophoblast biological behaviors; however, the underlying mechanisms remain poorly understood. Extracellular vesicles (EVs) were isolated from the supernatant of M1 macrophages inducted from THP-1 cells (M1-EVs) by ultracentrifugation, identified by transmission electron microscopy, nanoparticle tracking analysis, and western blotting, and their miRNA profile was characterized by miRNA sequencing. Scratch wound healing and transwell assays were used to investigate the effect of M1-EVs on trophoblast migration and invasion. RT-PCR, western blotting, and luciferase reporter assays were conducted to uncover the underlying mechanism. Finally, animal experiments were employed to explore the effect of M1-EVs on embryo absorption in mice. M1 macrophages suppressed trophoblast EMT to reduce their migration and invasion abilities by secreting EVs. Through miRNA sequencing, miR-146a-5p and miR-146b-5p were identified as the most upregulated miRNAs in trophoblasts treated with M1-EVs. Further functional experiments showed that M1-EVs inhibited trophoblast migration and invasion by transferring miR-146a-5p and miR-146b-5p. Mechanistically, EV miR-146a-5p and miR-146b-5p inhibited EMT of trophoblasts by directly suppressing TNF receptor-associated factor 6 (TRAF6) expression at the post-transcriptional level. Furthermore, M1-EVs aggravated embryo absorption in mice. Clinically, expression of miR-146a-5p, miR-146b-5p, and TRAF6 were aberrant in placental villous tissues from patients with RSA, and negative correlations were found between miR-146a-5p/miR-146b-5p and TRAF6 expression levels. Our findings indicate that miR-146a-5p and miR-146b-5p derived from EVs play important roles in intercellular communication between M1 macrophages and trophoblasts, illuminating a novel mechanism in M1 macrophage regulation of trophoblasts and their role in RSA.
越来越多的证据表明滋养细胞的迁移和侵袭不足在复发性自然流产（RSA）的发病机制中起关键作用。母胎界面的细胞间通讯对于维持滋养细胞的侵袭和迁移至关重要。M1 巨噬细胞是母胎界面重要的免疫细胞成分，据报道，在 RSA 患者的蜕膜组织中升高。最近的研究表明，M1 巨噬细胞调节滋养细胞的生物学行为；然而，其潜在机制仍知之甚少。 通过超速离心从 THP-1 细胞诱导的 M1 巨噬细胞上清液中分离出细胞外囊泡（EVs），通过透射电子显微镜、纳米颗粒跟踪分析和 Western blot 进行鉴定，并通过 miRNA 测序对其 miRNA 谱进行特征分析。划痕愈合和 Transwell 分析用于研究 M1-EVs 对滋养细胞迁移和侵袭的影响。进行 RT-PCR、Western blot 和荧光素酶报告基因分析以揭示潜在机制。最后，通过动物实验研究 M1-EVs 对小鼠胚胎吸收的影响。 M1 巨噬细胞通过分泌 EVs 抑制滋养细胞 EMT，从而降低其迁移和侵袭能力。通过 miRNA 测序，发现 miR-146a-5p 和 miR-146b-5p 是用 M1-EVs 处理的滋养细胞中上调最明显的 miRNA。进一步的功能实验表明，M1-EVs 通过转移 miR-146a-5p 和 miR-146b-5p 抑制滋养细胞的迁移和侵袭。从机制上讲，EV miR-146a-5p 和 miR-146b-5p 通过在转录后水平直接抑制 TNF 受体相关因子 6（TRAF6）的表达来抑制滋养细胞 EMT。此外，M1-EVs 加重了小鼠胚胎的吸收。临床上，RSA 患者胎盘绒毛组织中 miR-146a-5p、miR-146b-5p 和 TRAF6 的表达异常，并且 miR-146a-5p/miR-146b-5p 与 TRAF6 表达水平呈负相关。 我们的研究结果表明，源自 EV 的 miR-146a-5p 和 miR-146b-5p 在 M1 巨噬细胞与滋养细胞之间的细胞间通讯中发挥重要作用，揭示了 M1 巨噬细胞调节滋养细胞及其在 RSA 中的作用的新机制。
