Department of Obstetrics and Gynaecology, Semmelweis University, Budapest, Hungary.
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
Gene. 2019 Apr 15;692:138-144. doi: 10.1016/j.gene.2019.01.012. Epub 2019 Jan 17.
microRNAs (miRNAs) play important role in the regulation of placental development, and abnormal miRNA expression is associated with preeclampsia (PE). miRNAs are released from trophoblast cells to maternal blood flow, where they are highly stable, being encapsulated inside extracellular vesicles, like exosomes or bound to Argonaute proteins. In PE, placental dysfunction leads to aberrant extracellular miRNA secretion. hsa-miR-210 is a hypoxia-sensitive miRNA found to be upregulated in PE; however, it is unknown whether it is the cause or the consequence of the disease.
Our aim was to analyze the expression of several miRNAs, including hsa-miR-210 in placenta, exosome and Ago-bound fractions comparing normal (N) and PE pregnancies. We performed in vitro analyses of extracellular hsa-miR-210 secretion of trophoblast cell cultures (of villous and extravillous origin) under hypoxic condition.
PE and N placenta samples were collected from C-sections, and blood samples were drawn from each pregnant woman in the third trimester. HTR-8 and JAR cell lines were cultured in exosome-free media and treated with hypoxia-mimetic agents. Exosome and Ago-bound fractions were isolated by membrane affinity spin column method from plasma and cell media. Short RNAs were extracted from exosomes and vesicle-free fractions, and total-RNA was isolated from the placenta samples. The RNA purity and concentration were measured by spectrophotometry. Expression analysis was carried out by qPCR with specific primers to target and reference miRNAs.
The level of hsa-miR-210 was significantly higher in PE placentas, which could cause a minor increase of exosomal and a high elevation of Ago-bound miR-210 in circulation. Hypoxia lead to intracellular hsa-miR-210 upregulation in trophoblast cell lines. In extravillous cell (HTR-8) media, only the level of exosomal hsa-miR-210 was increased but no change in Ago-bound hsa-miR-210 level was observed. In contrast, in villous cell (JAR) media, the level of exosomal hsa-miR-210 was increased and enhanced release of Ago-bound hsa-miR-210 was also observed.
Based on our data, we postulate that in PE, exosomal hsa-miR-210 is secreted actively from the trophoblast, and by intercellular communication, it may have a role in disease etiology. In addition, there is a passive release of Ago-bound hsa-miR-210 into the circulation, which may represent by-products of cell-death and is thereby a possible consequence of the disease.
微小 RNA（miRNA）在胎盘发育调控中发挥重要作用，异常 miRNA 表达与子痫前期（PE）有关。miRNA 从滋养细胞释放到母体外周血流中，在那里它们高度稳定，被包裹在细胞外囊泡（如外泌体）或与 Argonaute 蛋白结合。在 PE 中，胎盘功能障碍导致异常的细胞外 miRNA 分泌。hsa-miR-210 是一种缺氧敏感的 miRNA，在 PE 中发现上调；然而，尚不清楚它是疾病的原因还是结果。
我们旨在分析包括 hsa-miR-210 在内的几种 miRNA 在胎盘、外泌体和 Ago 结合物中的表达，比较正常（N）和 PE 妊娠。我们在体外分析了缺氧条件下绒毛和绒毛外来源的滋养细胞培养物中外泌体 hsa-miR-210 的分泌情况。
从剖宫产中采集 PE 和 N 胎盘样本，并从每位孕妇的孕晚期抽取血液样本。HTR-8 和 JAR 细胞系在无外泌体培养基中培养，并接受缺氧模拟剂处理。通过膜亲和旋转柱法从血浆和细胞培养基中分离出外泌体和 Ago 结合物。从外泌体和无囊泡部分提取短 RNA，并从胎盘样本中提取总 RNA。通过分光光度法测量 RNA 纯度和浓度。使用靶向和参考 miRNA 的特异性引物进行 qPCR 表达分析。
PE 胎盘 hsa-miR-210 水平显著升高，可能导致外泌体中 miR-210 水平略有增加，而 Ago 结合物中 miR-210 水平显著升高。缺氧导致滋养细胞系中 hsa-miR-210 的细胞内上调。在绒毛外细胞（HTR-8）培养基中，仅观察到外泌体 hsa-miR-210 水平增加，而 Ago 结合物 hsa-miR-210 水平无变化。相比之下，在绒毛细胞（JAR）培养基中，外泌体 hsa-miR-210 水平增加，并观察到 Ago 结合物 hsa-miR-210 的增强释放。
根据我们的数据，我们假设在 PE 中，外泌体 hsa-miR-210 从滋养细胞主动分泌，并通过细胞间通讯，可能在疾病发病机制中发挥作用。此外，Ago 结合物 hsa-miR-210 被动释放到循环中，这可能代表细胞死亡的副产物，因此可能是疾病的结果。
