Departments of Molecular Medicine and Anatomy, Obstetrics and Gynecology, and Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Biol Reprod. 2009 Oct;81(4):717-29. doi: 10.1095/biolreprod.108.075481. Epub 2009 Jun 3.
In this study, we performed small RNA library sequencing using human placental tissues to identify placenta-specific miRNAs. We also tested the hypothesis that human chorionic villi could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation. By small RNA library sequencing, most placenta-specific miRNAs (e.g., MIR517A) were linked to a miRNA cluster on chromosome 19. The miRNA cluster genes were differentially expressed in placental development. Subsequent validation by real-time PCR and in situ hybridization revealed that villous trophoblasts express placenta-specific miRNAs. The analysis of small RNA libraries from the blood plasma showed that the placenta-specific miRNAs are abundant in the plasma of pregnant women. By real-time PCR, we confirmed the rapid clearance of the placenta-specific miRNAs from the plasma after delivery, indicating that such miRNAs enter into maternal circulation. By using the trophoblast cell line BeWo in culture, we demonstrated that miRNAs are indeed extracellularly released via exosomes. Taken together, our findings suggest that miRNAs are exported from the human placental syncytiotrophoblast into maternal circulation, where they could target maternal tissues. Finally, to address the biological functions of placenta-specific miRNAs, we performed a proteome analysis of BeWo cells transfected with MIR517A. Bioinformatic analysis suggests that this miRNA is possibly involved in tumor necrosis factor-mediated signaling. Our data provide important insights into miRNA biology of the human placenta.
在这项研究中，我们使用人胎盘组织进行了小 RNA 文库测序，以鉴定胎盘特异性 miRNA。我们还测试了这样一个假设，即人绒毛通过外泌体将 miRNA 分泌到细胞外，然后这些 miRNA 进入母系循环。通过小 RNA 文库测序，大多数胎盘特异性 miRNA（例如 MIR517A）与染色体 19 上的 miRNA 簇相关。miRNA 簇基因在胎盘发育过程中表达差异。随后通过实时 PCR 和原位杂交进行的验证表明，绒毛滋养层表达胎盘特异性 miRNA。对孕妇血浆中小 RNA 文库的分析表明，胎盘特异性 miRNA 在孕妇血浆中丰富。通过实时 PCR，我们证实了胎盘特异性 miRNA 在分娩后从血浆中迅速清除，表明这些 miRNA 进入母系循环。通过培养中的滋养层细胞系 BeWo，我们证明了 miRNA 确实是通过外泌体被细胞外释放的。总之，我们的研究结果表明，miRNA 从人胎盘合体滋养层输出到母系循环中，在母系循环中，它们可以靶向母体组织。最后，为了研究胎盘特异性 miRNA 的生物学功能，我们用 MIR517A 转染 BeWo 细胞进行了蛋白质组分析。生物信息学分析表明，该 miRNA 可能参与肿瘤坏死因子介导的信号通路。我们的数据为人类胎盘 miRNA 生物学提供了重要的见解。
