Department of Biology, York University, Toronto, ON, Canada.
J Cell Sci. 2012 Jul 1;125(Pt 13):3124-32. doi: 10.1242/jcs.096412. Epub 2012 Mar 27.
Nodal is a member of the transforming growth factor-β superfamily that plays crucial roles during embryogenesis. Recently, we have reported that Nodal inhibits trophoblast cell proliferation, migration and invasion, but induces apoptosis in the human placenta. In this study, we examined the regulation of Nodal by microRNAs. In silico analysis of Nodal 3'UTR revealed a potential binding site for miR-378a-5p. In luciferase reporter assays, we found that miR-378a-5p suppressed the luciferase activity of a reporter plasmid containing Nodal 3'UTR but this suppressive effect was completely abolished when the predicted target site was mutated. Western blot analysis showed that miR-378a-5p decreased whereas anti-miR-378a-5p increased Nodal protein levels. These results indicate that miR-378a-5p targets Nodal 3'UTR to repress its expression. Stable transfection of the miR-378a-5p precursor, mir-378a, into HTR8/SVneo cells enhanced cell survival, proliferation, migration and invasion. Transient transfection of mature miR-378a-5p mimic, and to a lesser extent, siRNA targeting Nodal, produced similar effects. However, anti-miR-378a-5p inhibited cell migration and invasion. In addition, overexpression of Nodal reversed the invasion-promoting effect of miR-378a-5p. Furthermore, miR-378a-5p enhanced, whereas anti-miR-378a-5p suppressed, the outgrowth and spreading of extravillous trophoblast cells in first trimester placental explants. Finally, miR-378a-5p was detected in human placenta throughout different stages of gestation and in preterm pregnancies, placental miR-378a-5p levels were lower in preeclamptic patients than in healthy controls. Taken together, these findings strongly suggest that miR-378a-5p plays an important role in human placental development by regulating trophoblast cell growth, survival, migration and invasion, and that miR-378a-5p exerts these effects, at least in part, through the suppression of Nodal expression.
节点是转化生长因子-β超家族的成员，在胚胎发生中发挥关键作用。最近，我们报道了节点抑制滋养细胞增殖、迁移和侵袭，但诱导人胎盘细胞凋亡。在这项研究中，我们检查了 microRNAs 对节点的调节。节点 3'UTR 的计算机分析显示出与 miR-378a-5p 潜在结合位点。在荧光素酶报告基因实验中，我们发现 miR-378a-5p 抑制含有节点 3'UTR 的报告质粒的荧光素酶活性，但当预测的靶位点发生突变时，这种抑制作用完全被消除。Western blot 分析表明，miR-378a-5p 降低而抗-miR-378a-5p 增加节点蛋白水平。这些结果表明 miR-378a-5p 靶向节点 3'UTR 以抑制其表达。miR-378a 的稳定转染，mir-378a，进入 HTR8/SVneo 细胞增强细胞存活、增殖、迁移和侵袭。成熟 miR-378a-5p 模拟物的瞬时转染，以及对 Nodal 的靶向 siRNA 的转染，产生了类似的效果。然而，抗-miR-378a-5p 抑制细胞迁移和侵袭。此外，节点的过表达逆转了 miR-378a-5p 的促进侵袭作用。此外，miR-378a-5p 增强，而抗-miR-378a-5p 抑制，在第一孕期胎盘外植体中绒毛外滋养细胞的生长和扩展。最后，miR-378a-5p 在不同孕期的人胎盘和早产妊娠中均有检测到，子痫前期患者胎盘 miR-378a-5p 水平低于健康对照组。总之，这些发现强烈表明 miR-378a-5p 通过调节滋养细胞的生长、存活、迁移和侵袭，在人类胎盘发育中发挥重要作用，并且 miR-378a-5p 通过抑制节点表达来发挥这些作用，至少部分如此。
