br Materials and methods br Results br Discussion
Materials and methods
Discussion It has been demonstrated that up-regulation of EPHB4 impaired trophoblast migration and invasion during placentation, which may contribute to the pathogenesis of preeclampsia , . However, the upstream regulating mechanism of EPHB4 expression in trophoblast Teicoplanin still remains largely unknown. Previous studies have verified that miR-17-family miRNAs (miR-17, miR-20a, and miR-20b) play key roles in trophoblast invasion during placental development via directly targeting the 3′ UTR of EPHB4 and regulating EPHB4 expression . In this study, we applied an integrative approach to analyze the public datasets of transcription factor binding, cis-regulatory elements, and transcription profiling of placenta specimens, and identified HOXA9 as the crucial modulator facilitating EPHB4 expression in tropholasts for the first time. Notably, HOXA9 is highly expressed and positively correlated with EPHB4 levels in preeclamptic placenta tissues, which strongly suggested an important role for HOXA9 in placental development and function. Next, our immunostaining results demonstrated that HOXA9 and EPHB4 were co-expressed in trophoblast cells of first-trimester villi, while EPHB4 hardly existed in normal preterm placenta with lower HOXA9 expression. As we know, villi are a multifunctional structure and the basis of placenta formation. Indeed, it would be more convincing if villous samples of different gestational age could be collected to know the dynamic expression of HOXA9 and EPHB4. However, we had no access to obtain enough samples due to clinical practice. Based on our and other\'s researches, we can largely infer that placental EPHB4 expression gradually decreased even appeared none as uncomplicated pregnancy went on. On the other hand, we can also explain this deduction from the perspective of the subcellular localization. EVT-dependent spiral artery remodeling is the key to placentation and EVTs role is of great value in this process . In our previous work, EPHB4 expression in decidual EVTs showed a distinct decrease compared to villous trophoblast . Consistently, we speculated that trophoblasts in villi differentiate into extravillous trophoblasts probably along with the down-regulation of EPHB4 during placentation. Herein, in this work, we adopted an immortalized EVTs cell line HTR8/SVneo to investigate the effect of EPHB4 expression on cell migration and invasion, since it was really difficult to extract and culture primary EVTs. Whereas HTR-8/SVneo cells respond to hypoxia differently with primary trophoblasts , we evaluated the changes of EVTs biological function by exogenously transfecting plasmids irrespective of hypoxia condition. Therefore, it seems to be feasible and meaningful to explore the upstream mechanism of EPHB4 regulation during placentation. Actually, amounting evidence indicated the expression of HOX genes in the normal and abnormal trophoblastic tissues and their role in the differentiation of trophoblast cells , , . However until now, few researches were reported on the expression and role of HOXA9 in the control of placental developmental processes. Emerging data showed HOXA9, as the functional targets of miR-210, was positively related with trophoblast migration and vascular remodeling . In addition, HOXA9 was demonstrated to be necessary for endometrial receptivity to blastocyst implantation . These results indicated the expression of HOXA9 is conductive to embryonic implantation, which was apparently in conflict with our observations in HTR-8/SVneo cells. Our current study indicated that HOXA9 inhibited the migration and invasion of HTR-8/SVneo cells in vitro, implicating the negative roles of HOXA9 during placentation. In particular, this contradiction may be due to the phenotypically heterogeneity of HTR-8/SVneo cells, the difference between specific cells, and the complexity of “forward” and “reverse” signaling between EPHB4 and its ligand , . For instance, EPHB/EFNB signaling in endothelial cells promoted cell adhesion, whereas the reverse signaling produced the opposite effect .