The Role of MicroRNA-378a-5p and its Target Genes in Differentiation and Fusion of BeWo Cells

MicroRNAs are the post-transcriptional modulators of gene expression and have been shown to play vital roles in the placental development. The placenta is a multifunctional organ that plays critical roles throughout the pregnancy and fetal development. Bathing in the maternal blood and formed from the fusion of mononucleated cytotrophoblast (CTBs), syncytiotrophoblasts (STBs) are the primary site of numerous placental functions such as exchange of respiratory gases and waste products, transport of nutrients and hormones production. We have previously shown that miR-378a-5p inhibits STB differentiation by targeting cyclin G2 and targets Nodal that inhibits trophoblast invasion in extravillous trophoblast cells (EVTs). In this study, we have further investigated the role of miR-378a-5p and its target genes in STB differentiation by using a choriocarcinoma cell line, BeWo. Using luciferase reporter assays, we showed that miR-378a-5p targets CREB. Transfection of miR-378a-5p decreased, while antimiR-378a-5p increased CREB protein and mRNA levels. We found that a miR-378a-5p target gene, Nodal, induced STB differentiation by activating CREB. Nodal is downregulated by miR-378a-5p but upregulated during Forskolin (an adenylate cyclase activator) induced STB differentiation. Treatment with recombinant Nodal (rhNodal) resulted in increased cell fusion and expression of several STB markers such as syncytin-1, syncytin-2, galactoside-binding soluble lectin13 (LGALS13) and chorionic gonadotropin beta (CGB). Treatment with rhNodal resulted in increased syncytin-1 and decreased E-cadherin protein expression. On the other hand, knockdown of Nodal inhibited cell fusion, decreased syncytin-1 protein expression and several STB markers but increased E-cadherin protein expression. Furthermore, silencing of CREB using siRNA attenuated the effect of Nodal, and overexpression of CREB and Nodal reversed the inhibitory effect of miR-378a-5p. We showed that miR-378a-5p target gene, cyclin G2, increased cell fusion and STB marker genes, while siRNA cyclin G2 decreased cell fusion and marker genes expression. Nodal induced cyclin G2 promoter activity, suggesting that downregulation of Nodal and cyclin G2 by miR-378a-5p modulate STB differentiation. In conclusion, our study demonstrates that miR-378a-5p exerts an inhibitory role in STB differentiation, in part by down-regulating CREB, Nodal and cyclin G2 expression.