miR-218-5p Induces Interleukin-1ß and Endovascular Trophoblast Differentiation by Targeting the Transforming Growth Factor ß-SMAD2 Pathway.

The acquisition of an endovascular trophoblast (enEVT) phenotype is essential for normal placental development and healthy pregnancy. MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating gene expression. We have recently reported that miR-218-5p promotes enEVT differentiation and spiral artery remodeling in part by targeting transforming growth factor ß2 (TGFß2). We also identified IL1B, which encodes interleukin 1ß (IL1ß), as one of the most highly upregulated genes by miR-218-5p. In this study, we investigated how miR-218-5p regulates IL1B expression and IL1ß secretion and the potential role of IL1ß in enEVT differentiation. Using two cell lines derived from extravillous trophoblasts (EVTs), HTR-8/SVneo and Swan 71, we found that stable overexpression of miR-218-5p precursor, mir-218-1, or transient transfection of miR-218-5p mimic, significantly increased IL1B mRNA and IL1ß protein levels in cells and conditioned media. We also showed that miR-218-5p directly interacted with SMAD2 3'UTR and reduced SMAD2 at mRNA and protein levels. Knockdown of SMAD2 induced IL1B expression and attenuated the inhibitory effect of TGFß2 on IL1B expression. On the other hand, overexpression of SMAD2 reduced IL1ß levels and blocked the stimulatory effects of miR-218-5p on IL1B expression, trophoblast migration and endothelial-like network formation. In addition, treatment of trophoblasts with IL1ß induced the formation of endothelial-like networks and the expression of enEVT markers in a dose-dependent manner. These results suggest that miR-218-5p inhibits the TGFß/SMAD2 pathway to induce IL1ß and enEVT differentiation. Finally, low doses of IL1ß also inhibited the expression of miR-218-5p, suggesting the existence of a negative feedback regulatory loop. Taken together, our findings suggest a novel interactive miR-218-5p/TGFß/SMAD2/IL1ß signaling nexus that regulates enEVT differentiation.