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

Abstract

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 β2 (TGFβ2). We also identified IL1B, which encodes interleukin 1β (IL1β), 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 IL1β secretion and the potential role of IL1β 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 IL1β 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 TGFβ2 on IL1B expression. On the other hand, overexpression of SMAD2 reduced IL1β 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 IL1β 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 TGFβ/SMAD2 pathway to induce IL1β and enEVT differentiation. Finally, low doses of IL1β 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/TGFβ/SMAD2/IL1β signaling nexus that regulates enEVT differentiation.

Keywords: IL1β; SMAD; TGFβ; endovascular trophoblast; miR-218-5p; placenta.

Figure 1

miR-218-5p induces IL1β expression and secretion. Stable transfection of mir-218-1 (left) in HTR-8/SVneo cells, transient transfection of miR-218-5p mimic in HTR-8/SVneo (middle) and Swan 71 (right) cells significantly increased IL1B mRNA (A) and IL1β protein levels in cell lysates (B) and conditioned media (C). Data are shown as mean ± SEM (n=3). **p < 0.01; ****p < 0.0001. EV, empty vector; NC, non-targeting control.

Figure 2

miR-218-5p inhibits SMAD2 by directly binding to its 3’UTR. (A) Reporter assay using two SMAD-responsive luciferase reporter vectors, pAR3-Lux and SBE4-Luc. Treatment with TGFβ or Activin A increased SMAD2/3 transcriptional activity but their effects were lower in mir-218-1-overexpressing HTR-8/SVneo cells than in control cells. (B) qPCR for SMAD2 and SMAD3 mRNA in control and mir-218-1-overexpressing HTR-8/SVneo cells. (C) qPCR for SMAD2 and SMAD3 mRNA in control and miR-218-5p mimic-treated HTR-8/SVneo and Swan 71 cells. (D) Western blotting for SMAD2 and SMAD3 in cells treated with miR-218-5p mimic. Data are representative of three independent experiments. (E) miR-218-5p targets SMAD2 3’UTR. A PCR fragment containing a predicted miR-218-5p binding site was cloned into the pMIR-REPORT vector downstream of the luciferase gene. miR-218-5p decreased the luciferase activity of the SMAD2 3’UTR reporter vector. Data are shown as mean ± SEM (n=3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. EV, empty vector; NC, non-targeting control.

Figure 3

TGFβ2 inhibits IL1β via SMAD2 signaling. (A) ELISA for IL1β in cell lysates harvested from HTR-8/SVneo and Swan 71 cells treated with recombinant TGFβ2 at various concentrations for 24 hr. (B) qPCR for TGFB2 mRNA in cells transfected with siTGFB2. (C) ELISA for IL1β in cell lysates harvested from cells treated with siTGFB2. (D) qPCR for IL1B mRNA in control, siSMAD2, and siSMAD3-treated cells, in the presence of recombinant TGFβ2 (1 ng/ml). Data are shown as mean ± SEM (n=3). **p < 0.01, ***p < 0.001, ****p < 0.0001. For (A, D), different letters above bars denote statistical significance.

Figure 4

SMAD2 blocks miR-218-5p-induced IL1B mRNA. (A) Western blotting for SMAD2 and SMAD3 in control and mir-218-1-overexpressing HTR-8/SVneo cells transiently transfected with Flag-tagged SMAD2 and/or SMAD3 expression constructs. Note that mir-218-1 overexpression reduced both endogenous and exogenous SMAD2 and SMAD3 protein levels. Data are representative of three independent experiments. (B) qPCR for IL1B mRNA in control and mir-218-1-overexpressing HTR-8/SVneo cells transfected with SMAD2 and/or SMAD3 vectors, showing that SMAD2 and SMAD3 exerted opposing effects on IL1B mRNA. Data are shown as mean ± SEM (n=3). Different letters above bars denote statistical significance.

Figure 5

SMAD2 inhibits miR-218-5p-induced cell migration and formation of endothelial-like networks. (A) Wound healing (left, n=6) and tube formation (right, n=5-6) assays in control or mir-218-1-overexpressing cells, transfected with SMAD2-expressing construct or its control pcDNA3.1 vector. (B, C) Wound healing (left, n=6) and tube formation (right, n=5-6) assays in HTR-8/SVneo and Swan71 cells, co-transfected with miR-218-5p mimic, SMAD2 construct, or their non-targeting control (NC) or empty vector, pcDNA3.1. Note that SMAD2 overexpression reversed the stimulatory effects of miR-218-5p on wound closure and the formation of endothelial-like networks. (D) Silencing of SMAD2 partially abolished the inhibitory effect of anti-miR-218-5p on endothelial-like network formation (n=6). Data are shown as mean ± SEM; scale bar = 800 µm. For wound healing assay in (A–C), the differences among all groups are significant (p < 0.05) starting at 10 hr.

Figure 6

IL1β promotes the acquisition of an enEVT-like phenotype. (A) Tube formation assay showing that IL1β enhanced the ability of HTR-8/SVneo and Swan 71 cells to form endothelial-like networks (n=6); scale bar = 800 µm. (B) qPCR for the expression of enEVT markers in cells treated with IL1β (n=3). Note that IL1β at low concentrations increased ITGA1, ITGA5, CDH5, and PECAM1 mRNA in a dose-dependent manner. Data are shown as mean ± SEM. Different letters above bars denote statistical significance.

Figure 7

Regulation of miR-218-5p by IL1β. (A) HTR-8/SVneo and Swan 71 cells were treated with different concentrations of IL1β for 24 hr. qPCR revealed that lower doses of IL1β reduced miR-218-5p level. Data are shown as mean ± SEM (n=3). Different letters above bars denote statistical significance. (B) A schematic outlining the interactions among miR-218-5p, IL1β, and the TGFβ/SMAD2 pathway and their potential roles in enEVT differentiation.