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


INTRODUCTION
The placenta is a multifunctional transient organ essential for nutrient and gas exchange between the mother and the fetus throughout the pregnancy (1). During placental development, cytotrophoblast progenitor cells differentiate into two lineages, syncytiotrophoblasts and extravillous trophoblasts (EVTs). EVTs acquire invasive properties and further differentiate into interstitial trophoblasts and endovascular trophoblasts (enEVTs). enEVTs invade the uterus and replace the endothelial cells lining the maternal spiral arteries, and transform them into high flow, low resistance vessels. Insufficient enEVT differentiation, invasion, and spiral artery remodeling can decrease blood flow to the placenta and cause oxidative stress, which is known to precipitate the early onset (<34 weeks of gestation) preeclampsia (PE). PE is a major pregnancy-related disorder characterized by hypertension and multi-organ damage (2). It is a leading cause of maternal and neonatal morbidity and mortality and affects approximately 3%-5% of pregnancies worldwide (3).
The transforming growth factor b (TGFb) superfamily plays a crucial role in the development and tissue homeostasis. Members of this family signal via heteromeric complexes of type I and type II receptors to activate receptor-regulated SMAD (R-SMAD), which form a complex with SMAD4 and translocate to the nucleus to regulate target gene transcription (4). Two R-SMADs, SMAD2 and SMAD3, are known to be activated by TGFb1, 2, 3, Activin, and Nodal. These SMADs, together with the TGFb ligands and receptors, are all expressed in trophoblasts (5)(6)(7). These signaling molecules regulate a variety of cellular functions, such as proliferation, migration, invasion, and apoptosis (8)(9)(10)(11)(12)(13), as well as hormone production (14), and their dysregulation is associated with PE (15)(16)(17)(18). Interestingly, we have recently found that SMAD2 and SMAD3 play differential roles in enEVT differentiation, in that activation of SMAD2 or inactivation of SMAD3 suppresses the acquisition of an enEVT-like phenotype (19).
MicroRNAs (miRNAs) are a class of small and highly conserved noncoding RNAs that are critically involved in numerous physiological and pathological events. In most cases, miRNAs interact with the 3′ untranslated region (3′UTR) of target mRNAs to induce their degradation and repress the translational process (20). The differential expression profiles of miRNAs in placentas from healthy and PE patients have been documented and some miRNAs have been reported to regulate trophoblast functions and placental development by modulating various signaling pathways, including the TGFb pathway (21)(22)(23)(24). For example, miR-195, downregulated in PE placental tissues, represses trophoblast invasion by targeting ACVR2A, a type II receptor for Activin and Nodal (25). miR-376c and miR-378-5 increase trophoblast proliferation, motility, and survival by inhibiting Activin receptor-like kinase (ALK) 5 (type I TGFb receptor)/ALK7 (type I Nodal receptor) and Nodal, respectively, both leading to compromised TGFb signaling (26,27). In addition, we and others have found that the expression of miR-18a and miR-218-5p is decreased in placentas from PE patients (21,28,29). These two miRNAs stimulate EVT differentiation, invasion, and spiral artery remodeling through the inhibition of SMAD2 and TGFb2, respectively (28,29).
Interleukin 1b (IL1b) is a proinflammatory cytokine that may play a role in implantation (30). Several studies have reported that IL1b increases the invasive capacity of trophoblasts (31,32) and enhances the secretion of IL8 from endometrial cells that subsequently stimulates survival and migration of first trimester villous cytotrophoblasts (33). However, IL1b may also have harmful effects on placental development, as serum IL1b levels are increased in gestational diseases, including PE and preterm labor (34)(35)(36), suggesting that a balanced IL1b expression/ activity is important for a healthy pregnancy. To date, whether IL1b modulates enEVT differentiation has not been reported, and this merits investigation.
Recently, we have reported that miR-218-5p stimulates enEVT differentiation and spiral artery remodeling by inhibiting TGFb2, and the IL1B mRNA is upregulated by miR-218-5p (28). In this study, we further investigated how miR-218-5p regulates IL1b and determined the potential role of IL1b in the acquisition of an enEVT-like phenotype. We hypothesized that miR-218-5p induces IL1b by targeting the TGFb signaling pathway and that IL1b contributes to the miR-218-5p-induced enEVT differentiation.

Quantitative Real-Time PCR (qPCR)
Total RNA was extracted from cells using TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. RNA was reverse transcribed with Moloney murine leukemia virus (M-MuLV) reverse transcriptase (New England Biolabs, Whitby, ON, Canada). RNA purity and concentration were examined by a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). All the samples had high purity, as indicated by an A260/A280 ratio of 2.01~2.1 and an A260/230 ratio >2. The integrity of RNA was confirmed using agarose gel electrophoresis. miRNA reverse transcription was performed using a TaqMan ® microRNA reverse transcription kit (Thermo Fisher Scientific) with a unique reverse primer. qPCR was carried out using BlasTaq 2×qPCR master mix (Applied Biological Materials, Richmond, BC, Canada) and gene specific primers ( Table 1). miR-218-5p and the internal control were measured using the hsa-miR-218-5p TaqMan miRNA kit and U6 snRNA TaqMan ® control miRNA kit (both from Thermo Fisher Scientific), respectively. All qPCR assays were performed on Rotor-Gene Q (Qiagen, Toronto, ON, Canada). The relative mRNA and miRNA levels were calculated using the 2 -DDCt method, normalized with b-actin and snRNA U6 as housekeeping control, respectively.

Enzyme-Linked Immunosorbent Assay (ELISA)
Conditioned media and cell lysates were collected 48 hr after transfection or 24 hr after treatment with TGFb2. The media

Wound Healing Assay
Cell migration was determined using an IncuCyte ® scratch wound healing approach. IncuCyte S3 (Sartorius, Gottingen, Germany) is a live-cell analysis system that can capture the images of cells in culture. At 12 hr post-transfection, 3×10 5 cells were seeded into a 96-well ImageLock plate (Sartorius) and cultured overnight. When cells reached 100% confluence, the ImageLock plate was placed into the WoundMaker (Sartorius) to create a scratch in each well. Cells were then cultured in a FBSfree medium, and the healing process was imaged every 2 or 3 hr with the IncuCyte S3 system. The relative migration rate at different time points was compared with that of 0 hr using the IncuCyte scratch wound analysis module.

Tube Formation Assay
The ability of trophoblasts to form endothelial-like networks was assessed using tube formation assay, as described previously (19). Briefly, cells were labeled with a green cell-tracking CMFDA dye (Sigma-Aldrich) and were then seeded into a 96-well plate precoated with Cultrex reduced growth factor base membrane extract (RGF-BME) (Trevigen). IncuCyte S3 was used to capture fluorescent images every 2 hr at 4X. Total network length was quantified by Angiogenesis analyzer, a plugin of ImageJ (41).

Luciferase Reporter Assay
The 3'UTR fragment (12738~13358 nt) of the human SMAD2 gene containing the putative binding site of miR-218-5p was amplified by PCR and cloned into the pMIR-REPORT luciferase plasmid vector (Thermo Fisher Scientific) at the SpeI and HindIII sites, downstream of a firefly luciferase gene. The sequences of primers for the cloning are listed in Table 1. The insertion of the fragment was confirmed by DNA sequencing. For luciferase reporter assay, cells were seeded into a 24-well plate and reached 70% confluence before transfection. Cells were co-transfected with 800 ng SMAD2 3'UTR reporter plasmid, 10 ng Renilla luciferase vector (pCMV-Renilla, Promega), and 80 nM miR-218-5p mimic or negative control (NC) (Shanghai GenePharma) for 6 hr, using Lipofectamine 2000 reagent. At 42 hr following the transfection, cell lysates were harvested, and the luciferase reporter activity was examined using a dual luciferase assay kit (GeneCopoeia, Rockville, MD, USA). Light emission was measured using a BioTek Synergy H4 hybrid multimode plate reader. TGFb/SMAD signaling activity was measured with pAR3-Lux (a gift from Dr. Jeff Wrana; Addgene plasmid # 24643) and SBE4-Luc (42) (Addgene plasmid #16495) reporter constructs. Control or mir-218-1-overexpressing cells were seeded into 12well plates and were co-transfected with 1 µg pAR3-Lux (or SBE4-Luc) reporter and 20 ng Renilla luciferase vector (pRL-TK, Promega) using Lipofectamine 2000. At 24 hr after transfection, cells were treated with recombinant human TGFb1, TGFb2, TGFb3 (10 ng/ml), or Activin A (50 ng/ml) for 30 min. These concentrations were chosen based on results from previous studies (10,14,43,44). Cell lysates were then collected, and the dual luciferase activity was examined as described above.

Statistical Analysis
All statistical analyses were performed using the GraphPad Prism 8 software. A two-tailed Student's t-test was applied to compare the difference between two groups. One-way ANOVA with Tukey's multiple comparison tests was used for comparisons among multiple groups. Two-way ANOVA with Tukey's multiple comparison tests was used in the wound healing assay. Most experiments were performed in triplicate but wound healing and tube formation experiments had n=5 or more. All experiments were repeated at least 2 times. The Shapiro-Wilk test was used to confirm that all data followed normal distribution before the t-test or ANOVA analysis. No outliers were identified using the ROUT method integrated with the software. Results were considered significant with a p-value less than 0.05.

miR-218-5p Induces IL1b Expression and Secretion
We have previously reported that in HTR-8/SVneo cells, miR-218-5p increased IL1b expression (28). Here, we first verified the upregulation of IL1b production by miR-218-5p. qPCR assay showed that the expression of IL1B mRNA was markedly elevated in HTR-8/SVneo cells stably transfected with mir-218-1, and in HTR-8/SVneo and Swan 71 cells transiently transfected with miR-218-5p mimic, compared to that of the control cells ( Figure 1A). ELISA was also performed to measure IL1b in cell lysates and conditioned media. As shown in Figures 1B, C, IL1b protein levels were increased in both lysates and media harvested from cells that had been transfected with mir-218-1 or miR-218-5p. These results suggest that miR-218-5p induces IL1b expression and secretion.
Using the bioinformatics tool miRanda (47), we identified a potential miR-218-5p binding site in the 3'UTR of the SMAD2 gene; however, no miR-218-5p binding sites were predicted in both the coding region and 3'UTR of SMAD3. We then generated a luciferase reporter construct by inserting a fragment of SMAD2 3'UTR containing the predicted miR-218-5p binding site into the pMIR-REPORT vector, downstream of the luciferase coding sequence. Reporter assays showed that transfection of miR-218-5p mimic inhibited the luciferase activity in both cell lines ( Figure 2E). These results suggest that miR-218-5p directly targets the SMAD2 gene.

IL1b Induces the Acquisition of an enEVT-Like Phenotype
IL1b can enhance the invasive ability of primary EVTs (32); however, whether it is involved in enEVT differentiation is unknown. Therefore, we explored the role of IL1b in the induction of an enEVT-like phenotype. We used recombinant IL1b at concentrations of 1 pg/ml-10 ng/ml in functional assays, a dosage range commonly used in previous studies (32,48,49). We found that IL1b enhanced the ability of trophoblasts to form endothelial-like network structures starting from 1 pg/ml ( Figure 6A). IL1b also elevated the mRNA levels of enEVT markers, including integrin subunit a1 (ITGA1), ITGA5, cadherin 5 (CDH5, also known as vascular endothelial cadherin, VE-Cadherin), and platelet endothelial cell adhesion molecule 1 (PECAM1) in the two cell lines ( Figure 6B) in a dosage range of 1 pg/ml-100 pg/ml. However, IL1b at higher concentrations (1 ng/ml-10 ng/ml) had little effect in the induction of these marker genes (except for CDH5). Taken together, these results suggest that IL1b may promote enEVT differentiation.
Although several studies have shown that IL1b increases trophoblast migration and viability (31)(32)(33), its role in EVT differentiation is unknown. Recently, we reported that miR-218-5p induces enEVT differentiation and increases IL1b expression (28). On the other hand, miR-210-3p inhibits the acquisition of an enEVT phenotype and also reduces IL1b expression (53). In this study, we showed that IL1b increased the expression of several enEVT differentiation-associated markers, such as ITGA1, ITGA5, CDH5, and PECAM1. IL1b also accelerated cell migration and the formation of endothelium-like networks. These findings suggest that IL1b functions as a positive regulator of enEVT differentiation. IL1b has been reported to be released from decidual uterine NK cells, stromal cells, and macrophages (54,55). Previous reports (19,56,57) and this study also revealed that both HTR-8/SVneo and Swan 71 cells expressed and secreted IL1b, supporting paracrine/autocrine effects of IL1b on the acquisition of an enEVT phenotype. Interestingly, we found that except for CDH5, IL1b significantly stimulated the expression of enEVT markers at 1 pg/ml and the maximal effect was observed at a dose of 100 pg/ml or 1 ng/ml, while higher Thus, IL1b likely promotes enEVT differentiation only under physiological conditions, yet a high-level IL1b may have adverse or even detrimental outcomes. Although inflammation is a critical component during normal pregnancies, maintaining a physiological balance of pro-and anti-inflammatory cytokines is essential for a successful pregnancy. As a major proinflammatory cytokine, high levels of IL1b may directly participate in the extensive inflammatory response that is correlated with pregnancy complications including PE (58,59). Further, IL1b is known to act as a potential mediator of endothelial dysfunction by inducing structural and functional alterations in endothelial cells (59)(60)(61), which is a hallmark of the maternal syndrome in PE.
We have previously reported that miR-218-5p expression is lower in PE placentas than in healthy controls and that this miRNA enhances enEVT differentiation and spiral artery remodeling by targeting TGFb2 ligand (28). In this study, overexpression of mir-218-1 or treatment with miR-218-5p mimic decreased SMAD2 at both mRNA and protein levels in HTR-8/SVneo and Swan 71 trophoblasts. The reporter assay verified the direct binding of miR-218-5p to the 3'UTR of the SMAD2 gene. Interestingly, in mir-218-1-overexpressing cells, both endogenous and transiently overexpressed exogenous SMAD2 protein levels were lower than those of the control cells. Since the SMAD2 expression construct does not contain a 3'UTR, this decrease cannot be explained by the binding of miR-218-5p to the SMAD2 3' UTR. Therefore, it is likely that miR- 218-5p also regulates the stability of SMAD2. Furthermore, we found that SMAD7, which can inhibit SMAD2/3 activation by the TGFb family (45,46), was significantly upregulated by miR-218-5p. Although the role of miR-218-5p in regulating SMAD7 expression and SMAD2 protein stability requires further investigation, these findings suggest that miR-218-5p inhibits SMAD2 activity via multiple direct and indirect actions. To validate that miR-218-5p modulates cellular behaviors of trophoblasts through inhibition of SMAD2, we performed a series of functional assays in the two cell lines. We found that SMAD2 overexpression reduced the migration and the ability to form endothelium-like networks in both control and miR-218-5p-treated trophoblasts. On the other hand, SMAD2 knockdown increased the formation of the endothelial networks in control and anti-miR-218-5p-treated cells. These data suggest that miR-218-5p stimulates the acquisition of an enEVT-like phenotype by targeting both TGFb2 and SMAD2, thus leading to impaired TGFb/SMAD2 signaling. In this study, we found that both HTR-8/SVneo and Swan 71 cells treated with miR-218-5p mimic or transfected with mir-218-1 transgene displayed increased expression/secretion of IL1b. In contrast, TGFb2 treatment reduced IL1b production, whereas siTGFB2 induced IL1b protein level. In a previous study, we showed that SMAD2 knockdown in HTR-8/SVneo cells stimulates the expression of several genes involved in trophoblast differentiation and function, such as MMP1, CDH5, IL8, and IL1B (19). Here, we confirmed that silencing of SMAD2 upregulated IL1B mRNA in two trophoblast cell lines. Further, we showed that SMAD2 knockdown attenuated the inhibitory effect of TGFb2, while SMAD2 overexpression abolished the stimulatory effect of miR-218-5p, on IL1B expression. These findings, together with the inhibition of TGFb2 and SMAD2 by miR-218-5p, indicate that miR-218-5p induces IL1B by downregulating the TGFb2/SMAD2 pathway.
Several studies have reported opposing actions of TGFb and IL1b, particularly in immune and hematopoietic systems (62)(63)(64). For example, TGFb1 inhibits IL1b-induced IL6 and IL17 in monocytes and CD4+ T cells, respectively (65,66). In mouse calvarial osteoblasts, TGFb abolishes the induction of cyclooxygenase 2 by IL1b (67). TGFb and IL1b also antagonistically modulate apoptosis of corneal myoblasts (68). In trophoblasts, TGFb and IL1b have inhibitory and stimulatory effects, respectively, on cell invasion (32,69,70). In this study, we showed that TGFb and IL1b displayed opposite effects not only on cell migration, but also on the expression of enEVT markers and the formation of endothelial-like networks. Additionally, we demonstrated that TGFb, signaling via SMAD2, inhibited IL1b expression. The mechanism by which TGFb/SMAD2 represses IL1b is not known and remains to be investigated in the future.
Consistent with our recent report (19), we observed that SMAD2 downregulated, while SMAD3 upregulated IL1b in trophoblasts. Although the two SMAD molecules share 92% amino acid sequence identity (71), they are not functionally equivalent and may play non-overlapping or even disparate roles in physiological and pathological conditions. For instance, SMAD3 differs from SMAD2 in static subcellular localization, the ability and sensitivity to transmit TGFb signal, and early lineage specification (72). In pancreatic cancer cells, Rac1 represses the TGFb1-mediated growth inhibition by suppressing SMAD2 but activating SMAD3 (73). Recently, we reported that SMAD2 blocks the acquisition of an enEVT-like phenotype but SMAD3 shows an opposite effect (19). Findings from the present study further support the differential functions of SMAD2 and SMAD3 in this process.
Although SMAD3 upregulates IL1b, a function similar to that of miR-218-5p, we found that SMAD3 mRNA and protein levels were also reduced by miR-218-5p. Unlike the SMAD2 gene that harbors a binding site of miR-218-5p in its 3'UTR, SMAD3 appears not a direct target as no predicted miR-218-5p binding sites were identified in SMAD3 3'UTR and coding region. Hence, miR-218-5p likely downregulates SMAD3 using some indirect unknown mechanisms, possibly via its other target genes. Using an antibody that detects both SMAD2 and SMAD3, we found that the endogenous SMAD2 protein level was much higher than SMAD3 in both trophoblast cell lines, indicating a differential abundance of the two SMAD proteins. It has been shown that the ratio of SMAD2 to SMAD3 is cell type-dependent and may be a determinant for the relative sensitivity of SMAD2 or SMAD3 to TGFb signals (74). For example, a decreased SMAD2/SMAD3 ratio enhances the SMAD3-dependent pathway in response to TGF-b (74). In this study, although SMAD3 upregulates IL1b, its expression is much lower than SMAD2. It is possible that the high SMAD2/SMAD3 ratio ensures that SMAD2, rather than SMAD3, predominantly mediates the innate TGFb signals.
Interestingly, while IL1b is stimulated by miR-218-5p, treatment with IL1b at lower dosages also reduced miR-218-5p expression in HTR-8/SVneo and Swan 71 cells, with the highest concentration tested (10 ng/ml) exhibiting no effect. These findings suggest that IL1b at physiological concentrations exerts negative feedback on miR-218-5p expression to limit its induction of IL1b. This self-regulatory property may be helpful to maintain IL1b at a moderate level to properly modulate trophoblast differentiation. On the other hand, an imbalanced IL1b overproduction, primarily induced under pathological conditions (e.g., infection), may lose its ability to inhibit miR-218-5p and is associated with harmful effects (such as extensive inflammation and endothelial dysfunction) that are implicated in the pathogenesis of PE.
In summary, we have demonstrated that miR-218-5p induces enEVT differentiation in part by inhibiting the TGFb2/SMAD2 pathway, leading to enhanced IL1b expression and secretion. We also identified IL1b-mediated negative feedback on miR-218-5p expression. These findings highlight a novel interactive miR-218-5p/TGFb/SMAD2/IL1b signaling nexus that plays an important role in the acquisition of an enEVT phenotype. To date, although preemptive administration with aspirin, calcium, or metformin can effectively prevent PE, there are no curative treatments for this progressive disorder, and once diagnosed, the only option is delivery (3). As such, understanding the signaling mechanism that underscores enEVT differentiation can facilitate the development of novel therapeutic strategies for the clinical intervention of PE.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.