Human Trophoblast Cell Invasion Research Articles

Growth differentiation factor-11 upregulates matrix metalloproteinase 2 expression by inducing Snail in human extravillous trophoblast cells

The human extravillous trophoblast (EVT) cell invasion is an important process during placentation. Although the placenta is normal tissue, the EVT cells exhibit some features common to cancer cells, including high migratory and invasive properties. Snail and Slug are transcription factors that mediate the epithelial-mesenchymal transition (EMT), a crucial event for cancer cell migration and invasion. It has been shown that GDF-11-induced matrix metalloproteinase 2 (MMP2) expression is required for EVT cell invasion. Whether GDF-11 can regulate Snail and Slug expression in human EVT cells remains unknown. If it does, the involvement of Snail and Slug in GDF-11-induced MMP2 expression and EVT cell invasion must also be defined. In the present study, using the immortalized human EVT cell line, HTR-8/SVneo, and primary cultures of human EVT cells as experimental models, our results show that GDF-11 upregulates Snail and Slug expression. ALK4 and ALK5 mediate the stimulatory effects of GDF-11 on Snail and Slug expression. In addition, we demonstrate that SMAD2 and SMAD3 are required for the GDF-11-upregulated Snail expression, while only SMAD3 is involved in GDF-11-induced Slug expression. Moreover, our results reveal that Snail mediates GDF-11-induced MMP2 expression and cell invasion but not Slug. This study increases our understanding of the biological function of GDF-11 in human EVT cells and provides a novel mechanism for regulating MMP2 and EVT cell invasion.

MiR-3074-5p Regulates Trophoblasts Function via EIF2S1/GDF15 Pathway in Recurrent Miscarriage.

Dysfunction of extravillous trophoblasts (EVTs) might cause early pregnancy failure by interfering with embryo implantation and/or placentation. We previously reported that the villus miR-3074-5p expression level was increased, whereas the peripheral level of GDF15, a predict target gene of miR-3074-5p, was decreased in recurrent miscarriages (RM) patients, and miR-3074-5p could enhance apoptosis but reduce invasion of human extravillous trophoblast cells (EVTs). The aim of this study was to further explore roles of miR-3074-5p/GDF15 pathway in regulation of EVTs function. It was validated that GDF15 was not the direct target of miR-3074-5p, whereas EIF2S1, an upstream regulator of GDF15 maturation and secretion, was the direct target of miR-3074-5p. The villus expression levels of GDF15 and EIF2S1 were significantly decreased in RM patients. Knockdown of GDF15 expression presented inhibitory effects on proliferation, migration, and invasion of HTR8/SVneo cells. Up-regulated miR-3074-5p expression led to the significant decreased GDF15 expression in HTR8/SVneo cells, and this effect could be efficiently reversed by the overexpression of EIF2S1. Meanwhile, the suppressive effects of miR-3074-5p on proliferation, migration, and invasion of HTR8/SVneo cells could be intercepted by the treatment of recombinant human GDF15 protein. Collectively, these data suggested that miR-3074-5p could reduce GDF15 production via targeting inhibition of EIF2S1 expression, and the deficiency in GDF15 function might lead to the early pregnancy loss by attenuating proliferation and invasion of EVTs.

Growth differentiation factor myostatin regulates epithelial-mesenchymal transition genes and enhances invasion by increasing serine protease inhibitors E1 and E2 in human trophoblast cells.

Placental insufficiency disorders, including preeclampsia and intrauterine growth restriction, are major obstetric complications that can have devastating effects on both the mother and the fetus. These syndromes have underlying poor placental trophoblast cell invasion into uterine tissues. Placental invasion is controlled by many hormones and growth factors. Myostatin (MSTN) is a transforming growth factor-β superfamily member recognized for its important role in muscle growth control. MSTN has also been shown to be secreted and functioning in the placenta, and its serum and/or placental levels were found to be upregulated in preeclampsia and intrauterine growth restriction. Considering that the mechanistic role of MSTN in placentation remains poorly understood, we hypothesized that MSTN uses ALK4/5-SMAD2/3/4 signaling to increase human trophoblast invasion through a group of epithelial-mesenchymal transition genes including SERPINE2, PAI-1, and SOX4. mRNA sequencing of control and MSTN-treated primary human trophoblast cells (n = 5) yielded a total of 610 differentially expressed genes (false discovery rate <0.05) of which 380 genes were upregulated and 230 were downregulated. These differentially expressed genes were highly enriched in epithelial-mesenchymal transition genes, and a subset including SERPINE2, PAI-1, and SOX4 was investigated for its role in MSTN-induced trophoblast cell invasion. We found that MSTN induced upregulation of SERPINE2 via ALK4/5-SMAD2/3/4 signaling; however, SMAD2 was not involved in MSTN-induced PAI-1 upregulation. SOX4 was involved in MSTN-induced upregulation of SERPINE2, but not PAI-1. Collectively, this study discovers novel molecular mechanisms of MSTN-induced human trophoblast cell invasion and provides insight into the functional consequences of its dysregulation in placental insufficiency disorders.

USP8 targeted by Mir-874-3p promotes trophoblastic cell invasion by stabilizing the expression of ENaC on trophoblast membrane

The aim of this study was to investigate the role of ubiquitin-specific peptidase 8 (USP8) in human trophoblast cells and its molecular mechanism. Based on the GSE30186 dataset, USP8 was identified as a downregulated gene in pre-eclampsia (PE). Analysis of clinical samples also revealed that USP8 expression at both the mRNA and protein levels in placental tissue from patients with PE was significantly lower than that from healthy pregnant women. Plate clone formation, scratch-wound healing, Transwell, tubule formation, and western blot assays collectively revealed that USP8 overexpression promoted the proliferation, migration, invasion, and pro-angiogenesis function of trophoblast cells, while USP8 knockdown induced the opposite effects. Bioinformatics analysis and luciferase reporter assay results indicated that the 3′ untranslated region of USP8 was targeted by miR-874-3p. USP8 expression in the placental tissue of patients with PE was significantly lower than that of healthy pregnant women. USP8 actively regulated the growth and invasion of human trophoblast cells and stabilized the epithelial sodium channel (ENaC) on the cell membrane. MiR-874 targeted USP8 in the trophoblast cells and upregulation of miR-874-3p resulted in a decrease in the proliferation, migration, invasion, and pro-angiogenesis ability of trophoblast cells. These results indicate that USP8 can reverse the above mentioned negative effects of miR-874-3p on trophoblast cells. USP8 targeted by miR-874-3p facilitates the invasion of trophoblastic cells by stabilizing the expression of the ENaC, which may be a possible therapeutic target for PE.

WNK1 mediates amphiregulin-induced MMP9 expression and cell invasion in human extravillous trophoblast cells

The invasion of human extravillous trophoblast (EVT) cells is a critical event required for a successful pregnancy. Amphiregulin, a ligand of the epidermal growth factor receptor (EGFR), has been shown to stimulate cell invasion in an immortalized human EVT cell line, HTR-8/SVneo. The with-no-lysine kinase 1 (WNK1) is involved in regulating cell invasion. It is known that WNK1 is expressed in the human placenta, but its role in human EVT cells remains unknown. In the present study, we show that AREG treatment phosphorylated WNK1 at Thr60 in both HTR-8/SVneo and primary human EVT cells. The stimulatory effect of AREG on WNK1 phosphorylation was mediated by the activation of PI3K/AKT, but not the ERK1/2 signaling pathway. AREG upregulated matrix metalloproteinase 9 (MMP9) but not MMP2. In addition, cell invasiveness was increased in response to the treatment of AREG. Using the siRNA-mediated knockdown approach, our results showed that the knockdown of WNK1 attenuated the AREG-induced upregulation of MMP9 expression and cell invasion. Moreover, the expression of WNK1 was downregulated in the placentas with preeclampsia, a disease resulting from insufficiency of EVT cell invasion during pregnancy. This study discovers the physiological function of WNK1 in human EVT cells and provides important insights into the regulation of MMP9 and cell invasion in human EVT cells.

Snail mediates GDF-8-stimulated human extravillous trophoblast cell invasion by upregulating MMP2 expression

BackgroundExtravillous trophoblast (EVT) cell invasion is a tightly regulated process that requires for a normal pregnancy. The epithelial-mesenchymal transition (EMT) has been implicated in EVT cell invasion. Growth differentiation factor-8 (GDF-8), a member of the transforming growth factor-beta (TGF-β) superfamily, is expressed in the human placenta and promotes EVT cell invasion by upregulating the expression of matrix metalloproteinase 2 (MMP2). However, the underlying molecular mechanism of GDF-8-induced MMP2 expression remains undetermined. Therefore, the present study aims to examine the role of Snail and Slug, the EMT-related transcriptional regulators, in GDF-8-stimulated MMP2 expression and cell invasion in HTR-8/SVneo human EVT cell line and primary cultures of human EVT cells.MethodsHTR-8/SVneo and primary cultures of human EVT cells were used to examine the effect of GDF-8 on MMP2 expression and explore the underlying mechanism. For gene silencing and overexpression, the HTR-8/SVneo cell line was used to make the experiments more technically feasible. The cell invasiveness was measured by Matrigel-coated transwell invasion assay.ResultsGDF-8 stimulated MMP2 expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effect of GDF-8 on MMP2 expression was blocked by the inhibitor of TGF-β type-I receptors, SB431542. Treatment with GDF-8 upregulated Snail and Slug expression in both HTR-8/SVneo and primary EVT cells. The stimulatory effects of GDF-8 on Snail and Slug expression were blocked by pretreatment of SB431542 and siRNA-mediated knockdown of SMAD4. Interestingly, using the siRNA knockdown approach, our results showed that Snail but not Slug was required for the GDF-8-induced MMP2 expression and cell invasion in HTR-8/SVneo cells. The reduction of MMP2 expression in the placentas with preeclampsia (PE) was also observed.ConclusionsThese findings discover the physiological function of GDF-8 in the human placenta and provide important insights into the regulation of MMP2 expression in human EVT cells.1EyNAiCz9UDZ9srU6xtfDxVideo

Blockade of stromal cell-derived factor-1 signaling disturbs the invasiveness of human extravillous trophoblast cells

Blockade of stromal cell-derived factor-1 signaling disturbs the invasiveness of human extravillous trophoblast cells

BPDE, the Migration and Invasion of Human Trophoblast Cells, and Occurrence of Miscarriage in Humans: Roles of a Novel lncRNA-HZ09.

Recurrent miscarriage (RM) affects 1%-3% of pregnancies. However, in almost 50% of cases, the cause is unknown. Increasing evidence have shown that benzo(a)pyrene [B(a)P], a representative of polycyclic aromatic hydrocarbons (PAHs), is correlated with miscarriage. However, the underlying mechanisms of B(a)P/benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-induced trophoblast cell dysfunctions and miscarriage remain largely unknown. The objective was to discover the role(s) of a novel lncRNA, lnc-HZ09, in the regulation of BPDE-inhibited migration and invasion of trophoblast cells and the occurrence of miscarriage. Human trophoblast cells were treated with 0, 0.25, 0.5, BPDE with or without corresponding lnc-HZ09 silencing or overexpression. Using these cells, we evaluated cell migration and invasion, the mRNA and protein levels of members of the PLD1/RAC1/CDC42 pathway, the regulatory roles of lnc-HZ09 in PLD1 transcription and mRNA stability, and lnc-HZ09 transcription and stability. Human villous tissues were collected from RM () group and their matched healthy control (HC, ) group. We evaluated the levels of BPDE-DNA adducts, lnc-HZ09, and the mRNA and protein expression of members of the PLD1/RAC1/CDC42 pathway, and correlated their relative expression levels. We further constructed 0, 0.05 or B(a)P-induced mouse miscarriage model (each ), in which the mRNA and protein expression of members of the Pld1/Rac1/Cdc42 pathway were measured. We identified a novel lnc-HZ09. Human trophoblast cells treated with lnc-HZ09 exhibited less cell migration and invasion. In addition, the levels of this lncRNA were higher in villous tissues from women with recurrent miscarriage than those from healthy individuals. SP1-mediated PLD1 mRNA levels were lower, and HuR-mediated PLD1 mRNA stability was less in trophoblast cells overexpressing lnc-HZ09. However, trophoblast cells treated with MSX1 had higher levels of lnc-HZ09, and METTL3-mediated m6A methylation on lnc-HZ09 resulted in greater lnc-HZ09 RNA stability. In BPDE-treated human trophoblast cells and in RM villous tissues, MSX1-mediated lnc-HZ09 transcription and METTL3-mediated lnc-HZ09 stability were both greater. In our mouse miscarriage model, B(a)P-treated mice had lower mRNA and protein levels of members of the Pld1/Rac1/Cdc42 pathway. These results suggest that in human trophoblast cells, BPDE exposure up-regulated lnc-HZ09 level, suppressed PLD1/RAC1/CDC42 pathway, and inhibited migration and invasion, providing new insights in understanding the causes and mechanisms of unexplained miscarriage. https://doi.org/10.1289/EHP10477.

EGF promotes human trophoblast cell invasion by downregulating CTGF expression via PI3K/AKT signaling.

Although the pro-invasive role of epidermal growth factor (EGF) has been reported in human trophoblast cells, the underlying mechanism remains largely unexplored. This work reveals that EGF-induced downregulation of connective tissue growth factor (CTGF) mediates the EGF-stimulated human trophoblast cell invasion. During the development of the placenta, trophoblast cell invasion must be carefully regulated. Although EGF has been shown to promote trophoblast cell invasion, the underlying mechanism remains largely undetermined. Our previous study using RNA-sequencing (RNA-seq) has identified that kisspeptin-1 is a downstream target of EGF in a human trophoblast cell line, HTR-8/SVneo, and mediates EGF-stimulated cell invasion. In the present study, after re-analysis of our previous RNA-seq data, we found that the CTGF was also downregulated in response to the EGF treatment. The inhibitory effects of EGF on CTGF mRNA and protein levels were confirmed in HTR-8/SVneo cells by reverse transcription quantitative real-time PCR and western blot, respectively. Treatment with EGF activated both PI3K/AKT and ERK1/2 signaling pathways. Using pharmacological inhibitors, our results showed that EGFR-mediated activation of PI3K/AKT signaling was required for the EGF-downregulated CTGF mRNA and protein levels. Matrigel-coated transwell invasion assays demonstrated that EGF treatment stimulated cell invasion. In addition, the invasiveness of HTR-8/SVneo cells was suppressed by treatment with recombinant human CTGF. By contrast, siRNA-mediated knockdown of CTGF increased cell invasion. Notably, the EGF-promoted HTR-8/SVneo cell invasion was attenuated by co-treatment with CTGF. This study provides important insights into the molecular mechanisms mediating EGF-stimulated human trophoblast cell invasion and increases the understanding of the biological functions of CTGF in the human placenta.

MiR-195-5p facilitates the proliferation, migration, and invasion of human trophoblast cells by targeting FGF2.

Preeclampsia (PE), the most significant adverse exposure to cardiovascular risk during pregnancy, is one of the three major factors contributing to maternal and fetal mortality and the leading cause of preterm birth. Recently, various miRNAs have been reported to participate in PE occurrence and development. Nevertheless, the regulatory impact of miR-195-5p in PE is still indistinct. Quantitative realtime-PCR (qRT-PCR), western blot, and fluorescence in situ hybridization (FISH) assay were performed to examine miR-195-5p and FGF2 expressions in PE serum samples or HTR-8/SVneo and TEV-1 cells. CCK8, flow cytometry, wound scratch, and transwell assays were conducted to determine cell viability, cycle, apoptosis, migration, and invasion. Dual-luciferase reporter assay unveiled the relationship between miR-195-5p and FGF2. Migration-related and invasion-related protein expressions were measured by western blot assay. miR-195-5p was prominently downregulated while FGF2 was increased in serum samples from PE patients and hypoxia-treated human trophoblast cells. FGF2 was predicted as a downstream target of miR-195-5p and targeted association was verified by dual-luciferase reporter assay. Functional experiments elaborated that miR-195-5p could facilitate trophoblast cell proliferation and metastasis but hinder cell cycle and apoptosis. Inversely, overexpressing of FGF2 could reverse the effects of miR-195-5p on trophoblast cell growth. miR-195-5p was decreased in PE serum samples and cell lines, serving as a potential biomarker in protecting PE exacerbation by targeting FGF2.

GDF-11 promotes human trophoblast cell invasion by increasing ID2-mediated MMP2 expression

BackgroundGrowth differentiation factor-11 (GDF-11), also known as bone morphogenetic protein-11, belongs to the transforming growth factor-beta superfamily. GDF-11 was first identified as an important regulator during embryonic development. Increasing evidence has demonstrated that GDF-11 regulates the development of various organs and its aberrant expressions are associated with the risk of cardiovascular diseases and cancers. Extravillous trophoblast (EVT) cells invasion is a critical event for placenta development and needs to be finely regulated. However, to date, the biological function of GDF-11 in the human EVT cells remains unknown.MethodsHTR-8/SVneo, a human EVT cell line, and primary cultures of human EVT cells were used to examine the effect of GDF-11 on matrix metalloproteinase 2 (MMP2) expression. Matrigel-coated transwell invasion assay was used to examine cell invasiveness. A series of in vitro experiments were applied to explore the underlying mechanisms that mediate the effect of GDF-11 on MMP2 expression and cell invasion.ResultsTreatment with GDF-11 stimulates MMP2 expression, in the HTR-8/SVneo and primary human EVT cells. Using a pharmacological inhibitor and siRNA-mediated knockdown approaches, our results demonstrated that the stimulatory effect of GDF-11 on MMP2 expression was mediated by the ALK4/5-SMAD2/3 signaling pathways. In addition, the expression of inhibitor of DNA-binding protein 2 (ID2) was upregulated by GDF-11 and that was required for the GDF-11-stimulated MMP2 expression and EVT cell invasion.ConclusionsThese findings discover a new biological function and underlying molecular mechanisms of GDF-11 in the regulation of human EVT cell invasion.Ede8tEoyGya1QPPVVUV7JYVideo

CircFBXW4 regulates human trophoblast cell proliferation and invasion via targeting miR-324–3p/TJP1 axis in recurrent spontaneous abortion

IntroductionIncreasing evidence has shown that circular RNAs (circRNAs) play vital roles in embryonic development. However, the function of circRNAs in recurrent spontaneous abortion (RSA) is largely unknown. This study aimed to investigate the expression profile of human circRNAs and their functional mechanisms in regulating RSA. MethodsThe profiles of circRNAs in placental villus tissues from women with RSA and healthy pregnancy with induced abortion were investigated using RNA-sequencing and bioinformatics. Nine circRNAs were verified in the 50 placental villus samples. RNase R digestion, actinomycin D treatment, and fluorescence in situ hybridization were performed to characterize circFBXW4. Furthermore, direct binding of circFBXW4 to miR-324–3p was confirmed by dual-luciferase reporter assay. The roles of circFBXW4 were determined by loss- and gain-of-function assays including cell proliferation, invasion, and apoptosis using CCK8 kit, transwell migration assay, and TUNEL kit in vitro, respectively. ResultsA total of 417 aberrantly expressed circRNAs was detected. circFBXW4, a circRNA significantly up-regulated in the RSA group, was further evaluated. circFBXW4 showed higher stability than FBXW4 mRNA and was localized in the cytoplasm and nucleus in HTR-8/SVneo cells. MiR-324–3p was lowly expressed in the RSA group, and directly regulated circFBXW4 and TJP1 expression in a targeted manner. Overexpression and knockdown of circFBXW4 and miR-324–3p mimic/inhibitor could increase or decrease HTR-8/SVneo cell proliferation and invasion. circFBXW4 regulated TJP1 expression, cell proliferation, and invasion by sponging miR-324–3p. DiscussionThe circFBXW4/miR-324–3p/TJP1 axis is involved in the occurrence and progression of RSA and may be a promising therapeutic target in RSA.

MiR-187 regulates the proliferation, migration and invasion of human trophoblast cells by repressing BCL6-mediated activation of PI3K/AKT signaling.

Recurrent miscarriage (RM), refers to two or more consecutive spontaneous miscarriage in a pregnant woman. RM is caused by many factors, and microRNAs play an important role in the development and pathology of RM. In the present study, we investigated the function of miR-187 in the pathogenesis of RM and its effects on human trophoblast cells. The localization of miR-187 in the human placenta in early pregnancy was determined by in situ hybridization. QRT-PCR was used to detect the expression of miR-187 in villi of normal early pregnancy induced abortion group and recurrent spontaneous miscarriage group. Then, HTR8/SVneo cells were used to investigated the effect of miR-187 on BCL6 expression and biological activity of trophoblasts. We found that the expression of miR-187 in villi of RM group was higher than that of normal abortion group and miR-187 inhibited the proliferation, migration, and invasion of HTR8 cells. We also found that miR-187 promoted apoptosis, inhibited EMT, and inhibited the PI3K/AKT pathway in HTR8 cells. In addition, we also found that BCL6 is a direct target of miR-187 and is negatively regulated by miR-187. In addition, BCL6 reversed the inhibitory effects of miR-187 on HTR8/SVneo cells. These data demonstrate that miR-187-induced repression of PI3K/AKT signaling is mediated by BCL6 in HTR8 cells. MiR-187 inhibits the proliferation, migration, and invasion of trophoblasts through a mechanism that involves regulation of BCL6.

Gene Expression Network Analysis Identifies Potential Targets for Prevention of Preeclampsia.

ObjectivePreeclampsia (PE) is a pregnancy-specific multisystem disease as well as an important cause of maternal and perinatal death. This study aimed to analyze the placental transcriptional data and clinical information of PE patients available in the published database and predict the target genes for prevention of PE.MethodsThe clinical information and corresponding RNA data of PE patients were downloaded from the GEO database. Cluster analysis was performed to examine the correlation between different genotyping genes and clinical manifestations. Then, bioinformatic approaches including GO, KEGG, WGCNA, and GSEA were employed to functionally characterize candidate target genes involved in pathogenesis of PE.ResultsTwo PE datasets GSE60438 and GSE75010 were obtained and combined, thereby providing the data of 205 samples in total (100 non-PE and 105 PE samples). After eliminating the batch effect, we grouped and analyzed the integrated data, and further performed GSEA analysis. It was found that the genes in group 1 and group 2 were different from those in normal samples. Moreover, WGCNA analysis revealed that genes in group 1 were up-regulated in turquoise module, including SASH1, PIK3CB and FLT-1, while genes in group 2 were up-regulated in the blue and brown modules. We further conducted GO and KEGG pathway enrichment analyses and found that the differential genes in turquoise module were mainly involved in biological processes such as small molecular catabolic process, while being highly enriched in pathways, including MAPK signaling pathway and Rap1 signaling pathway.ConclusionFLT-1 was conventionally used to predict PE risk, and sFLT-1 could also be used as an indicator to evaluate PE treatment effect. As a candidate biomarker for predicting PE, SASH1 may participate in proliferation, migration, invasion and epithelial mesenchymal transformation of human trophoblast cells by regulating MAPK pathway and Rap1 signaling pathway, thus affecting the progression of PE. The mechanism allowing PIK3CB to regulate PE development was not clear, while the gene could be another candidate biomarker for PE risk prediction. This is an exploratory study and our findings were still required verification in further studies.

TGF-\u03b21 inhibits human trophoblast cell invasion by upregulating kisspeptin expression through ERK1/2 but not SMAD signaling pathway

BackgroundTightly regulation of extravillous cytotrophoblast (EVT) cell invasion is critical for the placentation and establishment of a successful pregnancy. Insufficient EVT cell invasion leads to the development of preeclampsia (PE) which is a leading cause of maternal and perinatal mortality and morbidity. Transforming growth factor-beta1 (TGF-β1) and kisspeptin are expressed in the human placenta and have been shown to inhibit EVT cell invasion. Kisspeptin is a downstream target of TGF-β1 in human breast cancer cells. However, whether kisspeptin is regulated by TGF-β1 and mediates TGF-β1-suppressed human EVT cell invasion remains unclear.MethodsThe effect of TGF-β1 on kisspeptin expression and the underlying mechanisms were explored by a series of in vitro experiments in a human EVT cell line, HTR-8/SVneo, and primary cultures of human EVT cells. Serum levels of TGF-β1 and kisspeptin in patients with or without PE were measured by ELISA.ResultsTGF-β1 upregulates kisspeptin expression in HTR-8/SVneo cells and primary cultures of human EVT cells. Using pharmacological inhibitor and siRNA, we demonstrate that the stimulatory effect of TGF-β1 on kisspeptin expression is mediated via the ALK5 receptor. Treatment with TGF-β1 activates SMAD2/3 canonical pathways as well as ERK1/2 and PI3K/AKT non-canonical pathways. However, only inhibition of ERK1/2 activation attenuates the stimulatory effect of TGF-β1 on kisspeptin expression. In addition, siRNA-mediated knockdown of kisspeptin attenuated TGF-β1-suppressed EVT cell invasion. Moreover, we report that serum levels of TGF-β1 and kisspeptin are significantly upregulated in patients with PE.ConclusionsBy illustrating the potential physiological role of TGF-β1 in the regulation of kisspeptin expression, our results may serve to improve current strategies used to treat placental diseases.

Myostatin increases human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling

Placental insufficiency disorders are major obstetric complications that share a common phenomenon of poor placental trophoblast cell invasion and remodeling of uterine tissues. Myostatin is a transforming growth factor (TGF)-β superfamily member well known for its important role in muscle growth control. Myostatin is also produced in the placenta and has been shown to regulate some trophoblast functions. However, its roles in placental development are still poorly understood. In this study, we tested the hypothesis that myostatin increases trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling. Primary and immortalized (HTR8/SVneo) trophoblast cells were used as study models. Matrigel-coated transwell invasion assays were used to study the effects of recombinant human myostatin on trophoblast cell invasion. Reverse transcription quantitative real-time polymerase chain reaction and Western blot were used to measure myostatin effects on N-cadherin mRNA and protein levels, respectively. Small inhibitor molecules as well as siRNA-mediated knockdown were used to block myostatin receptor and downstream signaling, respectively. Data were analyzed either by unpaired Student T test or one-way analysis of variance followed by Newman Keuls test for multiple group comparisons. Myostatin significantly increased primary and HTR8/SVneo trophoblast cell invasion. Moreover, myostatin upregulated N-cadherin mRNA and protein levels in a time-dependent manner in both study models. These effects were blocked by inhibition of TGF-β type I receptors as well as siRNA-mediated knockdown of SMAD2/3 combined or common SMAD4. Importantly, myostatin-induced trophoblast cell invasion was abolished by knockdown of N-cadherin, SMAD2/3, or SMAD4. Myostatin may increase human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling.

TCDD-induced IL-24 secretion in human chorionic stromal cells inhibits placental trophoblast cell migration and invasion

Environmental pollutant dioxins are potentially harmful to pregnant women and can lead to severe adverse outcomes in pregnancy, such as spontaneous abortion and stillbirth. However, little is currently known about the underlying toxicological mechanism. Our previous study reported that the IL-24 gene is a dioxin response gene during 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) treatment. Here, we further tested the effect of TCDD on IL-24 expression in human chorionic stromal cells. We also investigated the effect of IL-24 on the behaviors of human placental trophoblast cells and predicted the potential mechanism underlying these behaviors using functional network analysis. We found that TCDD stimulates IL-24 expression in human chorionic stromal cells in an AhR (aromatic hydrocarbon receptor)-related manner. We also found that IL-24 inhibits the migration and invasion of human placental trophoblast cells, the possible mechanism of which involves thirteen key proteins and mitochondrial function. Our findings suggest that IL-24 is a potential factor induced by TCDD to regulate trophoblast cell invasion, which potentially involves in TCDD-induced abortion.

G protein-coupled estrogen receptor stimulates human trophoblast cell invasion via YAP-mediated ANGPTL4 expression

Insufficient invasion of trophoblast cells into the uterine decidua is associated with preeclampsia (PE). G protein-coupled estrogen receptor (GPER) is a membrane estrogen receptor involved in non-genomic estrogen signaling. GPER is expressed in human trophoblast cells and downregulated GPER levels are noted in PE. However, to date, the role of GPER in trophoblast cells remains largely unknown. Here, we applied RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to G1, an agonist of GPER, and identified angiopoietin-like 4 (ANGPTL4) as a target gene of GPER. Treatment of trophoblast cells with G1 or 17β-estradiol (E2) activated Yes-associated protein (YAP), the major downstream effector of the Hippo pathway, via GPER but in a mammalian STE20-like protein kinase 1 (MST1)-independent manner. Using pharmacological inhibitors as well as loss- and gain-of-function approaches, our results revealed that YAP activation was required for GPER-stimulated ANGPTL4 expression. Transwell invasion assays demonstrated that activation of GPER-induced ANGPTL4 promoted cell invasion. In addition, the expression levels of GPER, YAP, and ANGPTL4 were downregulated in the placenta of patients with PE. Our findings reveal a mechanism by which GPER exerts its stimulatory effect on human trophoblast cell invasion by upregulating YAP-mediated ANGPTL4 expression.

GDF-8 stimulates trophoblast cell invasion by inducing ALK5-SMAD2/3-mediated MMP2 expression.

Matrix metalloproteinases (MMPs) play a pivotal role in the regulation of cell invasion. Placental trophoblast cell invasion is a precisely regulated event. Dysregulation of MMPs has been linked to various placental diseases. Growth differentiation factor-8 (GDF-8), also known as myostatin, is a member of the transforming growth factor-beta (TGF-β) superfamily. GDF-8 and its putative receptors are expressed in human extravillous cytotrophoblast cells (EVTs). Although the pro-invasive effect of GDF-8 in human EVT cells has been recently reported, the underlying molecular mechanism remains largely unknown. In this study, we investigate the effects of GDF-8 on the expression of the two most important MMPs, MMP2 and MMP9, in the HTR-8/SVneo human EVT cell line. Our results show that GDF-8 significantly upregulates the expression of MMP2. The expression of MMP9 is not affected by GDF-8. Using a siRNA-mediated knockdown approach, we reveal that the stimulatory effect of GDF-8 on MMP2 expression is mediated by the ALK5-SMAD2/3 signaling pathway. Additionally, the knockdown of MMP2 attenuates the GDF-8-induced cell invasiveness. These findings deepen our understanding of the biological roles of GDF-8 in the regulation of human trophoblast cell invasion.

EGF stimulates human trophoblast cell invasion by downregulating ID3-mediated KISS1 expression

BackgroundDuring pregnancy, trophoblast cell invasion needs to be finely controlled. Aberrant trophoblast cell invasion is associated with placental diseases. Epidermal growth factor (EGF) and its receptor, EGFR, are expressed in trophoblast cells. Although the pro-invasive effect of EGF on trophoblast cells has been reported, the underlying mechanism remains largely unknown.ResultsIn the present study, we conducted an RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to EGF and identified KISS1 as a target gene of EGF. The human KISS1 gene encodes kisspeptin, also known as metastin, which can suppress tumor metastasis. Our results showed that EGF treatment downregulated KISS1 expression and secretion by activating the EGFR-mediated PI3K/AKT signaling pathway. In addition, the expression of inhibitor of DNA-binding protein 3 (ID3) was downregulated by EGF and that was required for the EGF-suppressed KISS1 expression. Functionally, transwell invasion assays demonstrated that EGF stimulated human trophoblast cell invasion by downregulating KISS1 expression. Preeclampsia (PE) is a placental disease characterized by insufficient trophoblast cell invasion. Our clinical results revealed that serum levels of EGF were downregulated while serum and placental levels of KISS1 were upregulated in PE patients.ConclusionsThis study demonstrates that downregulation of EGF can lead to poor trophoblast cell invasion by increasing KISS1 expression which subsequently contributes to the pathogenesis of PE.3yX-DcuH29NawtDrdPoN74Video