SVneo Cell Line Research Articles

Revealing ANXA6 as a Novel Autophagy-related Target for Pre-eclampsia Based on the Machine Learning

Background: Preeclampsia (PE) is a severe pregnancy complication associated with autophagy. Objective: This research sought to uncover autophagy-related genes in pre-eclampsia through bioinformatics and machine learning. Methods: GSE75010 from the GEO series was subjected to WGCNA to identify key modular genes in PE. Autophagy genes retrieved from the THANATOS overlapped with the modular genes to yield PErelated autophagy genes. Furthermore, the crucial step involved the utilization of two machine learning algorithms (LASSO and SVM-RFE) for dimensionality reduction. The candidate gene was further verified by quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry. Preliminary experiments were conducted on HTR-8/SVneo cell lines to explore the role of candidate genes in autophagy regulation. Results: WGCNA identified 291 genes from 5 hubs, and after overlapping with 1087 autophagy-related genes obtained from THANATOS, 42 PE-related ARGs were identified. ANXA6 was recognized as a potential target through SVM-RFE and LASSO analyses. The mRNA and protein expression of ANXA6 were verified in placenta samples. In HTR8/SVneo cells, modulating ANXA6 expression altered autophagy levels. Knocking down ANXA6 resulted in an anti-autophagy effect, which was reversed by treatment with CAL101, an inhibitor of PI3K, Akt, and mTOR. Conclusion: We observed that ANXA6 may serve as a possible PE action target and that autophagy may be crucial to the pathogenesis of PE.

IGF2BP3 participates in the pathogenesis of recurrent spontaneous abortion by regulating ferroptosis

The aberrant invasive capability of trophoblast cells is widely acknowledged as a primary mechanism underlying RSA. Recently, IGF2BP3 has been implicated in various cancers due to its influence on cellular invasion and migration. However, whether IGF2BP3 involve in the occurrence of RSA and the specific functions it assumes in the development of RSA remain elusive. In our study, we firstly collected villous tissues from RSA and those with normal pregnancies individuals to performed Protein sequencing and then detected the expression of IGF2BP3 through Western blot, qRT-PCR and immunohistochemistry. Secondly, we analyzed the single-cell data (GSE214607) to assess the expression of IGF2BP3 in invasive EVT trophoblasts. Thirdly, we utilized lentivirus technology to establish HTR-8/SVneo cell lines with stable IGF2BP3 knockdown and RNA-seq analysis was employed to investigate the GO functional pathway enrichment of IGF2BP3. Meanwhile, the effect of IGF2BP3 knockdown on trophoblast cells apoptosis, migration, and ferroptosis was evaluated through functional experiments. Additionally, LPS-induced abortion animal model was constructed to evaluate IGF2BP3 expression in placental tissues. A significant downregulation of IGF2BP3 was observed in the villous tissues of RSA patient, a finding corroborated by subsequent single cell sequencing results. Furthermore, it suggested that IGF2BP3 may be involved in the migration and apoptotic processes of trophoblast cells. Mechanistic research indicated that IGF2BP3 knockdown could compromise GPX4 mRNA stability, leading to the promotion of ferroptosis. Finally, our investigation observed the down-regulation of IGF2BP3 expression in placental villous tissues of an LPS-induced abortion animal model. Our findings revealed that IGF2BP3 was downregulated in the villous tissues of RSA patients. Mechanically, down-regulation of IGF2BP3 may induce RSA by promoting GPX4-mediated ferroptosis and inhibiting trophoblast invasion and migration. Our study may provide new targets and research directions for the pathogenesis of RSA.

Effects of individual drug and combination antiretroviral therapy on trophoblast proliferation

BackgroundCombination antiretroviral therapy (cART) has been reported to reduce perinatal transmission of human immunodeficiency virus (HIV) and improve maternal survival outcomes. Recent studies have associated in-utero exposure to cART drugs with adverse outcomes such as pre-eclampsia, preterm delivery, low birth weight and small-for-gestational-age births. However, the exact molecular mechanisms underlying cART-induced adverse pregnancy outcomes remain poorly defined. ObjectivesTo investigate the effects of cART drugs on trophoblast proliferation in the HTR-8/SVneo cell line. Study designHTR-8/SVneo cells were exposed to tenofovir (0.983–9.83 µM), emtricitabine (0.809–8.09 µM) and efavirenz (0.19–1.09 µM), the individual drugs of the first-line single tablet cART regimen termed ‘Atripla’, and zidovudine (1.12–1.12 µM), lamivudine (0.65–6.5 µM), lopinavir (0.32–3.2 µM) and ritonavir (0.69–6.9 µM), the individual drugs of the second-line single tablet cART regimen termed ‘Aluvia’. The cells were treated for 24, 48, 72 and 96 h, and trophoblast proliferation was assessed using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltretrazolium bromide assay. ResultsTwo-way analysis of variance showed a significant dose-dependent decrease (p < 0.05) in trophoblast proliferation in response to individual and combined drug components of first- and second-line antiretroviral therapy. ConclusionsFirst- and second-line cART drugs inhibit trophoblast proliferation, and may contribute to placenta-mediated adverse pregnancy outcomes in patients with HIV.

Mono-(2-ethylhexyl) phthalate induces trophoblast hypoxia and mitochondrial dysfunction through HIF-1α-miR-210-3p axis in HTR-8/SVneo cell line

The exposure to the ubiquitous phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) is connected to dysregulated trophoblast function and placenta health; however, the underlying mechanisms preluding this scenario remain to be elucidated. In this study, we explored the hypoxemic effects of MEHP on a human placental first-trimester trophoblast cell line (HTR-8/Svneo). MEHP-treated trophoblast cells displayed significantly increased levels of oxidative stress and hypoxia-inducible factor-1 alpha (HIF-1α) attributed by the induction of hypoxia. Further, HIF-1α exhibited higher DNA binding activity and upregulated gene expression of its downstream target vascular endothelial growth factor A (VEGFA). The hypoxia-induced microRNA miR-210-3p was also significantly increased upon MEHP treatment followed by disrupted mitochondrial ATP generation and membrane potential. This was identified to possibly be facilitated by lowered mitochondrial DNA copy number and inhibited expression of electron transport chain subunits, such as mitochondrial complex-IV. These results suggest potential adverse effects of MEHP exposure in a trophoblast cell line mediated by HIF-1α and the epigenetic modulator miR-210-3p. Chronic placental hypoxia and oxidative stress have long been implicated in the pathogenesis of pregnancy complications such as preeclampsia. As we’ve revealed genetic and epigenetic factors underscoring a potential mechanism induced by MEHP, this brings to light another significant implication of phthalate exposure on maternal and fetal health.

Comparative analysis of galectin-1, galectin-3, and galectin-8 signatures in normal and choriocarcinoma trophoblast cell lines

Galectins are found in the placenta during both normal and pathological conditions. Specifically, galectins -1, -3, and -8 have been identified in extravillous trophoblast cells, with galectins -1, and -3 playing notable roles in trophoblast cell function. Altered expression profiles of galectins are linked to various cancers. This study explores the expression and localization of galectins -1, -3, and - 8 in a normal extravillous HTR-8/SVneo cell line and two choriocarcinoma cell lines, JAR and JEG-3. Expression levels of these galectins at both mRNA and protein levels were evaluated using PCR, qPCR, and Western blotting, while their cellular localization and distribution were assessed through immunocytochemistry, subcellular fractionation, and Western blotting. Galectins -1, -3, and -8 were expressed at lower levels in JAR and JEG-3 cells compared to HTR-8/SVneo cells. Additionally, different splice variants of galectin-8 were detected across all cell lines. Galectin-1 was predominantly localized at the plasma membrane. Notable differences in galectin-3 localization and distribution were observed between HTR-8/SVneo cells and choriocarcinoma cells. These findings highlight variations in the expression, staining patterns, and subcellular distribution of galectins -1, -3, and -8, suggesting their potential involvement in the pathology of choriocarcinoma.

Effects of a combination of cannabidiol and delta-9-tetrahydrocannabinol on key biological functions of HTR-8/SVneo extravillous trophoblast cells

In recent years, cannabis use has increased among pregnant women. In addition, the phytocannabinoids cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) alone or in combination are being used for therapeutical applications. THC and CBD are able to cross the placenta and a lot remains unknown concerning their impact on angiogenesis and extravillous trophoblasts’ (EVTs) migration and invasion, which are essential processes for placentation. Thus, in this study, the HTR-8/SVneo cell line was employed to evaluate the effects of CBD, THC and of their combination (1:1, 2 µM). Cannabinoids affected epithelial-mesenchymal transition, as showed by increased expression of the epithelial protein marker E-cadherin for CBD and CBD plus THC treatments, and decrease of mesenchymal intermediate filament vimentin for all treatments. The gene expression of the metalloproteinases MMP2 and MMP9, and of their inhibitors TIMP1 and TIMP2 was increased, except the latter for THC treatment. Moreover, CBD reduced cell migration and invasion, an effect that was enhanced by its combination with THC. CBD with or without THC also upregulated the gene expression of PGF, while the anti-angiogenic factor sFLT1 was increased for all treatments. VEGFA and FLT1 were not affected. Alone or combined CBD and THC also decreased tube segments’ length. Additionally, ERK1/2 and STAT3 phosphorylation was increased in the CBD and CBD plus THC-treated cells, while THC only activated STAT3. AKT activation was only affected by CBD. This work demonstrates that the exposure to cannabinoid-based products containing CBD and/or THC, may interfere with key processes of EVTs differentiation. Therefore, crucial phases of placental development can be affected, compromising pregnancy success.

MiR 19a promotes proliferation and inhibits apoptosis of placental trophoblasts via PTEN pathway

Purpose: To screen the micro-ribonucleic acids (miRNAs) in the pathological processes of preeclampsia (PE), and to assess their effects on the proliferation and apoptosis of HTR-8/SVneo cells.&#x0D; Methods: The miRNAs in PE patients and healthy puerperae were screened out using miRNA microarray assay, and verified through low-throughput quantitative reverse transcription- polymerase chain reaction (qRT-PCR). The effects of expression level of miR-19a on proliferation and cycle of HTR-8/SVneo cells were determined using cell counting kit (CCK)-8 assay and propidium iodide (PI) staining, respectively. The influence of the expression level of miR-19a on apoptosis of HTR-8/SVneo cells was evaluated using Annexin V-fluorescein isothiocyanate (FITC)/PI staining. Subsequently, luciferase reporter gene assay, CCK-8 assay and Annexin V-FITC/PI staining were conducted, respectively.&#x0D; Results: Serum miR-19a was significantly decreased in PE patients, and a high level of miR-19a led to increase in the proliferation of HTR-8/SVneo cells. Decreased level of miR-19a arrested HTR-8/SVneo cell cycle at G1 phase, while increase in the miR-19a level repressed the apoptosis of HTR-8/SVneo cells. The PTEN was a direct target of miR-19a, and overexpression of miR-19a promoted the activation of Akt signaling pathway, thereby significantly decreasing the expression levels of downstream phosphorylated p21/p21 (p-p21/p21), p-p27/p27 and p-glycogen synthase kinase-3 beta (GSK3β)/GSK3β. Compared with those in cells transfected with empty vectors, the pro-proliferative and anti-apoptotic effects of miR-19a weakened in HTR-8/SVneo cell lines with overexpressed PTEN.&#x0D; Conclusion: MiR-19a targets PTEN in order to activate Akt signaling pathway, thereby promoting proliferation, and inhibiting apoptosis of HTR-8/SVneo cells. This provides new drug targets that can be investigated for future treatment of PE.

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

Reduction in CgA-Derived CST Protein Level in HTR-8/SVneo and BeWo Trophoblastic Cell Lines Caused by the Preeclamptic Environment.

One of the most dangerous complications of pregnancy is preeclampsia (PE), a disease associated with a high risk of maternal and fetal mortality and morbidity. Although its etiology remains unknown, the placenta is believed to be at the center of ongoing changes. One of the hormones produced by the placenta is chromogranin A (CgA). Thus far, its role in pregnancy and pregnancy-related disorders is enigmatic, yet it is known that both CgA and its derived peptide catestatin (CST) are involved in the majority of the processes that are disturbed in PE, such as blood pressure regulation or apoptosis. Therefore, in this study, the influence of the preeclamptic environment on the production of CgA using two cell lines, HTR-8/SVneo and BeWo, was investigated. Furthermore, the capacity of trophoblastic cells to secrete CST to the environment was tested, as well as the correlation between CST and apoptosis. This study provided the first evidence that CgA and CST proteins are produced by trophoblastic cell lines and that the PE environment has an impact on CST protein production. Furthermore, a strong negative correlation between CST protein level and apoptosis induction was found. Hence, both CgA and its derived peptide CST may play roles in the complex process of PE pathogenesis.

Designing Effective Multi-Target Drugs and Identifying Biomarkers in Recurrent Pregnancy Loss (RPL) Using In Vivo, In Vitro, and In Silico Approaches

Recurrent pregnancy loss (RPL) occurs in approximately 5% of women. Despite an abundance of evidence, the molecular mechanism of RPL's pathology remains unclear. Here, we report the protective role of polo-like kinase 1 (PLK1) during RPL. We aimed to construct an RPL network utilizing GEO datasets and identified hub high-traffic genes. We also investigated whether the expressions of PLK1 were altered in the chorionic villi collected from women with RPL compared to those from healthy early pregnant women. Gene expression differences were evaluated using both pathway and gene ontology (GO) analyses. The identified genes were validated using in vivo and in vitro models. Mice with PLK1-overexpression and PLK1-knockdown in vitro models were produced by transfecting certain plasmids and si-RNA, respectively. The apoptosis in the chorionic villi, mitochondrial function, and NF-κB signaling activity was evaluated. To suppress the activation of PLK1, the PLK1 inhibitor BI2536 was administered. The HTR-8/SVneo and JEG-3 cell lines were chosen to establish an RPL model in vitro. The NF-κB signaling, Foxo signaling, PI3K/AKT, and endometrial cancer signaling pathways were identified via the RPL regulatory network. The following genes were identified: PLK1 as hub high-traffic gene and MMP2, MMP9, BAX, MFN1, MFN2, FOXO1, OPA1, COX15, BCL2, DRP1, FIS1, TRAF2, and TOP2A. Clinical samples were examined, and the results demonstrated that RPL patients had tissues with decreased PLK1 expression in comparison to women with normal pregnancies (p < 0.01). In vitro, PLK1 knockdown induced the NF-κB signaling pathway and apoptosis activation while decreasing cell invasion, migration, and proliferation (p < 0.05). Furthermore, the in vivo model proved that cell mitochondrial function and chorionic villi development are both hampered by PLK1 suppression. Our findings revealed that the PLK1/TRAF2/NF-κB axis plays a crucial role in RPL-induced chorionic villi dysfunction by regulating mitochondrial dynamics and apoptosis and might be a potential therapeutic target in the clinic.

TMBIM4 Deficiency Facilitates NLRP3 Inflammasome Activation-Induced Pyroptosis of Trophoblasts: A Potential Pathogenesis of Preeclampsia

Impaired invasion of EVTs results in inadequate remodelling of arteries and poor placentation, leading to PE. TMBIM4 was found to promote the migration and invasion of human osteosarcoma U2-OS and breast cancer MCF7 cell lines. However, the effect of TMBIM4 on trophoblast biological behaviour and its relevance to PE pathophysiology remain unclear. In this study, we confirmed that TMBIM4 was highly expressed in cytotrophoblasts, syncytiotrophoblasts, and EVTs of the human placenta during early pregnancy. By comparing the expression levels of TMBIM4 in the placenta of women with normal-term pregnancy and PE, TMBIM4 was found to be significantly decreased in PE. Thereafter, we determined the expression of TMBIM4 in the LPS-treated first-trimester human trophoblast cell line HTR-8/SVneo (mimicking a PE-like cell model), and determined the effect of TMBIM4 on trophoblast function and its underlying mechanism. LPS treatment reduced the expression of TMBIM4 and induced NLRP3 inflammasome activity in HTR-8/SVneo cells. KO of TMBIM4 in the HTR-8/SVneo cell line impaired cell viability, migration, and invasion, which was more severe in the LPS/ATP-treated TMBIM4-KO cell line. Moreover, TMBIM4 deficiency enhanced NLRP3 inflammasome activity and promoted subsequent pyroptosis, with or without LPS/ATP treatment. The negative relationship between TMBIM4 expression and NLRP3 inflammatory activity was verified in PE placentas. Inhibiting the NLRP3 inflammasome with MCC950 in HTR-8/SVneo cells alleviated LPS/ATP-induced pyroptosis and damaged cell function in the TMBIM4-KO cell line. Overall, this study revealed a new PE-associated protein, TMBIM4, and its biological significance in trophoblast pyroptosis mediated by the NLRP3 inflammasome. TMBIM4 may serve as a potential target for the treatment of placental inflammation-associated PE.

Bioinformatics analysis combined with clinical sample screening reveals that leptin may be a biomarker of preeclampsia.

Introduction: Preeclampsia (PE) is a gestational hypertensive disease with unclear pathogenesis. This study aimed to identify the genes that play an important role in determining the pathogenesis of PE using bioinformatics analysis and fundamental researches. Materials and methods: Datasets from the Gene Expression Omnibus (GEO) database were used to screen for differentially expressed genes (DEGs). The NCBI, SangerBox, and other databases were used to analyze the functions of the DEGs. Targetscan7, miRWalk, ENCORI, DIANA TOOLS, CircBank databases, and the Cytoscape tool were used to construct the lncRNA/circRNA-miRNA- LEP network. SRAMP, RPISeq, RBPsuite, and catRPAID were used to analyze the RNA modifications of LEP. Immune cell infiltration was analyzed using the dataset GSE75010. Placental tissues from normal pregnant women and PE patients were collected, screened for gene expression using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The results were further verified in HTR-8/SVneo cell line hypoxia model and PE mouse model. Results: Our analyses revealed that LEP was significantly upregulated in eight datasets. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses indicated that LEP was involved in the JAK/STAT signaling pathway, angiogenesis, and placental development. Immune cell infiltration analysis showed that M1 and M2 macrophages differed between normal pregnancies and those in PE patients. A competing endogenous RNA (ceRNA) network was constructed, and proteins interacting with LEP were identified. RNA modification sites of LEP were also identified. Finally, the overexpression of LEP in PE was confirmed in clinical samples, HTR-8/SVneo cell line and PE mouse model. Conclusion: Our results indicate that LEP overexpression is associated with PE and may be a potential diagnostic marker and therapeutic target.

Identifying novel regulators of placental development using time-series transcriptome data.

The placenta serves as a connection between the mother and the fetus during pregnancy, providing the fetus with oxygen, nutrients, and growth hormones. However, the regulatory mechanisms and dynamic gene interaction networks underlying early placental development are understudied. Here, we generated RNA-sequencing data from mouse fetal placenta at embryonic days 7.5, 8.5, and 9.5 to identify genes with timepoint-specific expression, then inferred gene interaction networks to analyze highly connected network modules. We determined that timepoint-specific gene network modules were associated with distinct developmental processes, and with similar expression profiles to specific human placental cell populations. From each module, we identified hub genes and their direct neighboring genes, which were predicted to govern placental functions. We confirmed that four novel candidate regulators identified through our analyses regulate cell migration in the HTR-8/SVneo cell line. Overall, we predicted several novel regulators of placental development expressed in specific placental cell types using network analysis of bulk RNA-sequencing data. Our findings and analysis approaches will be valuable for future studies investigating the transcriptional landscape of early development.

MiR-101-3p contributes to the progression of preeclampsia by suppressing WDR5-mediated proliferation and invasion of trophoblast.

Decreased proliferation and invasion of trophoblast were proven to be involved in the pathogenesis of preeclampsia (PE). However, the regulatory network has not been clarified yet. This study aimed to explore the role of miR-101-3p in the progression of PE. miR-101-3p expression in placentas of pregnant women with or without PE was analyzed by real-time quantitative PCR (RT-qPCR). Trophoblastic HTR-8/SVneo and HPT-8 cell lines were cultured and underwent hypoxia/reoxygenation (H/R) treatment to mimic PE in vitro. Cell proliferation and invasion were analyzed in gain-of and loss-of-function assays. Finally, we undertook in vivo studies to explore effects of miR-101-3p in the PE model. Compared to placentas from patients without PE, miR-101-3p expressed significantly higher in placentas from PE patients, and its level was positively correlated with the severity of patients. In vitro studies found that overexpression of miR-101-3p significantly suppressed cell proliferation and invasion, while knockdown of miR-101-3p reversed the impacts of H/R treatment. Further research showed that the expression of WD repeat domain 5 (WDR5) was significantly lower in placentas from patients with PE, and its level was negatively associated with the severity of patients. In vitro and in vivo studies confirmed that miR-101-3p promoted PE progression through the regulation of WD WDR5 expression. Increased expression of miR-101-3p in placenta contributes to the development of PE by suppressing WDR5-mediated proliferation and invasion of trophoblast.

Regulatory Effects of Fyn on Trophoblast Cell Behaviors and Function.

Fyn has been proven to be involved in various cell behaviors and pathophysiological processes. However, the expression and roles of Fyn in trophoblasts remain unclear. Here, we aimed to evaluate the participation of Fyn in trophoblast behavior and function, and the related mechanisms were briefly explored. Fyn expression in the HTR-8/SVneo, JEG-3, and JAR cell lines was evaluated by immunofluorescence, quantitative real-time PCR and western blotting. Fyn expression in human hydatidiform moles was also determined by immunohistochemistry and western blot. To explore the effects of Fyn, HTR-8/SVneo and JEG-3 cells were transfected with Fyn shRNA or overexpression plasmid or treated with the Fyn activity inhibitor SU6656 or ERK1/2 inhibitor U0126. The migration, proliferation, and apoptosis of trophoblast cells were assessed using transwell assays, flow cytometry, and cell counting kit-8 assays, respectively. The production of primary inflammatory cytokines, HLA-G and active matrix metallopeptidase (MMP) 2/9, and the phosphorylation of ERK1/2 and STAT3 were evaluated by ELISA, western blot, or gelatin zymography. The results showed that Fyn was expressed by trophoblast cells, mainly in the cytoplasm and membrane. Fyn expression and activity levels both increased in order from HTR-8/SVneo and JAR to JEG-3. The overexpression of Fyn promoted the proliferation and migration of trophoblast cells and inhibited their apoptosis, while the opposite effects were observed for Fyn knockdown and inhibition. Fyn regulated inflammatory cytokine production in trophoblast cells by promoting TGF-β and IL-4 secretion while inhibiting IFN-γ and TNF-α secretion. Moreover, HLA-G expression in JEG-3 was positively regulated by Fyn. Fyn also facilitated the expression of active MMP2/9 and the activation of ERK1/2 and STAT3. Besides, it was confirmed that Fyn regulated trophoblast cell activities through ERK1/2 signal pathway by using U0126. Our study first detected the expression of Fyn in trophoblast cells. Fyn played pivotal roles in trophoblast cell behaviors and function, ERK1/2 was one of its targets, and MMP2/9 and STAT3 may also be involved in the regulatory mechanism.

Dose–response mapping of MEHP exposure with metabolic changes of trophoblast cell and determination of sensitive markers

Mono(2-ethylhexyl) phthalate (MEHP) is a metabolite of DEHP which is one of phthalic acid esters (PAEs) widely used in daily necessities. Moreover, MEHP has been proven to have stronger biological toxicity comparing to DEHP. In particular, several recent population-based studies have reported that intrauterine exposure to MEHP results in adverse pregnancy outcomes. To explore the mechanisms and metabolic biomarkers of MEHP exposure, we examined the metabolic status of HTR-8/Svneo cell lines exposed to different doses of MEHP (0, 1.25, 5.0, 20 μM). Global and dose–response metabolomics tools were used to identify metabolic perturbations and sensitive markers associated with MEHP. Only 22 metabolic features (accounted for <1 %) were significantly changed when exposed to 1.25 μM. However, when the exposure dose was increased to 5 or 20 μM, the number of significantly changed metabolic features exceeded 300 (approximately 10 %). In particular, amino acid metabolism, pyrimidine metabolism and glutathione metabolism were widely affected according to the enrich analysis of those significant altered metabolites, which has and have previously been reported to be closely related to fetal development. Moreover, 5′-UMP and N-acetylputrescine with the lowest effective concentrations (EC−10 = 0.1 μM and EC+10 = 0.11 μM, respectively) were identified as sensitive endogenous biomarkers of MEHP exposure.

HADHA Inhibits the Migration and Invasion of HTR-8/SVneo Cells by Regulating PI3K/AKT Signaling Pathway

To explore the effects of hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) on the migration and invasion of HTR-8/SVneo cells, a human trophoblast cell line, and its potential mechanism of action. Immunofluorescence staining was done to evaluate the expression levels of HADHA in samples of normal villi and recurrent spontaneous abortion (RSA) villi at 6-8 weeks. Lentiviral infection system was used to construct stable HTR-8/SVneo cell lines with HADHA overexpression and knockdown. Western blot, qRT-PCR, Wound-healing assay, and Transwell assay were used to determine the effect of HADHA on the migration and invasion of HTR-8/SVneo cells and the expression of relevant genes. Transcriptome sequencing and bioinformatics analysis were done to screen for the potential target genes and signaling pathways regulated by HADHA. The specific molecular mechanism of how HADHA regulates the migration and invasion of HTR-8/SVneo cells was examined by adding the inhibitor of protein kinase B (PKB/AKT). HADHA was highly expressed in extravillous trophoblasts (EVT) of RSA villus samples as compared with samples from the normal control group. In HTR-8/SVneo cells overexpressing HADHA, the expression levels of migration and invasion-related genes, including HLA-G, MMP2, MMP9, and NCAD, were decreased (P<0.01,P<0.05), and the migration and invasion abilities of HTR-8/SVneo cells were weakened (P<0.05). HADHA knockdown increased the expression levels of HLA-G, MMP2, MMP9, and NCAD (P<0.01, P<0.05), and promoted the migration and invasion of HTR-8/SVneo cells (P<0.05). In addition, HADHA overexpression decreased the phosphorylation levels of PI3K and AKT (P<0.05) and inhibited the PI3K/AKT signaling pathway. HADHA knockdown activated the PI3K/AKT signaling pathway. When MK-2206, an AKT inhibitor, was added to stable HTR-8/SVneo cell lines with HADHA knockdown, the migration and invasion of the cells were significantly reduced. HADHA inhibits the migration and invasion of HTR-8/SVneo cells by inhibiting the PI3K/AKT signaling pathway.

METTL3-Mediated m6A RNA Methylation of ZBTB4 Interferes With Trophoblast Invasion and Maybe Involved in RSA.

N6-methyladenosine (m6A) was the most abundant modification of mRNA and lncRNA in mammalian cells and played an important role in many biological processes. However, whether m6A modification was associated with recurrent spontaneous abortion (RSA) and its roles were still unclear. Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was used to study the global m6A modification pattern in RSAs and controls. RNA sequencing (RNA-Seq) was used to study the level of global mRNA in two groups. Real-time quantitative PCR (RT-qPCR) was used to verify the level of mRNA of METTL3 and ZBTB4. MeRIP–qPCR was conducted to test the level of ZBTB4 m6A modification in two groups. In order to further explore whether ZBTB4 was the substrate of METTL3, the HTR-8/SVneo (HTR-8) cell line was selected for the knockdown and overexpression of METTL3. To study whether METTL3 regulated the ZBTB4 expression by recognizing ZBTB4 mRNA m6A motifs in coding sequences (CDS), dual-luciferase reporter assay was conducted. RNA stability assays using actinomycin D were conducted to study the RNA stability of the HTR-8 cell line with METTL3 overexpression and knockdown. To illustrate the role of METTL3 in the invasion of trophoblast, matrigel invasion assays and transwell migration assays were conducted using the HTR-8 cell line with METTL3 overexpression and knockdown. Results: A total of 65 genes were found with significant differences both in m6A modification and mRNA expression. We found m6A methyltransferase METTL3 was significantly down-regulated in the RSA group. Through gene function analysis, RT-qPCR, MeRIP–qPCR validation experiment, knockdown, and overexpression of METTL3 in the HTR-8 cell line, ZBTB4 was selected as one target of METTL3. Furthermore, we clarified that METTL3 regulated the expression of ZBTB4 by recognizing ZBTB4 mRNA m6A motifs in the CDS using the dual-luciferase reporter assay and METTL3 regulated the invasion of trophoblast by altering the stability and expression of ZBTB4 by RNA stability, matrigel invasion, and transwell migration assays. Conclusion: Our study revealed the mechanism by which METTL3 regulated the stability and expression of ZBTB4 and the trophoblast migration ability of RSA. A new perspective was provided for exploring the mechanism of embryonic development in RSA patients.

Folic Acid Levels During Pregnancy Regulate Trophoblast Invasive Behavior and the Possible Development of Preeclampsia.

BackgroundOne of the unique features of placentation is its similarity to tumorigenesis yet being very well regulated. It allows rapid proliferation, migration, and invasion of mononuclear trophoblast cells into the maternal uterus and remodeling the maternal vasculature. This pseudomalignant nature of trophoblastic cells is strictly regulated and its importance becomes evident in abnormal pregnancies that are characterized by aberrant trophoblast proliferation/invasion like preeclampsia. In addition to this, the importance of folic acid supplementation during pregnancy is well documented. We aimed to analyze the molecular and epigenetic regulation of the pseudomalignant nature of placentation via folic acid levels.MethodsPlacental tissue samples were collected from different pregnancies in three different gestational stages. We estimated the impact of folic acid levels on global methylation, LINE1 methylation, and expression of DNMTs in all three gestational stages in pregnant women and preeclampsia pregnancies. We also analyzed the effect of folic acid supplementation on trophoblastic invasion using placental derived cells viz, JEG-3 and HTR-8/SVneo cell line and verified the molecular and epigenetic mechanisms involved in this regulation.ResultsDevelopment of preeclampsia was observed to be associated with lower folate levels in placental tissue, higher global methylation level, and higher expression of DNMT1and DNMT3A. Folic acid supplementation was found to increase the invasive potential of placental trophoblasts by almost two folds which were associated with the decreased expression of tumor suppressor genes and tissue inhibitors of matrix metalloproteinases; and increased expression of oncogenes, telomerase gene, and matrix metalloproteinases. These folic acid-mediated changes were observed to be regulated by CpG methylation in the case of many genes. Folic acid supplementation was also observed to significantly decrease global methylation in placental trophoblasts related to decreasing expression of DNMT1 and DNMT3A.ConclusionLower folic acid levels are associated with preeclampsia development and folic acid supplementation regulates the invasive potential of placental trophoblasts as mediated by various epigenetic changes in the placenta suggesting the protective effect of folic acid against preeclampsia.

Toxicity assessments of selected trichloroethylene and perchloroethylene metabolites in three in vitro human placental models

Residential and occupational exposures to the industrial solvents perchloroethylene (PERC) and trichloroethylene (TCE) present public health concerns. In humans, maternal PERC and TCE exposures can be associated with adverse birth outcomes. Because PERC and TCE are biotransformed to toxic metabolites and placental dysfunction can contribute to adverse birth outcomes, the present study compared the toxicity of key PERC and TCE metabolites in three in vitro human placenta models. We measured cell viability and caspase 3 + 7 activity in the HTR-8/SVneo and BeWo cell lines, and caspase 3 + 7 activity in first trimester villous explant cultures. Cultures were exposed for 24 h to 5–100 µM S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), or 5–200 µM trichloroacetate (TCA) and dichloroacetate (DCA). DCVC significantly reduced cell viability and increased caspase 3 + 7 activity in HTR-8/SVneo cells at a lower concentration (20 µM) compared with concentrations toxic to BeWo cells and villous explants. Similarly, TCVC reduced cell viability and increased caspase 3 + 7 activity in HTR-8/SVneo cells but not in BeWo cells. TCA and DCA had only negligible effects on HTR-8/SVneo or BeWo cells. This study advances understanding of potential risks of PERC and TCE exposure during pregnancy by identifying metabolites toxic in placental cells and tissues.