Establishment and comparison of human term placenta-derived trophoblast cells†.

Research on the biology of fetal-maternal barriers has been limited by access to physiologically relevant cells, including trophoblast cells. In this study, we describe the development of a human term placenta-derived cytotrophoblast immortalized cell line (hPTCCTB) derived from the basal plate. Human-term placenta-derived cytotrophoblast immortalized cell line cells are comparable to their primary cells of origin in terms of morphology, marker expression, and functional responses. We demonstrate that these can transform into syncytiotrophoblast and extravillous trophoblasts. We also compared the hPTCCTB cells to immortalized chorionic trophoblasts (hFM-CTC), trophoblasts of the chorionic plate, and BeWo cells, choriocarcinoma cell lines of conventional use. Human-term placenta-derived cytotrophoblast immortalized cell line and hFM-CTCs displayed more similarity to each other than to BeWos, but these differ in syncytialization ability. Overall, this study (1) demonstrates that the immortalized hPTCCTB generated are cells of higher physiological relevance and (2) provides a look into the distinction between the spatially distinct placental and fetal barrier trophoblasts cells, hPTCCTB and hFM-CTC, respectively.

Essential oil of oregano (Origanum vulgare L.) reduces infection and proliferation of Toxoplasma gondii in BeWo cells with induction of autophagy and death of tachyzoites through a mechanism similar to necrosis.

Toxoplasmosis poses a global health threat, ranging from asymptomatic cases to severe, potentially fatal manifestations, especially in immunocompromised individuals and congenital transmission. Prior research suggests that oregano essential oil (OEO) exhibits diverse biological effects, including antiparasitic activity against Toxoplasma gondii. Given concerns about current treatments, exploring new compounds is important. This study was to assess the toxicity of OEO on BeWo cells and T. gondii tachyzoites, as well as to evaluate its effectiveness in in vitro infection models and determine its direct action on free tachyzoites. OEO toxicity on BeWo cells and T. gondii tachyzoites was assessed by MTT and trypan blue methods, determining cytotoxic concentration (CC50), inhibitory concentration (IC50), and selectivity index (SI). Infection and proliferation indices were analyzed. Direct assessments of the parasite included reactive oxygen species (ROS) levels, mitochondrial membrane potential, necrosis, and apoptosis, as well as electron microscopy. Oregano oil exhibited low cytotoxicity on BeWo cells (CC50: 114.8 µg/mL ± 0.01) and reduced parasite viability (IC50 12.5 ± 0.06 µg/mL), demonstrating 9.18 times greater selectivity for parasites than BeWo cells. OEO treatment significantly decreased intracellular proliferation in infected cells by 84% after 24 h with 50 µg/mL. Mechanistic investigations revealed increased ROS levels, mitochondrial depolarization, and lipid droplet formation, linked to autophagy induction and plasma membrane permeabilization. These alterations, observed through electron microscopy, suggested a necrotic process confirmed by propidium iodide labeling. OEO treatment demonstrated anti-T. gondii action through cellular and metabolic change while maintaining low toxicity to trophoblastic cells.

HTRA1 in Placental Cell Models: A Possible Role in Preeclampsia.

The HtrA serine peptidase 1 (HTRA1) is a multidomain secretory protein with serine-protease activity involved in the regulation of many cellular processes in both physiological and pathological conditions. HTRA1 is normally expressed in the human placenta, and its expression is higher in the first trimester compared to the third trimester, suggesting an important role of this serine protease in the early phases of human placenta development. The aim of this study was to evaluate the functional role of HTRA1 in in vitro models of human placenta in order to define the role of this serine protease in preeclampsia (PE). BeWo and HTR8/SVneo cells expressing HTRA1 were used as syncytiotrophoblast and cytotrophoblast models, respectively. Oxidative stress was induced by treating BeWo and HTR8/SVneo cells with H2O2 to mimic PE conditions in order to evaluate its effect on HTRA1 expression. In addition, HTRA1 overexpression and silencing experiments were performed to evaluate the effects on syncytialization, cell mobility, and invasion processes. Our main data showed that oxidative stress significantly increased HTRA1 expression in both BeWo and HTR8/SVneo cells. In addition, we demonstrated that HTRA1 has a pivotal role in cell motility and invasion processes. In particular, HTRA1 overexpression increased while HTRA1 silencing decreased cell motility and invasion in HTR8/SVneo cell model. In conclusion, our results suggest an important role of HTRA1 in regulating extravillous cytotrophoblast invasion and motility during the early stage of placentation in the first trimester of gestation, suggesting a key role of this serine protease in PE onset.

Uptake of antiepileptic drugs in forskolin-induced differentiated BeWo cells: alteration of gabapentin transport.

Previous studies have indicated that the expression levels of several transporters are altered during placental trophoblast differentiation. However, changes in the transport activities of therapeutic agents during differentiation must be comprehensively characterised. Antiepileptic drugs, including gabapentin (GBP), lamotrigine (LTG), topiramate, and levetiracetam, are increasingly prescribed during pregnancy. The objective of this study was to elucidate differences in the uptake of antiepileptic drugs during the differentiation process.Human placental choriocarcinoma BeWo cells were used as trophoblast models. For differentiation into syncytiotrophoblast-like cells, cells were treated with forskolin.The uptake of GBP and LTG was lower in differentiated BeWo cells than in undifferentiated cells. In particular, the maximum uptake rate of GBP transport was decreased in differentiated BeWo cells. Furthermore, GBP transport was trans-stimulated by the amino acids His and Met. We investigated the profiles of amino acids in undifferentiated and differentiated BeWo cells. Supplementation with His and Met, which demonstrated trans-stimulatory effects on GBP uptake, restored GBP uptake in differentiated cells. The findings of this study suggest that drug transport in BeWo cells can be altered before and after differentiation, and that the altered GBP uptake could be mediated by the intracellular amino acid status.

Effects of polycyclic aromatic hydrocarbons on gestational hormone production in a placental cell line: Application of passive dosing to in vitro tests.

Air pollution includes polycyclic aromatic hydrocarbons (PAHs), which have been correlated to endocrine disruptor pathways during early pregnancy. PAHs have been found in the placenta and cord blood, which may affect the hormones involved in placental development. We studied the effects of some airborne PAHs on beta human chorionic gonadotropin (ß-hCG) and progesterone production by using a syncytial BeWo cell line as a placental model. PAH congeners were spiked in silicon rubber membrane (SRMs) and were then introduced into the cell medium by the passive dosing method to reach a freely dissolved concentration for BeWo cell exposure. Ultrahigh-performance liquid chromatography coupled with a diode array detector was used to analyze the PAHs, and electrochemiluminescence was used to test the hormone levels. Our results showed that passive dosing can deliver low levels of PAH congeners in the cell medium, which allowed us to calculate the individual release constants at equilibrium and to estimate their effects. Benzo[a]pyrene was released quickly from the SRMs to the cell medium, which can be attributed to its lipophilic properties. The PAHs were shown to decrease the ß-hCG level in the short term and progesterone level in the long term, so they may serve as a pathway for endocrine disorder in trophoblastic cells. This approximation may explain observations of impaired endometrium receptivity and placental dysfunction, which enhance adverse pregnancy outcomes such as embryonic mortality and intrauterine growth restriction.

Differences and Interactions in Placental Manganese and Iron Transfer across an In Vitro Model of Human Villous Trophoblasts.

Manganese (Mn) as well as iron (Fe) are essential trace elements (TE) important for the maintenance of physiological functions including fetal development. However, in the case of Mn, evidence suggests that excess levels of intrauterine Mn are associated with adverse pregnancy outcomes. Although Mn is known to cross the placenta, the fundamentals of Mn transfer kinetics and mechanisms are largely unknown. Moreover, exposure to combinations of TEs should be considered in mechanistic transfer studies, in particular for TEs expected to share similar transfer pathways. Here, we performed a mechanistic in vitro study on the placental transfer of Mn across a BeWo b30 trophoblast layer. Our data revealed distinct differences in the placental transfer of Mn and Fe. While placental permeability to Fe showed a clear inverse dose-dependency, Mn transfer was largely independent of the applied doses. Concurrent exposure of Mn and Fe revealed transfer interactions of Fe and Mn, indicating that they share common transfer mechanisms. In general, mRNA and protein expression of discussed transporters like DMT1, TfR, or FPN were only marginally altered in BeWo cells despite the different exposure scenarios highlighting that Mn transfer across the trophoblast layer likely involves a combination of active and passive transport processes.

Modeling Preeclampsia In Vitro: Polymorphic Variants of STOX1-A/B Genes Can Downregulate CD24 in Trophoblast Cell Lines.

CD24 is a mucin-like immunosuppressing glycoprotein whose levels increase during pregnancy and decrease in the syncytio- and cytotrophoblasts in early and preterm preeclampsia. We used two modified cell lines that mimic in vitro features of preeclampsia to identify if this phenomenon could be reproduced. Our model was the immortalized placental-derived BeWo and JEG-3 cell lines that overexpress the STOX1 A/B transcription factor gene that was discovered in familial forms of preeclampsia. BeWo and JEG-3 cells stably transduced with the two major isoforms of STOX1-A/B or by an empty vector (control), were propagated, harvested, and analyzed. CD24 mRNA expression was determined by quantitative real-time polymerase nuclear chain reaction (qRT-PCR). CD24 protein levels were determined by Western blots. In STOX1-A/B overexpressing in BeWo cells, CD24 mRNA was downregulated by 91 and 85%, respectively, compared to the control, and by 30% and 74%, respectively in JEG-3 cells. A 67% and 82% decrease in CD24 protein level was determined by immunoblot in BeWo overexpressing STOX1-A/B, respectively, while the reduction in JEG-3 cells was between 47 and 62%. The immortalized BeWo and JEG-3 cell lines overexpressing STOX1-A/B had reduced CD24. Although both cell lines were affected, BeWo appears to be more susceptible to downregulation by STOX-1 than JEG-3, potentially because of their different cell origin and properties. These results strengthen the in vivo results of reduced CD24 levels found in early and preterm preeclampsia. Accordingly, it implies the importance of the reduced immune tolerance in preeclampsia, which was already demonstrated in vivo in the STOX1-A/B model of preeclampsia, and is now implied in the in vitro STOX-1 model, a subject that warrants further investigations.

Comparative Proteomic Analysis of Drug Trichosanthin Addition to BeWo Cell Line.

Trichosanthin (TCS) is a traditional Chinese herbal medicine used to treat some gynecological diseases. Its effective component has diverse biological functions, including antineoplastic activity. The human trophoblast cell line BeWo was chosen as an experimental model for in vitro testing of a drug screen for anticancer properties of TCS. The MTT method was used in this study to get a primary screen result. The result showed that 100 mM had the best IC50 value. Proteomics analysis was then performed for further investigation of the drug effect of TCS on the BeWo cell line. In this differential proteomic expression analysis, the total proteins extracted from the BeWo cell line and their protein expression level after the drug treatment were compared by 2DE. Then, 24 unique three-fold differentially expressed proteins (DEPs) were successfully identified by MALDI-TOF/TOF MS. Label-free proteomics was run as a complemental method for the same experimental procedure. There are two proteins that were identified in both the 2DE and label-free methods. Among those identified proteins, bioinformatics analysis showed the importance of pathway and signal transduction and gives us the potential possibility for the disease treatment hypothesis.

Dietary Antioxidant Curcumin Mitigates CuO Nanoparticle-Induced Cytotoxicity through the Oxidative Stress Pathway in Human Placental Cells.

The placenta is an important organ that maintains a healthy pregnancy by transporting nutrients to the fetus and removing waste from the fetus. It also acts as a barrier to protect the fetus from hazardous materials. Recent studies have indicated that nanoparticles (NPs) can cross the placental barrier and pose a health risk to the developing fetus. The high production and widespread application of copper oxide (CuO) NPs may lead to higher exposure to humans, raising concerns of health hazards, especially in vulnerable life stages, e.g., pregnancy. Oxidative stress plays a crucial role in the pathogenesis of adverse pregnancy outcomes. Due to its strong antioxidant activity, dietary curcumin can act as a therapeutic agent for adverse pregnancy. There is limited knowledge on the hazardous effects of CuO NPs during pregnancy and their mitigation by curcumin. This study aimed to investigate the preventive effect of curcumin against CuO NP-induced toxicity in human placental (BeWo) cells. CuO NPs were synthesized by a facile hydrothermal process and characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and photoluminescence techniques. We observed that curcumin did not induce toxicity in BeWo cells (1-100 µg/mL for 24 h), whereas CuO NPs decreased the cell viability dose-dependently (5-200 µg/mL for 24 h). Interestingly, CuO NP-induced cytotoxicity was effectively mitigated by curcumin co-exposure. The apoptosis data also exhibited that CuO NPs modulate the expression of several genes (p53, bax, bcl-2, casp3, and casp9), the activity of enzymes (caspase-3 and -9), and mitochondrial membrane potential loss, which was successfully reverted by co-treatment with curcumin. The mechanistic study suggested that CuO-induced reactive oxygen species generation, lipid peroxidation, and higher levels of hydrogen peroxide were significantly alleviated by curcumin co-exposure. Moreover, glutathione depletion and the lower activity of antioxidant enzymes (superoxide dismutase, glutathione peroxidase, and catalase) were effectively mitigated by curcumin. We believe this is the first report exhibiting that CuO-induced toxicity in BeWo cells can be effectively alleviated by curcumin. The pharmacological potential of dietary curcumin in NP-induced toxicity during pregnancy warrants further investigation.

Dicalcin suppresses in vitro trophoblast attachment in human cell lines.

Implantation is a highly organized process that involves an interaction between a competent blastocyst and a receptive uterus. Despite significant research efforts, the molecular mechanisms governing this complex process remain elusive. Here, we investigated the effect of dicalcin, an S100-like Ca2+-binding protein, on the attachment of choriocarcinoma cells (BeWo cells) onto a monolayer of endometrial carcinoma cells (Ishikawa cells). Extracellularly administered dicalcin bound to both BeWo and Ishikawa cells. Pretreatment of BeWo spheroids with dicalcin reduced the attachment ratio of the spheroids onto the monolayer, whereas that of Ishikawa cells showed no apparent change. We identified the partial amino acid sequence of human dicalcin that exhibited maximum suppression for BeWo spheroid attachment. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide caused a dilation of the intercellular junction between BeWo and ISK cells. Peptide treatment of BeWo spheroids downregulated the expression of integrinαvß3 in BeWo cells, and induced alterations in their phalloidin-staining pattern, as measured by the length of each F-actin fiber and the thickness of the cortical stress fiber. Thus, dicalcin affects reorganization of the intracellular actin meshwork and subsequently the intensity of attachment, functioning as a novel suppressor of implantation.

Chemical Induction of Trophoblast Hypoxia by Cobalt Chloride Leads to Increased Expression of DDIT3.

Choriocarcinoma cells BeWo b30 are used to model human placental trophoblast hypoxia using cobalt (II) chloride and hydroxyquinoline derivative (HD) as chemical inducers of hypoxia-inducible factor (HIF). In this study, it was shown that both substances activate the hypoxic pathway and the epithelial-mesenchymal transition and inhibit the pathways of cell proliferation. However, CoCl2 caused activation of the apoptosis pathway, increased the activity of effector caspases 3 and 7, and increased the expression of the unfolded protein response target DDIT3. The mTORC1 pathway was activated upon exposition to CoCl2, while HD suppressed this pathway, as it happens during real trophoblast hypoxia. Thus, effect of CoCl2 on BeWo cells can be a model of severe hypoxia with activation of apoptosis, while HD mimics moderate hypoxia.

Small molecule inhibitors provide insights into the relevance of LAT1 and LAT2 in materno-foetal amino acid transport.

The placenta supplies the foetus with critical nutrients such as essential amino acids (AA, eg leucine) for development and growth. It also represents a cellular barrier which is formed by a polarized, differentiated syncytiotrophoblast (STB) monolayer. Active Na+ -independent leucine transport across the placenta is mainly attributed to the System L transporters LAT1/SLC7A5 and LAT2/SLC7A8. This study explored the influence of trophoblast differentiation on the activity of LAT1/LAT2 and the relevance of LAT1/LAT2 in leucine uptake and transfer in trophoblasts by applying specific small molecule inhibitors (JPH203/JG336/JX009). L-leucine uptake (total dose = 167 µmol/L) was sensitive to LAT1-specific inhibition by JPH203 (EC50  = 2.55 µmol/L). The inhibition efficiency of JPH203 was increased by an additional methoxy group in the JPH203-derivate JG336 (EC50  = 1.99 µmol/L). Interestingly, JX009 showed efficient System L inhibition (EC50  = 2.35 µmol/L) and was the most potent inhibitor of leucine uptake in trophoblasts. The application of JPH203 and JX009 in Transwell® -based leucine transfer revealed LAT1 as the major accumulative transporter at the apical membrane, but other System L transporters such as LAT2 as rate-limiting for leucine efflux across the basal membrane. Therefore, differential specificity of the applied inhibitors allowed for estimation of the contribution of LAT1 and LAT2 in materno-foetal AA transfer and their potential impact in pregnancy diseases associated with impaired foetal growth.

The supression of DOCK family members by their specific inhibitors induces the cell fusion of human trophoblastic cells.

PURPOSE: Among the members of the DOCK family, DOCK1-5 function as guanine-nucleotide exchange factors for small GTPase Rac1, which regulates the actin cytoskeleton. It has been reported that in model organisms the Dock-Rac axis is required for myoblast fusion. We examined the role of DOCK1-5 in trophoblast fusion herein. METHODS: We used a quantitative polymerase chain reaction (qPCR) to examine the mRNA expressions of DOCK1-5 and differentiation-related genes, i.e., fusogenic genes, in human trophoblastic cell lines, BeWo and JEG-3. We treated BeWo cells with TBOPP and C21 to inhibit DOCK1 and DOCK5. Cell dynamics and cell fusion were assessed by live imaging and immunostaining. The signaling pathways induced by DOCK1/5 inhibition were examined by western blotting. RESULTS: DOCK1 and DOCK5 were expressed in BeWo cells. The inhibition of DOCK1 or DOCK5 did not prevent the cell fusion induced by forskolin (a common reagent for cell fusion); it induced cell fusion. DOCK1 inhibition induced cell death, as did forskolin. DOCK1 and DOCK5 inhibition for 24 and 48 h increased the expression of the genes ASCT2 and SYNCYTIN2, which code responsive proteins of trophoblast cell fusion, respectively. CONCLUSION: DOCK1 and DOCK5 inhibition participates in BeWo cell fusion, probably via pathways independent from forskolin-mediated pathways.

A comparative study of key physiological stem cell parameters between three human trophoblast cell lines.

Human trophoblast stem cells (TSCs) play a key role in the placenta. These cells are proliferative, undifferentiated, and can differentiate into mature trophoblast cell types. However, primary human TSCs are difficult to obtain. In our previous study, we established TSCs from human induced pluripotent stem cells (TShiPSC). Here, we aimed to characterize the identity of these TShiPSC cells by comparing them with BeWo choriocarcinoma cells and primary TSCs (CT cells). Compared with BeWo cells, CT and TShiPSC cells showed high secretion of human chorionic gonadotrophin (hCG) and syncytiotrophoblast differentiation ability. Global gene microarray analysis results showed that CT and TShiPSC cells, unlike BeWo cells, could be classified in the same group. Compared with BeWo cells, CT and TShiPSC cells showed high expression levels of TSC-specific genes and low expression of cancer adhesion and invasion genes. Analysis of placental barrier integrity showed that TShiPSC cells could form a good barrier. Prospective studies using TShiPSC cells hold great promise for elucidating the pathogenesis of infertility due to trophoblast defects.

Establishment of an in vitro placental barrier model cultured under physiologically relevant oxygen levels.

The human placental barrier facilitates many key functions during pregnancy, most notably the exchange of all substances between the mother and fetus. However, preclinical models of the placental barrier often lacked the multiple cell layers, syncytialization of the trophoblast cells and the low oxygen levels that are present within the body. Therefore, we aimed to design and develop an in vitro model of the placental barrier that would reinstate these factors and enable improved investigations of barrier function. BeWo placental trophoblastic cells and human umbilical vein endothelial cells were co-cultured on contralateral sides of an extracellular matrix-coated transwell insert to establish a multilayered barrier. Epidermal growth factor and forskolin led to significantly increased multi-nucleation of the BeWo cell layer and increased biochemical markers of syncytial fusion, for example syncytin-1 and hCGß. Our in vitro placental barrier possessed size-specific permeability, with 4000-Da molecules experiencing greater transport and a lower apparent permeability coefficient than 70 000-Da molecules. We further demonstrated that the BeWo layer had greater resistance to smaller molecules compared to the endothelial layer. Chronic, physiologically low oxygen exposure (3-8%) increased the expression of hypoxia-inducible factor 1α and syncytin-1, further increased multi-nucleation of the BeWo cell layer and decreased barrier permeability only against smaller molecules (457 Da/4000 Da). In conclusion, we built a novel in vitro co-culture model of the placental barrier that possessed size-specific permeability and could function under physiologically low oxygen levels. Importantly, this will enable future researchers to better study the maternal-fetal transport of nutrients and drugs during pregnancy.

The Effects of 5,6,7,8,3',4'-Hexamethoxyflavone on Apoptosis of Cultured Human Choriocarcinoma Trophoblast Cells.

5,6,7,8,3,4'-Hexamethoxyflavone, also called nobiletin (NOB), widely found in the citrus peel, is one of the main byproducts in citrus processing. NOB is considered safe, but its safety for women during pregnancy is unknown. Therefore, the effect of NOB on apoptosis in human choriocarcinoma trophoblast cells (BeWo cells) was evaluated. Cells were divided into four groups and cultured with different concentrations of NOB (0, 10, 33, and 100 µM) for 12, 24, 36, and 48 h respectively. Cell viability was detected by CCK-8 assay, cell morphology was detected by a Cell Imaging Multi-Mode Reader, and cell cycle and apoptosis were detected by flow cytometry. Cleaved PARP level, the expressions of B cell lymphoma 2 (BCL2) family proteins, and p53 pathway proteins were detected by Western blot. The results showed that after 48 h of cell culture, the cell viability was decreased significantly, but apoptosis was significantly increased. Compared to the cells without NOB treatment, the cells treated with NOB at 10 or 33 µΜ showed no significant differences in the number of suspended cells or late apoptosis rate, except the increase of cell viability. Treatment of NOB at the concentration of 100 µM improved cell viability, attenuated apoptosis, decreased suspended cells, and did not alter the G1 phase arrest, compared with the non-NOB-treated group after 48 h of culturing. The 100 µΜ NOB treatment increased the levels of BCL2 and BCLXL, and decreased p53 accumulation in BeWo cells at 48 h, but had no effect on the expression of BAX, BAK, BAD, p21, and G1 phase arrest. These findings provide evidence that NOB (10, 33, and 100 µΜ) was safe for BeWo cells. NOB at the concentration of 100 µΜ could attenuate apoptosis in BeWo cells, which might be helpful to prevent pregnancy-related diseases caused by apoptosis.

Factors Involved in miRNA Processing Change Its Expression Level during Imitation of Hypoxia in BeWo b30 Cells.

One of the main complications of pregnancy and causes of maternal and perinatal mortality is preeclampsia. The pathophysiology of preeclampsia is associated with the development of placenta and fetal hypoxia and secretion of a number of effective molecules. The human choriocarcinoma cell line BeWo b30 is often used as a model of the placental barrier. It was shown that oxyquinoline derivatives can mimic hypoxia by suppressing HIF-prolyl hydroxylases and the accumulation of HIF-1α. This effect also leads to a change in the expression of microRNAs and their target genes. However, with hypoxia in cells, not only the level of individual miRNAs but also the ratio of miRNA isoforms (isomiRs) can change, presumably due to inaccuracies in the work of the Drosha and Dicer enzymes. In this work, we showed a change in the expression of the factors involved in the maturation of miRNAs when simulating hypoxia in BeWo b30 cells with an oxyquinoline derivative, which may be one of the causes for the change in the ratio of miRNA isoforms.

lncRNA GHET1 has effects in development of pre-eclampsia.

To explain long noncoding RNA (lncRNA) gastric carcinoma high expressed transcript 1 (GHET1) affects the mechanism in development of pre-eclampsia. The pathological changes of normal, mild, and severe pre-eclampsia were evaluated by hematoxylin and eosin staining and measured the lncRNA GHET1 expression in different tissues by reverse-transcription polymerase chain reaction. In the cell experiment, the BeWo cells were randomly divided into three groups: normal control (NC) group, model group, and lncRNA group. The JEG3 cells of the model and lncRNA groups were cultured in the hypoxia condition. The JEG3 cells invasion and migration abilities were measured by Tanswell and wound-healing assays. The relative protein expressions of different groups were evaluated by Western blot (WB) assay. Compared with normal puerperal, the lncRNA GHET1 gene expression of pre-eclampsia was significantly downregulated (P < 0.05, respectively). In the cell experiment, the invasion cell number and wound-healing rate of the model group were significantly suppressed compared with the NC group (P < 0.05, respectively). However, the invasion cell number and wound-healing rate of lncRNA group were enhanced by lncRNA GHET1 overexpression (P < 0.05, respectively). In WB assay, the E-cadherin, fibronectin, and vimentin proteins expression showed significant differences between the model and lncRNA groups (P < 0.05, respectively). lncRNA GHET1 overexpression had restored cell invasion and migration abilities reduced by hypoxia in pre-eclampsia.

Multiple Drug Transporters Contribute to the Placental Transfer of Emtricitabine.

Emtricitabine (FTC) is a first-line antiviral drug recommended for the treatment of AIDS during pregnancy. We hypothesized that transporters located in the placenta contribute to FTC transfer across the blood-placenta barrier. BeWo cells, cell models with stable or transient expression of transporter genes, primary human trophoblast cells (PHTCs), and small interfering RNAs (siRNAs) were applied to demonstrate which transporters were involved. FTC accumulation in BeWo cells was reduced markedly by inhibitors of equilibrative nucleoside transporters (ENTs), concentrative nucleoside transporters (CNTs), organic cation transporters (OCTs), and organic cation/carnitine transporter 1 (OCTN1) and increased by inhibitors of breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs). ENT1, CNT1, OCTN1, MRP1/2/3, and BCRP, but not ENT2, CNT3, OCTN2, or multidrug resistance protein 1 (MDR1), were found to transport FTC. FTC accumulation in PHTCs was decreased significantly by inhibitors of ENTs and OCTN1. These results suggest that ENT1, CNT1, and OCTN1 probably contribute to FTC uptake from maternal circulation to trophoblasts and that ENT1, CNT1, and MRP1 are likely involved in FTC transport between trophoblasts and fetal blood, whereas BCRP and MRP1/2/3 facilitate FTC transport from trophoblasts to maternal circulation. Coexistence of tenofovir or efavirenz with FTC in the cell medium did not influence FTC accumulation in BeWo cells or PHTCs.

Transcriptomic profiling of trophoblast fusion using BeWo and JEG-3 cell lines.

In human placenta, alteration in trophoblast differentiation has a major impact on placental maintenance and integrity. However, little is known about the mechanisms that control cytotrophoblast fusion. The BeWo cell line is used to study placental function, since it forms syncytium and secretes hormones after treatment with cAMP or forskolin. In contrast, the JEG-3 cell line fails to undergo substantial fusion. Therefore, BeWo and JEG-3 cells were used to identify a set of genes responsible for trophoblast fusion. Cells were treated with forskolin for 48 h to induce fusion. RNA was extracted, hybridised to Affymetrix HuGene ST1.0 arrays and analysed using system biology. Trophoblast differentiation was evaluated by real-time PCR and immunocytochemistry analysis. Moreover, some of the identified genes were validated by real-time PCR and their functional capacity was demonstrated by western blot using phospho-specific antibodies and CRISPR/cas9 knockdown experiments. Our results identified a list of 32 altered genes in fused BeWo cells compared to JEG-3 cells after forskolin treatment. Among these genes, four were validated by RT-PCR, including salt-inducible kinase 1 (SIK1) gene which is specifically upregulated in BeWo cells upon fusion and activated after 2 min with forskolin. Moreover, silencing of SIK1 completely abolished the fusion. Finally, SIK1 was shown to be at the center of many biological and functional processes, suggesting that it might play a role in trophoblast differentiation. In conclusion, this study identified new target genes implicated in trophoblast fusion. More studies are required to investigate the role of these genes in some placental pathology.