Human Choriocarcinoma Cell Line Research Articles

Regulation of Human Trophoblast GLUT3 Glucose Transporter by Mammalian Target of Rapamycin Signaling.

Glucose transporter isoform-3 (GLUT3), one of the primary placental facilitative glucose transporters responsible for basal glucose transport, has a crucial role in glucose transport and fetal growth during early pregnancy. A GLUT3 mutation in mice has been reported to cause loss of early pregnancy or late-gestational fetal growth restriction. However, the underlying mechanisms that regulate the placental GLUT3 transporter in humans are largely unknown. In the present study, we used the JEG-3 human choriocarcinoma cell line, which resembles a first trimester placental model, to study the role of the mammalian target of rapamycin complex 1 (mTORC1) in the regulation of placental GLUT3. We combined rapamycin treatment and small interfering (si) RNA-mediated silencing approaches with mRNA and protein expression/localization studies to investigate the alteration of GLUT3 expression and localization following mTORC1 inhibition in JEG-3 trophoblasts. Inhibition of mTORC1 signaling by silencing raptor decreased GLUT3 mRNA expression (−41%) and protein expression (−50%). Similar effects were obtained in cells in which mTORC1 was inhibited by rapamycin. Immunofluorescence analysis revealed that GLUT3 expression was markedly reduced in the cell surface and cytoplasm of JEG-3 cells in response to mTORC1 silencing. Because placental mTORC1 activity and GLUT3 expression are decreased in human intrauterine growth restriction, our data suggested one possible mechanism for the abnormal fetal growth in this pregnancy complication.

The Role of Megalin in the Transport of Gentamicin Across BeWo Cells, an In Vitro Model of the Human Placenta.

Aminoglycosides (AG) are known to readily cross the placenta, although the mechanisms responsible for placental transport have not been characterized. Megalin is expressed in human placenta, and it is reasonable to speculate, given its role in renal AG uptake, that it is similarly involved in placental transport. However, the role of megalin in placental AG uptake has not been established. An in vitro model to study megalin-mediated placental transport has also not been previously described. The objectives of this study, therefore, were to evaluate the human choriocarcinoma (BeWo) cell line as a model to study megalin-mediated placental transport and to assess the uptake kinetics of gentamicin, an AG antibiotic, using this in vitro model. BeWo cells were grown on Transwell® plates, and megalin expression and functional activity were assessed. Uptake of (3)H-gentamicin was also evaluated in the presence and absence of megalin inhibitors. Expression of megalin protein and mRNA in BeWo cells were confirmed via immunoblot and qPCR analysis. Uptake of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) (a megalin substrate) was time-, concentration-, and temperature-dependent consistent with a transporter-mediated process. FITC-BSA uptake was also significantly reduced in the presence of unlabeled gentamicin (a megalin substrate) and sodium maleate (to induce megalin shedding) suggesting that megalin is functionally active in BeWo cells. Gentamicin uptake exhibited time and temperature dependence, saturability and Michaelis-Menten kinetics, all of which suggest a transporter-mediated process. Gentamicin uptake was also significantly reduced in the presence of the megalin inhibitors RAP and EDTA suggesting that megalin is likely involved in gentamicin uptake.

Epidermal growth factor-like domain 7 promotes migration and invasion of human trophoblast cells through activation of MAPK, PI3K and NOTCH signaling pathways.

Epidermal growth factor-like domain 7 (Egfl7) is a gene that encodes a partially secreted protein and whose expression is largely restricted to the endothelia. We recently reported that EGFL7 is also expressed by trophoblast cells in mouse and human placentas. Here, we investigated the molecular pathways that are regulated by EGFL7 in trophoblast cells. Stable EGFL7 overexpression in a Jeg3 human choriocarcinoma cell line resulted in significantly increased cell migration and invasiveness, while cell proliferation was unaffected. Analysis of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways showed that EGFL7 promotes Jeg3 cell motility by activating both pathways. We show that EGFL7 activates the epidermal growth factor receptor (EGFR) in Jeg3 cells, resulting in downstream activation of extracellular regulated kinases (ERKs). In addition, we provide evidence that EGFL7-triggered migration of Jeg3 cells involves activation of NOTCH signaling. EGFL7 and NOTCH1 are co-expressed in Jeg3 cells, and blocking of NOTCH activation abrogates enhanced migration of Jeg3 cells overexpressing EGFL7. We also demonstrate that signaling through EGFR and NOTCH converged to mediate EGFL7 effects. Reduction of endogenous EGFL7 expression in Jeg3 cells significantly decreased cell migration. We further confirmed that EGFL7 stimulates cell migration by using primary human first trimester trophoblast (PTB) cells overexpressing EGFL7. In conclusion, our data suggest that in trophoblast cells, EGFL7 regulates cell migration and invasion by activating multiple signaling pathways. Our results provide a possible explanation for the correlation between reduced expression of EGFL7 and inadequate trophoblast invasion observed in placentopathies.

Genomic Alterations in Ethanol-Exposed Trophoblast Cell Lines Induced by Chronic Ethanol Treatment

Background: Ethanol consumption during pregnancy results in a broad spectrum of damage, but the knowledge of its mechanism is lacking. Objective: The aim of this study is to determine ethanol-caused genomic alterations in placental cell lines after a repeated ethanol treatment in order to describe new genomic targets of cell damage. Methods: A model of sustained exposure to standard doses of ethanol on two in vitro human choriocarcinoma cell lines, JEG-3 and BeWo, was used. Chromosomic abnormalities and copy number alterations (CNAs) were assessed by G-Banding cytogenetics and oligonucleotide Single Nucleotide Polymorphism-Array analysis (CytoScan, Affymetrix). Results: Chromosomal abnormalities did not change despite ethanol exposure except for the presence of a derivative chromosome 4 [add(4)(p14)] in exposed BeWo cells. Regarding SNP-Array analysis, a total of 21 CNAs were found to be caused by ethanol exposure, 16 in JEG-3 cell line and 5 in BeWo cell line, which were not found in controls. There was no coincidence between JEG-3 and BeWo regions affected by ethanol. Conclusion: Trophoblast cell lines exposed repetitively to ethanol presented genomic instability resulting in CNAs. However, no region has been equally altered in both models to consider it an ethanol exposure target area. So, further studies involving different models and approaches that target gene regulation are required.

Chemometric strategy for untargeted lipidomics: Biomarker detection and identification in stressed human placental cells

A lipidomic study was developed in a human placental choriocarcinoma cell line (JEG-3) exposed to tributyltin (TBT) and to a mixture of perfluorinated chemicals (PFCs). The method was based on the application of multivariate curve resolution-alternating least squares (MCR-ALS) to data sets obtained by ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC–TOF-MS) using an untargeted approach. Lipids from exposed JEG-3 cells were solid–liquid extracted and analyzed by UHPLC–TOF-MS in full scan mode, together with control samples. Raw UHPLC–TOF-MS data of the different cell samples were subdivided into 20 distinct chromatographic windows and each window was further organized in a column-wise augmented data matrix, where data from every sample was in an individual data matrix. Then, the 20 new augmented data matrices were modeled by MCR-ALS. A total number of 86 components were resolved and a statistical comparative study of their elution profiles showed distinct responses for the lipids of exposed versus control cells, evidencing a lipidome disruption attributed to the presence of the xenobiotics. Results from one-way ANOVA followed by a multiple comparisons test and from discriminant partial least squares (PLS-DA) analysis were compared as usual strategies for the determination of potential biomarkers. Identification of 24 out of the 33 proposed biomarkers contributed to the better understanding of the effects of PFCs and TBT in the lipidome of human placental cells. Overall, this study proposes an innovative untargeted LC–MS MCR-ALS approach valid for -omic sciences such as lipidomics.

The endocannabinoid anandamide induces apoptosis in cytotrophoblast cells: Involvement of both mitochondrial and death receptor pathways

IntroductionA balanced proliferation, apoptosis and differentiation in trophoblast cells of the human placenta is crucial for a proper placental development. Alteration in trophoblast apoptosis and differentiation are associated with gestational-related complications, such as preeclampsia, intrauterine growth restriction or miscarriages. The endocannabinoids (eCBs) have been recognized as new interveners in pregnancy events such as implantation and decidualization. However, their importance in placentation is poorly understood. We hypothesise that these novel lipid mediators may intervene in cytotrophoblast apoptosis and, concomitantly, have a role during placental development. Methodsprimary human cytotrophoblasts (hCTs) and the human trophoblast-like choriocarcinoma cell line BeWo cells were exposed to Anandamide (AEA). It was investigated the cellular pathways involved in cell death, by the assessment of cell morphology, caspases activity, mitochondrial membrane potential (Δψm), reactive oxygen/nitrogen species (ROS/RNS) and western blot of cleaved Poly (ADP-ribose) polymerase 1 (PARP-1), truncated Bid (t-Bid) and IκB-α. ResultsAEA decreased hCTs viability and induced morphological features of apoptosis (chromatin condensation and fragmentation), caspase-3/7 activation and PARP-1 cleavage. In BeWo, AEA also increased the activities of caspase-3/7 and 9, induced loss in Δψm and production of ROS/RNS. These effects were reversed by either CB1 or CB2 antagonists, whereas the increase in caspase-3/7 activity was only reversed with CB2 blockage. AEA-treated cells showed increased caspase-8 activation and formation of t-Bid, suggesting the interplay between intrinsic and extrinsic apoptotic pathways. AEA also increased IκB-α expression, a NF-κB regulatory protein. ConclusionOur results highlight the importance of eCBs in cytotrophoblast cell apoptosis and indicate that a crosstalk between intrinsic and extrinsic apoptotic pathways is involved in AEA-induced effects.

Role of the vasohibin family in the regulation of fetoplacental vascularization and syncytiotrophoblast formation.

Vasohibin-1 (VASH1) and vasohibin-2 (VASH2), the 2 members of the vasohibin family, have been identified as novel regulators of angiogenesis. VASH1 ceases angiogenesis, whereas VASH2 stimulates sprouting. Here we characterized their functional role in the placenta. Immunohistochemical analysis of human placental tissue clarified their distinctive localization; VASH1 in endothelial cells and VASH2 in trophoblasts. We then used a mouse model to explore their function. Wild-type, Vash1(−/−), and Vash2(−/−) mice on a C57BL6 background were used in their first pregnancy. As expected, the fetal vascular area was increased in the Vash1(−/−) mice, whereas it was decreased in the Vash2(−/−) mice relative to wild-type. In addition, we noticed that the Vash2(−/−) mice at 18.5dpc displayed thinner villi of the labyrinth and larger maternal lacunae. Careful observation by an electron microscopy revealed that the syncytiotrophoblast formation was defective in the Vash2(−/−) mice. To test the possible involvement of VASH2 in the syncytiotrophoblast formation, we examined the fusion of BeWo cells, a human trophoblastoid choriocarcinoma cell line. The forskolin treatment induced the fusion of BeWo cells, and the knockdown of VASH2 expression significantly inhibited this cell fusion. Conversely, the overexpression of VASH2 by the infection with adenovirus vector encoding human VASH2 gene significantly increased the fusion of BeWo cells. Glial cell missing-1 and endogenous retrovirus envelope glycoprotein Syncytin 1 and Syncytin 2 are known to be involved in the fusion of trophoblasts. However, VASH2 did not alter their expression in BeWo cells. These results indicate that VASH1 and VASH2 showed distinctive localization and opposing function on the fetoplacental vascularization. Moreover, our study shows for the first time that VASH2 expressed in trophoblasts is involved in the regulation of cell fusion for syncytiotrophoblast formation.

Expression of C1QBP gene and its correlation with drug resistance in human resistance choriocarcinoma cell line

To examine the complement component 1 Q subcomponent-binding protein (C1QBP) gene expression in human resistance choriocarcinoma cell lines and its parental cell line JeG-3, and to investigate whether silence C1QBP by small interference RNA could reverse the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs. Expression of C1QBP mRNA and protein in cells were detected by real- time fluorogenic quantitative PCR and western blot, respectively. The difference of C1QBP expression was compared between human resistance choriocarcinoma cell lines and its parental cell line JeG-3. Sub-cellular location was proved by confocal immunofluorescence microscopy. A lentiviral vector containing short hairpin RNA (shRNA) targeting C1QBP was constructed and cotransfected with the packaging plasmid mixture into 293T cells by lipofectamine 2000. The human resistance choriocarcinoma cell lines were infected with the packaged lentivirus. Real- time fluorogenic quantitative PCR and western blot were used to validate whether the C1QBP gene expression was silenced. The cell counting kit 8 (CCK8) was used to determine the drug sensitivity. (1) The C1QBP mRNA expression levels among four human resistance choriocarcinoma cell lines [JeG-3/floxuridiuum (FUDR), JeG- 3/methotrexate (MTX), JeG-3/etoposide (VP), JeG-3/dactinomycin (KSM)] were 2.520 ±0.680, 1.770 ± 0.230, 1.940 ± 0.090 and 1.740 ± 0.350 folds compared to that in JeG- 3 cells. The C1QBP protein was higher expression level in human resistance choriocarcinoma cell lines than that in JeG-3. The immunofluorescence methods and confocal analysis showed that C1QBP localized predominantly in the mitochondrial matrix. (2) The C1QBP mRNA expression in JeG-3/FUDR cells after infected with lentiviral vector were decreased by 93.1% (P < 0.01). The protein expression of C1QBP in JeG-3/FUDR cells after infected with lentiviral vector were almost completely suppressed. The resistance indexes of four human resistance choriocarcinoma cell lines (JeG-3/FUDR, JeG-3/MTX, JeG-3/VP, JeG-3/KSM) were respectively 86.3% , 93.9% , 92.8% and 89.9%, which were decreased remarkably by knockdown the C1QBP expression (P < 0.05). C1QBP is overexpressed in human resistance choriocarcinoma cell lines compared with parental cell line JeG-3. Inhibition of C1QBP by lentivirus- mediated small interference RNA could effectively reverses the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs.

Modulating drug resistance by targeting BCRP/ABCG2 using retrovirus-mediated RNA interference.

BackgroundThe BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications.Methodology/Principal FindingsTo target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor.Conclusions/SignificanceThe goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi) to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors.

Soluble endoglin production is upregulated by oxysterols but not quenched by pravastatin in primary placental and endothelial cells

IntroductionPreeclampsia is a serious pregnancy complication. Soluble endoglin (sEng) is released from the placenta and contributes to the maternal endothelial dysfunction seen in preeclampsia. Recently oxysterols, which activate the Liver X Receptor (LXR), have been implicated in producing sEng, by upregulating matrix metalloproteinase-14 (MMP14; cleaves endoglin to produce sEng) and down-regulating tissue inhibitor of metalloproteinase-3 (TIMP-3; inhibitor of MMP14). The functional experiments in that study were performed on JAR cells (human choriocarcinoma cell line) and placental explants. MethodsWe characterized LXR in severe preeclamptic placentas, and assessed whether oxysterols increase release of sEng from primary human umbilical vein endothelial cells (HUVECs), primary trophoblasts and placental explants. Given pravastatin is thought to block oxysterol production and inhibit the LXR, we examined whether pravastatin reduces sEng release. ResultsLXRα and β were localized to the syncytiotrophoblast and villous tips and were significantly up-regulated in preeclamptic placenta. Oxysterols upregulated sEng production in HUVECs and placental explants although the increases were far more modest than that recently reported. Oxysterols did not upregulate sEng in primary trophoblasts. Furthermore, mRNA expression of MMP14 and TIMP-3 were not altered by oxysterols in any tissue. Surprisingly, pravastatin did not decrease oxysterol-induced upregulation of sEng. DiscussionLXR is up-regulated in preeclamptic placenta. Oxysterols upregulate sEng production from human tissues, but the increase is modest, suggesting this may not be the main mechanism for the very significant elevations in sEng seen in preeclampsia. Pravastatin does not decrease sEng production. ConclusionOxysterols modestly up-regulate sEng production which is not quenched by pravastatin.

MicroRNA-218 inhibits the proliferation of human choriocarcinoma JEG-3 cell line by targeting Fbxw8

MicroRNAs (miRNAs) are endogenous 19–25 nucleotide noncoding single-stranded RNAs that regulate gene expression by blocking the translation or decreasing the stability of mRNAs. In this study, we showed that miR-218 expression levels were decreased while Fbxw8 expression levels were increased in human choriocarcinoma cell lines, and identified Fbxw8 as a novel direct target of miR-218. Overexpression of miR-218 inhibited cell growth arrest at G2/M phase, suppressed the protein levels of cyclin A and up-regulated the expression levels of p27 through decreasing the levels of Fbxw8. On the other hand, forced expression of Fbxw8 partly rescued the effect of miR-218 in the cells, attenuated cell proliferation decrease the percentage of cells at G2/M phase, induced cyclin A protein expression and suppressed the protein level of p27 through up-regulating the levels of Fbxw8. Taken together, these findings will shed light the role to mechanism of miR-218 in regulating JEG-3 cells proliferation via miR-218/Fbxw8 axis, and miR-218 may serve as a novel potential therapeutic target in human choriocarcinoma in the future.

Methylation pattern of H19 exon 1 is closely related to preeclampsia and trophoblast abnormalities

Preeclampsia (PE) is a pregnancy-induced disorder characterized by the overproliferation of trophoblasts. Hydatidiform moles, which are associated with a high risk of developing PE, are characterized by the excessive proliferation of trophoblastic tissue. H19 is highly expressed in placental tissue; however, its biological function remains unclear. Afundamental modification of the H19 gene is DNA methylation, which typically occurs in CG-rich regions at the promoter or the first exon region. In this study, in order to investigate the DNA methylation pattern of the H19 exon1 region in placental tissues and trophoblast cells, placental specimens were collected from women in the first trimester of pregrancy (FTP) and the third trimester of pregnancy(TTP), as well as from from women with severe preeclampsia (sPE). We found that the DNA methylation levels of H19 exon1 were significantly higher in the tissues obtained from women in TTP than from those obtained from women in FFP. The methylation status of CpG1 sites within exon1 of H19 was markedly higher in the placental tissues obtained from women with sPE than in the tissues obtained from women in TTP. In addition, we used the human choriocarcinoma cell line, JEG-3, and treated the cells with the methylation inhibitor, 5-aza-2'-deoxycytidine (5-Aza‑Dc). Following treatment with 5-Aza-Dc, the methylation levels at this CpG site showed marked hypomethylation. In addtion, the cell proliferative, migratory and invasive capacities of the cells were remarkably inhibited. Our data suggest that hypermethylation at individual CpG sites within exon1 of H19 may be involved in the dysfunction of trophoblasts and the pathogenesis of PE.

Progranulin shows cytoprotective effects on trophoblast cells in vitro but does not antagonize TNF-α-induced apoptosis.

The glycoprotein progranulin directly binds to TNF-receptors and thereby can antagonize the inflammatory effects of TNF-α. Here we analyzed the impact of both cytokines on cytotoxicity and viability of trophoblast cells. Isolated villous first trimester human trophoblast cells and the human choriocarcinoma cell line BeWo were treated with recombinant human progranulin and TNF-α. Analyses were performed by LDH- and MTT-assay and measurement of caspase-8-activity. Progranulin treatment showed some cytoprotective effects on isolated trophoblast cells. However, TNF-α-induced apoptosis was not antagonized by addition of progranulin. Effects were similar, but more pronounced in BeWo cells. The cytoprotective activity of progranulin on trophoblast cells in vitro was only weak and of doubtful biologic relevance. It was not able to antagonize TNF-α. Future studies should focus on possible paracrine activities of progranulin.

Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma cell lines and human first trimester/term trophoblast cells

Trophoblast invasion into uterine tissues represents a hallmark of first trimester placental development. As expression of serum amyloid A4 (SAA4) occurs in tumorigenic and invasive tissues we here investigated whether SAA4 is present in trophoblast-like human AC1-M59/Jeg-3 cells and trophoblast preparations of human first trimester and term placenta. SAA4 mRNA was expressed in non-stimulated and cytokine-treated AC1-M59/Jeg-3 cells. In purified trophoblast cells SAA4 mRNA expression was upregulated at weeks 10 and 12 of pregnancy. Western-blot and immunohistochemical staining of first trimester placental tissue revealed pronounced SAA4 expression in invasive trophoblast cells indicating a potential role of SAA4 during invasion.

Perfluorinated chemicals: Differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells

The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs – PFOS, PFDoA, PFNA, PFOA – showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647μM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA>PFOS≫PFNA>PFOA>PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57–80μM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6μM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells.

Differential response to lipopolysaccharide by JEG-3 and BeWo human choriocarcinoma cell lines

ObjectiveTo determine the effect of lipopolysaccharide (LPS) on NF-κB gene expression and proinflammatory cytokine release from trophoblast cell models, JEG-3 and BeWo human choriocarcinoma cells. Study designSerum-starved JEG-3 and BeWo cells were treated with LPS (from Escherichia coli serotype 0111:B4) for 24 or 48h. Cell culture medium was collected and assayed for interleukin (IL)-1β, IL-6, IL-8, and transforming necrosis factor (TNF)-α cytokine release using enzyme-linked immunosorbent assays. RNA was extracted from the cells and real-time PCR was performed to measure NF-κB mRNA expression. All results were analyzed by one-way analysis of variance tests followed by Sidak's post hoc analysis. p<0.05 was considered statistically significant. ResultsLPS triggered an inflammatory response in JEG-3 cells by inducing a 1.5-fold increase in NF-κB mRNA expression and TNF-α release (0μg/mL: 15.13±2.14, 1μg/mL: 14.94±0.75, 10μg/mL: 23.05±4.50, p<0.05) and a 2-fold elevation in IL-6 secretion (0μg/mL: 12.54±5.44, 1μg/mL: 25.54±0.91, 10μg/mL: 24.28±4.43, p<0.05). In contrast, BeWo cells were not as sensitive to LPS exposure; NF-κB mRNA expression was unchanged between LPS-treated and control cells, whereas a small but significant 1.3-fold increase in TNF-α release was found (TNF-α: 15.45±1.53pg/mL, control: 12.24±1.00pg/mL, p<0.05). The inflammatory pathways in BeWo cells were found to be active given that treatment of these cells with IL-1β and TNF-α induced IL-6 secretion. Interestingly, 1μg/mL LPS appeared to decrease IL-6 and TNF-α release from BeWo cells. IL-1β and IL-8 secretion were not detected from either cell lines. ConclusionLPS activates the NF-κB pathway in JEG-3 but not BeWo human choriocarcinoma cells and this may be the reason for their differential inflammatory response to LPS exposure.

A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation

Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines.

Inhibition of phosphodiesterase 4 promotes differentiation and fusion of bewo choriocarcinoma cells

Inhibition of phosphodiesterase 4 promotes differentiation and fusion of bewo choriocarcinoma cells

Effect of hepatitis B virus infection on apoptosis of a human choriocarcinoma cell line in vitro

To investigate the effect and mechanism of hepatitis B virus (HBV) infection on the human choriocarcinoma cell line, JEG-3, in relation to apoptosis and intrauterine infection. HBV-DNA serum was used to infect the choriocarcinoma cell line, JEG-3, in vitro. Real-time fluorescence quantitative PCR (RT-PCR) was then employed to detect intracellular replication of HBV DNA. Cells were also stained with Annexin-V and propidium iodide (PI) to identify the stages of apoptosis following infection. In addition, reverse transcription PCR was used to detect intracellular HBx mRNA levels, and Western blotting and immunohistochemistry were used to detect changes in the intracellular expression of HBxAg and phosphatidylinositol kinase 3 (PI3K). Flow cytometry was also used to detect the intracellular levels of phosphorylated AKT (pAKT). After JEG-3 cells were infected with HBV in vitro, HBV DNA was detected. The percentage of cells in early and late stage apoptosis also decreased significantly. Expression of HBx mRNA and HBxAg were detected, and intracellular levels of PI3K and pAKT were observed to significantly increase. HBV infected JEG-3 cells in vitro, resulting in an inhibition of early and late stage apoptosis. In addition, the HBxAg/PI3K/pAKT pathway is a possible mechanism mediating this inhibition of apoptosis, and the infection of the placenta by HBV.

Lentivirus-mediated RNA interference targeting the H19 gene inhibits cell proliferation and apoptosis in human choriocarcinoma cell line JAR

BackgroundH19 is a paternally imprinted gene that has been shown to be highly expressed in the trophoblast tissue. Results from previous studies have initiated a debate as to whether noncoding RNA H19 acts as a tumor suppressor or as a tumor promotor in trophoblast tissue. In the present study, we developed lentiviral vectors expressing H19-specific small interfering RNA (siRNA) to specifically block the expression of H19 in the human choriocarcinoma cell line JAR. Using this approach, we investigated the impact of the H19 gene on the proliferation, invasion and apoptosis of JAR cells. Moreover, we examined the effect of H19 knockdown on the expression of insulin-like growth factor 2 (IGF2), hairy and enhancer of split homologue-1 (HES-1) and dual-specific phosphatase 5 (DUSP5) genes.ResultsH19 knockdown inhibited apoptosis and proliferation of JAR cells, but had no significant impact on cell invasion. In addition, H19 knockdown resulted in significant upregulation of HES-1 and DUSP5 expression, but not IGF2 expression in JAR cells.ConclusionsThe finding that H19 downregulation could simultaneously inhibit proliferation and apoptosis of JAR cells highlights a putative dual function for H19 in choriocarcinoma and may explain the debate on whether H19 acts as a tumor suppressor or a tumor promotor in trophoblast tissue. Furthermore, upregulation of HES-1 and DUSP5 may mediate H19 downregulation-induced suppression of proliferation and apoptosis of JAR cells.