BaP/BPDE exposure causes human trophoblast cell dysfunctions and induces miscarriage by up-regulating lnc-HZ06-regulated IL1B.

Exposure to environmental BaP or its metabolite BPDE causes trophoblast cell dysfunctions to induce miscarriage (abnormal early embryo loss), which might be generally regulated by lncRNAs. IL1B, a critical inflammatory cytokine, is closely associated with adverse pregnancy outcomes. However, whether IL1B might cause dysfunctions of BaP/BPDE-exposed trophoblast cells to induce miscarriage, as well as its specific epigenetic regulatory mechanisms, is completely unexplored. In this study, we find that BPDE-DNA adducts, trophoblast cell dysfunctions, and miscarriage are closely associated. Moreover, we also identify a novel lnc-HZ06 and IL1B, both of which are highly expressed in BPDE-exposed trophoblast cells, in villous tissues of recurrent miscarriage patients, and in placental tissues of BaP-exposed mice with miscarriage. Both lnc-HZ06 and IL1B suppress trophoblast cell migration/invasion and increase apoptosis. In mechanism, lnc-HZ06 promotes STAT4-mediated IL1B mRNA transcription, enhances IL1B mRNA stability by promoting the formation of METTL3/HuR/IL1B mRNA ternary complex, and finally up-regulates IL1B expression levels. BPDE exposure promotes TBP-mediated lnc-HZ06 transcription, and thus up-regulates IL1B levels. Knockdown of either murine lnc-hz06 (which down-regulates Il1b levels) or murine Il1b could alleviate miscarriage in BaP-exposed mice. Collectively, this study not only discovers novel biological mechanisms and pathogenesis of unexplained miscarriage but also provides novel potential targets for treatment against BaP/BPDE-induced miscarriage.

Environmental BaP/BPDE suppressed trophoblast cell invasion/migration and induced miscarriage by down-regulating lnc-HZ01/MEST/VIM axis.

Environmental benzo(a)pyrene (BaP) and itsmetabolite benzo(a)pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE), classic endocrine disrupting chemical and persistent organic pollutant, could cause miscarriage. However, the detailed mechanisms are still largely unclear and should be further explored. In this study, we discovered that exposure of trophoblast cells with BPDE could suppressed cell invasion/migration by inhibiting MEST/VIM (Vimentin) pathway. Moreover, BPDE exposure also increased lnc-HZ01 expression level, which further inhibited MEST/VIM pathway and then suppressed invasion/migration. Knockdown of lnc-HZ01 or overexpression of MEST could efficiently rescue invasion/migration of BPDE-exposed Swan 71 cells. Furthermore, lnc-HZ01 was highly expressed and MEST/VIM were lowly expressed in recurrent miscarriage (RM) villous tissues compared with healthy control (HC) group. Finally, we also found that BaP exposure inhibited murine Mest/Vim pathway in placental tissues and induced miscarriage in BaP-exposed mice. Therefore, the regulatory mechanisms were similar in BPDE-exposed human trophoblast cells, RM villous tissues, and placental tissues of BaP-exposed mice with miscarriage, building a bridge to connect BaP/BPDE exposure, invasion/migration, and miscarriage. This study provided novel insights in the toxicological effects and molecular mechanisms of BaP/BPDE-induced miscarriage, which is helpful for better elucidating the toxicological risks of BaP/BPDE on female reproduction.

Spheroid formation induces chemokine production in trophoblast-derived Swan71 cells.

PROBLEM: In the cell column of anchoring villi, the cytotrophoblast differentiates into extravillous trophoblast (EVT) and invades the endometrium in contact with maternal immune cells. Recently, chemokines were proposed to regulate the decidual immune response. To investigate the roles of chemokines around the anchoring villi, we examined the expression profiles of chemokines in the first-trimester trophoblast-derived Swan71 cells using a three-dimensional culture model. METHOD OF STUDY: The gene expressions in the spheroid-formed Swan71 cells were examined by microarray and qPCR analyses. The protein expressions were examined by immunochemical staining. The chemoattractant effects of spheroid-formed Swan71 cells were examined by migration assay using monocyte-derived THP-1 cells. RESULTS: The expressions of an EVT marker, laeverin, and matrix metalloproteases, MMP2 and MMP9, were increased in the spheroid-cultured Swan71 cells. Microarray and qPCR analysis revealed that mRNA expressions of various chemokines, CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, and CXCL10, in the spheroid-cultured Swan71 cells were up-regulated as compared with those in the monolayer-cultured Swan71 cells. These expressions were significantly suppressed by hypoxia. Migration assay showed that culture media derived from the spheroid-formed Swan71 cells promoted THP-1 cell migration. CONCLUSION: This study indicated that chemokine expressions in Swan71 cells increase under a spheroid-forming culture and the culture media have chemoattractant effects. Since three-dimensional cell assembling in the spheroid resembles the structure of the cell column, this study also suggests that chemokines play important roles in the interaction between EVT and immune cells in their early differentiation stage.

Low Susceptibility of Rubella Virus in First-Trimester Trophoblast Cell Lines.

We recently published an article about myelin oligodendrocyte glycoprotein-independent rubella infection of keratinocytes in vitro, in which first-trimester trophoblast cells were shown as rubella virus (RuV)-resistant. Given an incident rate as high as 90% of congenital rubella syndrome in the first eight weeks of pregnancy, the RuV infection of first-trimester trophoblasts is considered key to opening the gate to transplacental transmission mechanisms. Therefore, with this study, we aimed to verify the susceptibility/resistance of first-trimester trophoblast cell lines, HTR-8/SVneo and Swan.71, against RuV. Cells cultured on multi-well plates were challenged with a RuV clinical strain at a multiplicity of infection from 5 to 10 for 3 h. The infectivity was investigated by immunofluorescence (IF) assay and flow cytometry (FCM) analysis. Supernatants collected during the post-infection period were used to determine virus-progeny production. The scattered signaling of RuV infection of these cells was noted by IF assay, and the FCM analysis showed an average of 4-5% of gated cells infected with RuV. In addition, a small but significant production of virus progeny was also observed. In conclusion, by employing appropriate approaches, we determined the low infectivity of RuV in first-trimester trophoblast cell lines but not resistance as in our previous report.

Lnc-HZ05 regulates BPDE-inhibited human trophoblast cell proliferation and affects the occurrence of miscarriage by directly binding with miR-hz05.

Approximately 15-25% pregnant women end with miscarriage in the world. Environmental BaP (benzo(a)pyrene) and its terminal metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) may result in the dysfunctions of trophoblast cells, which might further lead to RM (recurrent miscarriage). However, potential mechanisms remain unelucidated. In this work, we identified a novel lnc-HZ05 highly expressed and a novel miR-hz05 lowly expressed in both trophoblast cells exposed to BPDE and human RM tissues. MiR-hz05 reduces FOXO3a mRNA level by weakening its mRNA stability. Lnc-HZ05 increases the expression of FOXO3a by acting as a ceRNA for miR-hz05, and then increases P21 level and reduces CDK2 level. Thus, cell cycle is arrested at G0/G1 phase and trophoblast proliferation is inhibited. Lnc-HZ05 harboring wild-type binding site for miR-hz05, but not its mutant site, could upregulate FOXO3a expression. In normal trophoblast cells, relatively less lnc-HZ05 and more miR-hz05 activate FOXO3a/P21/CDK2 pathway and promote trophoblast proliferation, giving normal pregnancy. In RM tissues and BPDE-treated human trophoblast cells, lnc-HZ05 is increased and miR-hz05 is reduced, both of which suppress this pathway and inhibit cell proliferation, and finally lead to miscarriage. Thus, lnc-HZ05 and miR-hz05 simultaneously regulate cell cycle and proliferation of BPDE-exposed trophoblast cells and miscarriage, providing new perspectives and clinical understandings in the occurrence of unexplained miscarriage.

Upregulated lnc-HZ02 and miR-hz02 inhibited migration and invasion by downregulating the FAK/SRC/PI3K/AKT pathway in BPDE-treated trophoblast cells.

BPDE (benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide), a metabolite of environmental carcinogenic BaP, weakens the migration and invasion of human villous trophoblast cells and may further induce miscarriage. However, the underlying mechanisms remain largely unknown. In this study, we identified that in trophoblast Swan 71 and HTR-8/SVneo cells, miR-hz02 upregulates the level of lnc-HZ02, which inhibits the expression of an RNA-binding protein HuR. HuR could interact with FAK mRNA and promote its mRNA stability, thus upregulating the FAK level and the FAK/SRC/PI3K/AKT pathway, and finally maintaining the normal migration and invasion of trophoblast cells. If trophoblast cells are exposed to BPDE, both miR-hz02 and lnc-HZ02 are upregulated, which reduce the level of HuR, weaken the interactions of HuR with FAK mRNA, downregulate FAK level and the FAK/SRC/PI3K/AKT pathway, and finally inhibit cell migration and invasion. This study provides a novel scientific understanding of the dysfunctions of human trophoblast cells.

Depletion of CTCF disrupts PSG gene expression in the human trophoblast cell line Swan 71.

Pregnancy-specific glycoproteins (PSGs) are fetal proteins secreted by the placenta during pregnancy. The PSG level in maternal serum is an indicator of risk for pregnancy complications. However, little is known about the molecular mechanisms underlying PSG gene expression. Recently, the importance of epigenetic regulation of placental genes has been emphasized in the study of developmental defects and placental disease. In this study, the role of the CCCTC-binding factor (CTCF) in regulation of PSG expression was investigated to better understand the epigenetic regulatory mechanisms of the PSG genes. Inhibition of CTCF expression disturbed transcription of several PSG genes: PSG1, PSG2, PSG4, PSG5, PSG8, and PSG9 were upregulated and PSG6 and PSG11 were downregulated. These transcriptional changes were correlated with decreased CTCF binding and changes in histone modification at the PSG promoters. Our data demonstrate that CTCF is a potential mediator in the regulation of PSG gene expression.

SIRT1 negatively regulates invasive and angiogenic activities of the extravillous trophoblast.

PROBLEM: Dysregulation of extravillous trophoblast (EVT) invasion leads to pregnancy complications, such as pre-eclampsia, fetal growth restriction, and placenta accreta. The aim of this study was to explore the role of SIRT1 in EVT invasion and its underlying mechanism. METHOD OF STUDY: SIRT1-specific siRNA was transfected into Swan 71 cells, an immortalized first trimester trophoblast cell line. The Boyden chamber invasion assay, the scratch wound healing assay, and cell proliferation assay were performed. The expression levels of epithelial-to-mesenchymal transition (EMT) markers, matrix metalloproteinase-2 (MMP-2), MMP-9, p-Akt, Akt, p-p38MAPK, p38MAPK, p-ERK, ERK, p-JNK, JNK, Fas, and Fas ligand (FasL) were examined by western blot. Tube formation assay was conducted by using Matrigel. RESULTS: SIRT1 knockdown by siRNA significantly enhanced invasion and migration as well as the expression of MMP-2, MMP-9, and EMT markers in Swan 71 cells, but reduced proliferation. The effects of SIRT1 knockdown on invasion, migration, proliferation, and endothelial-like tube formation in Swan 71 cells were reversely regulated by blockade of Akt and p38MAPK signaling. In addition, SIRT1 knockdown markedly promoted colocalization of Swan 71 cells to human umbilical vein endothelial cell (HUVEC) networks and induced reduction in Fas and enhancement of FasL. Conditioned media of SIRT1 knockdown-Swan 71 cells caused reduction in cell proliferation and augmentation of cytotoxicity along with increased Fas expression in HUVECs. CONCLUSION: Our results suggest that SIRT1 may be associated with placental development by controlling EVT invasion and spiral artery remodeling via modulation of EMT, MMP-2, MMP-9, Akt/p38MAPK signaling, and Fas/FasL.

Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide induces human trophoblast Swan 71 cell dysfunctions due to cell apoptosis through disorder of mitochondrial fission/fusion.

Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE) is an endocrine disrupter and ultimate carcinogenic product of benzo(a)pyrene (BaP). Numerous studies have shown that BPDE causes trophoblast-related diseases, such as preeclampsia, growth restriction or miscarriages. However, the underlying mechanism, especially the mitochondria-related BPDE-induced trophoblast dysfunction remains unknown. In this study, we examined mitochondrial functions in BPDE-induced human trophoblast cell line Swan 71. BPDE decreased cell ability, attenuated cell invasion and HCG secretion, induced cell apoptosis, decreased mitochondrial membrane potential, increased reactive oxygen species (ROS) and MDA, and decreased SOD activity in a dose-dependent manner. In the mechanism, BPDE significantly increased pro-apoptosis protein (P53 and Bak1) and decreased anti-apoptosis protein (Bcl-2). Furthermore, the protein expression levels of mitochondrial fusion genes (Mfn1, Mfn2, and OPA1) were decreased and those of fission genes (Fis1 and Drp1) were increased with increasing concentrations of BPDE and incubation time, resulting in the release of Cyt c and activation of Caspase 3, which irreversibly induced trophoblast cell apoptosis. This study reveals the mechanism of dysfunction of trophoblast cells through cell apoptosis due to the disorder of mitochondrial fission/fusion after exposure to BPDE, providing a further experimental understanding the adverse effects of BaP on trophoblast cells in early pregnancy.

Granulocyte colony-stimulating factor (G-CSF) upregulates ß1 integrin and increases migration of human trophoblast Swan 71 cells via PI3K and MAPK activation.

Multiple cytokines and growth factors expressed at the fetal-maternal interface are involved in the regulation of trophoblast functions and placental growth, but the role of G-CSF has not been completely established. Based on our previous study showing that G-CSF increases the activity of matrix metalloproteinase-2 and the release of vascular endothelial growth factor in Swan 71 human trophoblast cells, in this work we explore the possible contribution of G-CSF to cell migration and the G-CSF-triggered signaling pathway. We found that G-CSF induced morphological changes on actin cytoskeleton consistent with a migratory cell phenotype. G-CSF also up-regulated the expression levels of ß1 integrin and promoted Swan 71 cell migration. By using selective pharmacological inhibitors and dominant negative mutants we showed that PI3K, Erk 1/2 and p38 pathways are required for promoting Swan 71 cell motility. It was also demonstrated that PI3K behaved as an upstream regulator of Erk 1/2 and p38 MAPK. In addition, the increase of ß1 integrin expression was dependent on PI3K activation. In conclusion, our results indicate that G-CSF stimulates ß1 integrin expression and Swan 71 cell migration by activating PI3K and MAPK signaling pathways, suggesting that G-CSF should be considered as an additional regulatory factor that contributes to a successful embryo implantation and to the placenta development.