RESUMO
Placental development involves coordinated expansion and differentiation of trophoblast cell lineages possessing specialized functions. Among the differentiated trophoblast cell lineages are invasive trophoblast cells, which exit the placenta and invade the uterus, where they restructure the uterine parenchyma and facilitate remodeling of uterine spiral arteries. The rat exhibits deep intrauterine trophoblast cell invasion, a feature shared with human placentation, and is also amenable to gene manipulation using genome-editing techniques. In this investigation, we generated a conditional rat model targeting the invasive trophoblast cell lineage. Prolactin family 7, subfamily b, member 1 (Prl7b1) is uniquely and abundantly expressed in the rat invasive trophoblast cell lineage. Disruption of Prl7b1 did not adversely affect placental development. We demonstrated that the Prl7b1 locus could be effectively used to drive the expression of Cre recombinase in invasive trophoblast cells. Our rat model represents a new tool for investigating candidate genes contributing to the regulation of invasive trophoblast cells and their roles in trophoblast-guided uterine spiral artery remodeling.
Assuntos
Placenta , Placentação , Gravidez , Ratos , Feminino , Animais , Humanos , Placenta/metabolismo , Placentação/genética , Trofoblastos , Útero , Linhagem da Célula/genética , Modelos AnimaisRESUMO
The binding of 17ß-oestradiol to oestrogen receptor alpha (ERα) plays a crucial role in the control of reproduction, acting through both nuclear and membrane-initiated signalling. To study the physiological role of membrane ERα in the reproductive system, we used the C451A-ERα mouse model with selective loss of function of membrane ERα. Despite C451A-ERα mice being described as sterile, daily weighing and ultrasound imaging revealed that homozygous females do become pregnant, allowing the investigation of the role of ERα during pregnancy for the first time. All neonatal deaths of the mutant offspring mice resulted from delayed parturition associated with failure in pre-term progesterone withdrawal. Moreover, pregnant C451A-ERα females exhibited partial intrauterine embryo arrest at about E9.5. The observed embryonic lethality resulted from altered expansion of Tpbpa-positive spiral artery-associated trophoblast giant cells into the utero-placental unit, which is associated with an imbalance in expression of angiogenic factors. Together, these processes control the trophoblast-mediated spiral arterial remodelling. Hence, loss of membrane ERα within maternal tissues clearly alters the activity of invasive trophoblast cells during placentogenesis. This previously unreported function of membrane ERα could open new avenues towards a better understanding of human pregnancy-associated pathologies.
Assuntos
Receptor alfa de Estrogênio , Trofoblastos , Animais , Estradiol/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Fertilidade , Humanos , Camundongos , Placenta/metabolismo , Gravidez , Progesterona/metabolismo , Receptores de Estrogênio/metabolismo , Trofoblastos/metabolismoRESUMO
Preeclampsia (PE) is a life-threatening pregnancy-specific complication with controversial mechanisms and no effective treatment except delivery is available. Currently, increasing researchers suggested that PE shares pathophysiologic features with protein misfolding/aggregation disorders, such as Alzheimer disease (AD). Evidences have proposed defective autophagy as a potential source of protein aggregation in PE. Endoplasmic reticulum-selective autophagy (ER-phagy) plays a critical role in clearing misfolded proteins and maintaining ER homeostasis. However, its roles in the molecular pathology of PE remain unclear. We found that lncRNA DUXAP8 was upregulated in preeclamptic placentae and significantly correlated with clinical indicators. DUXAP8 specifically binds to PCBP2 and inhibits its ubiquitination-mediated degradation, and decreased levels of PCBP2 reversed the activation effect of DUXAP8 overexpression on AKT/mTOR signaling pathway. Function experiments showed that DUXAP8 overexpression inhibited trophoblastic proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Moreover, pathological accumulation of swollen and lytic ER (endoplasmic reticulum) was observed in DUXAP8-overexpressed HTR8/SVneo cells and PE placental villus trophoblast cells, which suggesting that ER clearance ability is impaired. Further studies found that DUXAP8 overexpression impaired ER-phagy and caused protein aggregation medicated by reduced FAM134B and LC3II expression (key proteins involved in ER-phagy) via activating AKT/mTOR signaling pathway. The increased level of FAM134B significantly reversed the inhibitory effect of DUXAP8 overexpression on the proliferation, migration, and invasion of trophoblasts. In vivo, DUXAP8 overexpression through tail vein injection of adenovirus induced PE-like phenotypes in pregnant rats accompanied with activated AKT/mTOR signaling, decreased expression of FAM134B and LC3-II proteins and increased protein aggregation in placental tissues. Our study reveals the important role of lncRNA DUXAP8 in regulating trophoblast biological behaviors through FAM134B-mediated ER-phagy, providing a new theoretical basis for understanding the pathogenesis of PE.
Assuntos
Autofagia , Retículo Endoplasmático , Pré-Eclâmpsia , Proteínas Proto-Oncogênicas c-akt , RNA Longo não Codificante , Transdução de Sinais , Serina-Treonina Quinases TOR , Trofoblastos , Adulto , Animais , Feminino , Humanos , Gravidez , Ratos , Autofagia/genética , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Retículo Endoplasmático/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Serina-Treonina Quinases TOR/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patologia , MasculinoRESUMO
In humans, the uterus undergoes a dramatic transformation to form an endometrial stroma-derived secretory tissue, termed decidua, during early pregnancy. The decidua secretes various factors that act in an autocrine/paracrine manner to promote stromal differentiation, facilitate maternal angiogenesis, and influence trophoblast differentiation and development, which are critical for the formation of a functional placenta. Here, we investigated the mechanisms by which decidual cells communicate with each other and with other cell types within the uterine milieu. We discovered that primary human endometrial stromal cells (HESCs) secrete extracellular vesicles (EVs) during decidualization and that this process is controlled by a conserved HIF2α-RAB27B pathway. Mass spectrometry revealed that the decidual EVs harbor a variety of protein cargo, including cell signaling molecules, growth modulators, metabolic regulators, and factors controlling endothelial cell expansion and remodeling. We tested the hypothesis that EVs secreted by the decidual cells mediate functional communications between various cell types within the uterus. We demonstrated that the internalization of EVs, specifically those carrying the glucose transporter 1 (GLUT1), promotes glucose uptake in recipient HESCs, supporting and advancing the decidualization program. Additionally, delivery of HESC-derived EVs into human endothelial cells stimulated their proliferation and led to enhanced vascular network formation. Strikingly, stromal EVs also promoted the differentiation of trophoblast stem cells into the extravillous trophoblast lineage. Collectively, these findings provide a deeper understanding of the pleiotropic roles played by EVs secreted by the decidual cells to ensure coordination of endometrial differentiation and angiogenesis with trophoblast function during the progressive phases of decidualization and placentation.
Assuntos
Decídua , Vesículas Extracelulares , Trofoblastos , Diferenciação Celular , Decídua/citologia , Decídua/fisiologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Vesículas Extracelulares/fisiologia , Feminino , Humanos , Neovascularização Fisiológica , Gravidez , Células Estromais/citologia , Células Estromais/fisiologia , Trofoblastos/citologia , Trofoblastos/fisiologiaRESUMO
The alterations in DNA methylation and transcriptome in trophoblast cells under conditions of low oxygen and oxidative stress have major implications for pregnancy-related disorders. However, the exact mechanism is still not fully understood. In this study, we established models of hypoxia (H group) and oxidative stress (HR group) using HTR-8/SVneo trophoblast cells and performed combined analysis of genome-wide DNA methylation changes using reduced representation bisulphite sequencing and transcriptome expression changes using RNA sequencing. Our findings revealed that the H group exhibited a higher number of differentially methylated genes and differentially expressed genes than the HR group. In the H group, only 0.90% of all differentially expressed genes displayed simultaneous changes in DNA methylation and transcriptome expression. After the threshold was expanded, this number increased to 6.29% in the HR group. Notably, both the H group and HR group exhibited concurrent alterations in DNA methylation and transcriptome expression within Axon guidance and MAPK signalling pathway. Among the top 25 differentially methylated KEGG pathways in the promoter region, 11 pathways were commonly enriched in H group and HR group, accounting for 44.00%. Among the top 25 KEGG pathways in transcriptome with significant differences between the H group and HR group, 10 pathways were consistent, accounting for 40.00%. By integrating our previous data on DNA methylation from preeclamptic placental tissues, we identified that the ANKRD37 and PFKFB3 genes may contribute to the pathogenesis of preeclampsia through DNA methylation-mediated transcriptome expression under hypoxic conditions.
Assuntos
Hipóxia Celular , Metilação de DNA , Estresse Oxidativo , Transcriptoma , Trofoblastos , Humanos , Trofoblastos/metabolismo , Estresse Oxidativo/genética , Transcriptoma/genética , Hipóxia Celular/genética , Linhagem Celular , Feminino , Gravidez , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismoRESUMO
Recurrent spontaneous abortion (RSA) has various causes, including chromosomal abnormalities, prethrombotic state, and abnormal uterine anatomical factors. However, the pathogenesis of RSA is still unclear. Surprisingly, non-coding RNA can stably express at the maternal-fetal interface and regulate immune cells' proliferation, apoptosis, invasion, metastasis, and angiogenesis. Accumulating evidence suggests that the competing endogenous RNA (ceRNA) regulatory network between non-coding RNAs complicates RSA's pathological process and maybe a new starting point for exploring RSA. In this review, we mainly discuss the regulatory network and potential significance of non-coding RNA in the immune microenvironment of RSA patients. In addition, the cellular interactions of non-coding RNA transported through vesicles were introduced from aspects of trophoblast function and immune regulation. Finally, we analyze previous studies and further discuss that the stable expression of non-coding RNA may be used as a biomarker of some disease states and a prediction target of RSA.
Assuntos
Aborto Habitual , Aborto Espontâneo , Gravidez , Feminino , Humanos , Útero/metabolismo , Trofoblastos/metabolismo , Aberrações Cromossômicas , RNA não Traduzido/genética , RNA não Traduzido/metabolismoRESUMO
Gestational diabetes mellitus (GDM) is a common disorder in the clinic, which may lead to severe detrimental outcomes both for mothers and infants. However, the underlying mechanisms for GDM are still not clear. In the present study, we performed label-free proteomics using placentas from GDM patients and normal controls. Vitronectin caused our attention among differentially expressed proteins due to its potential role in the pathological progression of GDM. Vitronectin was increased in the placentas of GDM patients, which was confirmed by Western blot analysis. Vitronectin represses insulin signal transduction in trophoblast cells, whereas the knockdown of vitronectin further potentiates insulin-evoked events. Neutralization of CD51/61 abolishes the repressed insulin signal transduction in vitronectin-treated trophoblast cells. Moreover, vitronectin activates JNK in a CD51/61-depedent manner. Inhibition of JNK rescues impaired insulin signal transduction induced by vitronectin. Overall, our data indicate that vitronectin binds CD51/61 in trophoblast cells to activate JNK, and thus induces insulin resistance. In this regard, increased expression of vitronectin is likely a risk factor for the pathological progression of GDM. Moreover, blockade of vitronectin production or its receptors (CD51/61) may have therapeutic potential for dealing with GDM.
RESUMO
Adequate proliferation and migration of placental trophoblasts is the prerequisite of a successful pregnancy. Peroxiredoxin2 (Prdx2) is a multi-functional gene involved in various signal events to maintain essential biological functions and normal cellular homeostasis. In this study, substantially lower Prdx2 levels were found in the first trimester cytotrophoblasts of women who suffered from recurrent miscarriage (RM). Prdx2 downregulation inhibited trophoblast proliferation and migration. We demonstrated that histone deacetylase2 (HDAC2) acts downstream of Prdx2 in regulating trophoblast proliferation and migration. HDAC2 deacetylates histone-3-lysine-9 in E-cadherin (E-cad) promoter and reduces the transcription of E-cad epigenetically, whereas it promotes the expression of Slug and Snail genes. These molecular changes may contribute to the trophoblast epithelial-mesenchymal transition. We further verified whether Prdx2 modulated the expression of HDAC2 through SPIB. SPIB could bind to the HDAC2 promoter PU-box region and induce HDAC2 expression. In RM, down-regulated Prdx2 suppresses SPIB-HDAC2 pathway, leading to increased E-cad and decreased Slug and Snail, and eventually restrains trophoblast proliferation and migration. Our study unveils the role of Prdx2-regulated SPIB-HDAC2 pathway in the pathology of RM and provides diagnostic and therapeutic targets for RM as well as other "great obstetrical syndromes" including preeclampsia and intrauterine growth restriction.
Assuntos
Aborto Habitual , Peroxirredoxinas , Trofoblastos , Feminino , Humanos , Gravidez , Aborto Habitual/genética , Movimento Celular/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Placenta/metabolismo , Fatores de Transcrição/metabolismo , Trofoblastos/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismoRESUMO
Arginine, which is metabolized into ornithine, proline, and nitric oxide, plays an important role in embryonic development. The present study was conducted to investigate the molecular mechanism of arginine in proliferation, differentiation, and physiological function of porcine trophoblast cells (pTr2) through metabolic pathways. The results showed that arginine significantly increased cell viability (P < 0.05). The addition of arginine had a quadratic tendency to increase the content of progesterone (P = 0.06) and protein synthesis rate (P = 0.03), in which the maximum protein synthesis rate was observed at 0.4 mM arginine. Arginine quadratically increased (P < 0.05) the intracellular contents of spermine, spermidine and putrescine, as well as linearly increased (P < 0.05) the intracellular content of NO in a dose-dependent manner. Arginine showed a quadratic tendency to increase the content of putrescine (P = 0.07) and a linear tendency to increase NO content (P = 0.09) in cell supernatant. Moreover, increasing arginine activated (P < 0.05) the mRNA expressions for ARG, ODC, iNOS and PCNA. Furthermore, inhibitors of arginine metabolism (L-NMMA and DFMO) both inhibited cell proliferation, while addition of its metabolites (NO and putrescine) promoted the cell proliferation and cell cycle, the mRNA expressions of PCNA, EGF and IGF-1, and increased (P < 0.05) cellular protein synthesis rate, as well as estradiol and hCG secretion (P < 0.05). In conclusion, our results suggested that arginine could promote cell proliferation and physiological function by regulating the metabolic pathway. Further studies showed that arginine and its metabolites modulate cell function mainly through ß-catenin and mTOR pathways.
Assuntos
Arginina , Diferenciação Celular , Proliferação de Células , Serina-Treonina Quinases TOR , Trofoblastos , beta Catenina , Animais , Arginina/farmacologia , Arginina/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Suínos , Proliferação de Células/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Diferenciação Celular/efeitos dos fármacos , beta Catenina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Óxido Nítrico/metabolismo , Linhagem CelularRESUMO
Copper pollution has attracted global environmental concern. Widespread Cu pollution results in excessive Cu accumulation in human. Epidemiological studies and animal experiments revealed that Cu exposure might have reproductive toxicity. Cuproptosis is a recently reported Cu-dependent and programmed cell death pattern. However, the mechanism by which copper exposure might cause cell cuproptosis is largely unknown. We chose trophoblast cells as cell model and found that copper exposure causes trophoblast cell cuproptosis. In mechanism, copper exposure up-regulates lnc-HZ11 expression levels, which increases intracellular Cu2+ levels and causes trophoblast cell cuproptosis. Knockdown of lnc-HZ11 efficiently reduces intracellular Cu2+ levels and alleviate trophoblast cell cuproptosis, which could be further alleviated by co-treatment with DC or TEPA. These results discover novel toxicological effects of copper exposure and also provide potential target for protection trophoblast cells from cuproptosis in the presence of excessive copper exposure.
Assuntos
Cobre , Trofoblastos , Regulação para Cima , Trofoblastos/efeitos dos fármacos , Cobre/toxicidade , Humanos , Regulação para Cima/efeitos dos fármacos , Linhagem Celular , Poluentes Ambientais/toxicidade , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: Endometrial receptivity (ER), a pivotal event for successful embryo implantation, refers to the capacity of endometrium to allow the adhesion of the trophectoderm of the blastocyst to endometrial cells. In this paper, we set to elucidate whether the peptides encoded by lncRNAs could influence trophoblast cells' adhesion to endometrial cells. METHODS: WGCNA construction and bioinformatics were used to find out the ER-related lncRNAs with coding potential. Protein analysis was done by immunoblotting and immunofluorescence (IF) microscopy. CCK-8 and Calcein-AM/PI double staining assays were employed to evaluate cell viability. The effect of the peptide on trophoblast spheroids' adhesion to endometrial cells was evaluated. The RNA sequencing (RNA-seq) analysis was applied to identify downstream molecular processes. RESULTS: lncRNA LINC00339 was found to be related to ER development and it had been predicted to have protein-coding potential. LINC00339 had high occupancy of ribosomes and was confirmed to encode a 49-aa peptide (named LINC00339-205-49aa). LINC00339-205-49aa could promote the attachment of JAR trophoblast spheroids to Ishikawa endometrial cells in vitro. LINC00339-205-49aa also upregulated the expression of E-cadherin in Ishikawa cells. Mechanistically, MAPK and PI3K-Akt signaling pathways were involved in the modulation of LINC00339-205-49aa, which were activated by LINC00339-205-49aa in Ishikawa cells. CONCLUSION: These data demonstrate that a previously uncharacterized peptide encoded by lncRNA LINC00339 has the ability to enhance JAR trophoblast spheroids' adhesion to Ishikawa endometrial cells, highlighting a new opportunity for the development of drugs to improve ER.
Assuntos
RNA Longo não Codificante , Feminino , Humanos , RNA Longo não Codificante/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Endométrio/metabolismo , Implantação do Embrião/genética , Trofoblastos/metabolismo , Linhagem Celular Tumoral , Transdução de Sinais/genética , Adesão Celular/genética , PeptídeosRESUMO
OBJECTIVE: This study aimed to study the correlation between preeclampsia (PE) and lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), and to examine the molecular mechanisms behind the development of PE. METHODS: 30 PE and 30 normal pregnant women placental samples were assessed the levels of NEAT1 and miR-217 by quantitative real-time PCR (qRT-PCR). The trophoblast cell line HTR8/SVneo was used for silencing NEAT1 or miR-217 inhibitor in the absence or presence of an inhibitor and H2O2. Cell counting Kit 8 (CCK-8), flow cytometry, and Transwell were used to detect cell proliferation, apoptosis, migration, and invasion. Luciferase reporter gene assay was utilized to verify the binding between miR-217 and Wnt family member 3 (Wnt3), and between the miR-217 and NEAT1. Proteins related to the Wnt/ß-catenin signaling pathway were detected using western blotting. RESULTS: The PE group exhibited a significantly downregulated expression of miR-217 and a significantly upregulated expression of NEAT1. NEAT1 targeted miR-217, and Wnt is a miR-217 target gene. siRNA-NEAT1 inhibited the apoptosis of trophoblast cells, but promoted their invasion, migration, and proliferation. MiR-217 inhibitor could partially reverse the effects of siRNA-NEAT1. The expression of the Wnt/ß-catenin signaling pathway-related proteins, WNT signaling pathway inhibitor 1 (DKK1), cyclin-D1 and ß-catenin, was significantly increased after siRNA-NEAT1. CONCLUSIONS: NEAT1 could reduce trophoblast cell invasion and migration by suppressing miR-217/Wnt signaling pathway, leading to PE.
RESUMO
Preeclampsia (PE) is a pregnancy-specific disorder associated with shallow invasion of the trophoblast cells and insufficient remodeling of the uterine spiral artery. Protein glycosylation plays an important role in trophoblast cell invasion. However, the glycobiological mechanism of PE has not been fully elucidated. In the current study, employing the Lectin array, we found that soybean agglutinin (SBA), which recognizes the terminal N-acetylgalactosamine α1,3-galactose (GalNAc α1,3 Gal) glycotype, was significantly increased in placental trophoblast cells from PE patients compared with third-trimester pregnant controls. Upregulating the expression of the key enzyme α1,3 N-acetylgalactosaminyl transferase (GTA) promoted the biosynthesis of terminal GalNAc α1,3 Gal and inhibited the migration/invasion of HTR8/SVneo trophoblast cells. Moreover, the methylation status of GTA promoter in placental tissues from PE patients was lower than that in the third trimester by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis. Elevated GTA expression in combination with the DNA methylation inhibitor 5-azacytidine (5-AzaC) treatment increased the glycotype biosynthesis and impaired the invasion potential of trophoblast cells, leading to preeclampsia. This study suggests that elevated terminal GalNAc α1,3 Gal biosynthesis and GTA expression may be applied as the new markers for evaluating placental function and the auxiliary diagnosis of preeclampsia.
Assuntos
Movimento Celular , N-Acetilgalactosaminiltransferases , Pré-Eclâmpsia , Trofoblastos , Humanos , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Trofoblastos/metabolismo , Trofoblastos/patologia , Feminino , Gravidez , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilgalactosaminiltransferases/genética , Adulto , Metilação de DNA , Regiões Promotoras Genéticas , Linhagem Celular , Placenta/metabolismoRESUMO
Dysregulation of noncoding RNAs has been reported to have a close correlation with preeclampsia(PE)development. TCL6 was upregulated in patients with PE. In this study, we examined the impacts of TCL6 on modulating HTR-8/SVneo cells induced by LPS. LPS (100 and 200 ng/ml) was applied to induce inflammation in trophoblast cells HTR-8/SVneo. Cell viability, apoptosis, and transwell experiments were conducted. The ELISA methods were used for pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α. MDA, GSH, and GPX kits were employed. Transfection was performed for expression regulation of TCL6, miR-485-5p, and TFRC in cells. Bioinformatic online tools were used to predict the targeting sites. Luciferase and RNA immunoprecipitation-qPCR were done to verify the interactions of TCL6, miR-485-5p, and TFRC. RNA expression levels were measured using RT-qPCR, and protein expression of TFRC and GPX4 was detected using a western blot. The free Fe (II) contents were measured. LPS decreased viability, invasion, and migration but enhanced apoptosis, ferroptosis, and inflammation. TCL6 expression was enhanced by LPS induction. The knockdown of TCL6 increased HTR-8/SVneo cell viability and invasion but inhibited cell apoptosis, inflammation, and ferroptosis while inhibition of miR-485-5p could reverse this through TFRC regulation. Moreover, miR-485-5p was sponged by TCL6 and bound to TFRC. TCL6 protected trophoblast cells from LPS-induced injury through the TFRC pathway.
Assuntos
Ferroptose , MicroRNAs , RNA Longo não Codificante , Feminino , Humanos , Gravidez , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo , Ferroptose/genética , Lipopolissacarídeos/toxicidade , MicroRNAs/genética , MicroRNAs/metabolismo , Trofoblastos/metabolismoRESUMO
BACKGROUND: Insulin resistance (IR) is an important determinant of glucose metabolic disturbance and placental dysplasia in gestational diabetes mellitus (GDM). Calcium/calmodulin dependent protein kinase IV (CAMK4) improves insulin IR induced by a high-fat diet (HFD). The current study sought to elucidate the role and potential mechanism of CAMK4 in GDM. METHODS: A GDM model was established in female C57BL/6J mice via HFD feeding for one week before mating and throughout gestation. The IR was elicited by 10-6 M insulin treatment for 48 h in HTR-8/SVneo cells and mouse primary trophoblast cells. The function of CAMK4 was investigated by transfection of overexpression plasmid in HTR-8/SVneo cells and infection of lentivirus loaded with CAMK4 encoding sequence in primary trophoblast cells. Real-time PCR, western blot, cell counting kit-8, transwell, wound healing, dual-luciferase reporter assay, and liquid chromatography/mass spectrometry-based untargeted metabolomics were performed to confirm the effects of CAMK4 on trophoblast cells. RESULTS: Decreased CAMK4 expression was found in the placenta of GDM mice. CAMK4 overexpression ameliorated IR-induced viability impairment, migratory and invasive capacity inhibition, autophagy blocking, insulin signaling inactivation and glucose uptake disorder in trophoblast cells. CAMK4 also transcriptionally activated orphan nuclear receptor NUR77, and the effects of CAMK4 were abrogated by silencing of NUR77. Metabolomics analysis revealed that CAMK4 overexpression caused alterations of amino acid, lipid and carbohydrate metabolism, which were important in GDM. CONCLUSION: Our results indicated that CAMK4/NUR77 axis may provide novel potential targets in GDM treatment.
Assuntos
Diabetes Gestacional , Resistência à Insulina , Insulinas , Animais , Feminino , Humanos , Camundongos , Gravidez , Cálcio , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Metabolômica , Camundongos Endogâmicos C57BL , Placenta , TrofoblastosRESUMO
OBJECTIVE: To determine the influence of ultrasound/microbubble-mediated miR-424-5p delivery on trophoblast cells and the underlying mechanism. METHODS: Blood pressure and 24-h proteinuria of patients with preeclampsia (PE) were measured as well as the levels of miR-424-5p and amine oxidase copper containing 1 (AOC1) in placental tissues. HTR-8/Svneo and TEV-1 cells were subjected to cell transfection or ultrasonic microbubble transfection for determination of the expression of miR-424-5p, AOC1, ß-catenin and c-Myc as well as cell proliferation, apoptosis, migration and invasiveness. The concentrations of placental growth factor (PLGF), human chorionic gonadotropin (ß-hCG) and tumor necrosis factor-α (TNF-α) were measured in HTR-8/Svneo and TEV-1 cells. RNA immunoprecipitation (RIP) and dual luciferase reporter assay detected the binding of miR-424-5p to AOC1. A PE mouse model was induced by subcutaneous injection of L-NAME, where the influence of ultrasound/microbubble-mediated miR-424-5p delivery was evaluated. RESULTS: miR-424-5p was downregulated while AOC1 was upregulated in the placental tissues from PE patients. Overexpression of miR-424-5p activated Wnt/ß-catenin signaling pathway and promoted the proliferation of HTR-8/Svneo and TEV-1 cells as well as enhanced the migratory and invasive behaviors. AOC1 overexpression partly eliminated the effects of miR-424-5p on HTR-8/Svneo and TEV-1 cells. Ultrasound and microbubble mediated gene delivery enhanced the transfection efficiency of miR-424-5p and further promoted the effects of miR-424-5p in trophoblast cells. Ultrasound/microbubble-mediated miR-424-5p delivery alleviated experimental PE in mice. CONCLUSION: Ultrasound and microbubble-mediated miR-424-5p delivery targets AOC1 and activates Wnt/ß-catenin signaling pathway, thus promoting the aggressive phenotype of trophoblast cells, which indicating that miR-424-5p/AOC1 axis might be involved with PE pathogenesis.
RESUMO
Trophoblast plays a crucial role in gestation maintenance and embryo implantation, partly due to the synthesis of progesterone. It has been demonstrated that hypoxia regulates invasion, proliferation, and differentiation of trophoblast cells. Additionally, human trophoblasts display rhythmic expression of circadian clock genes. However, it remains unclear if the circadian clock system is present in goat trophoblast cells (GTCs), and its involvement in hypoxia regulation of steroid hormone synthesis remains elusive. In this study, immunofluorescence staining revealed that both BMAL1 and NR1D1 (two circadian clock components) were highly expressed in GTCs. Quantitative real-time PCR analysis showed that several circadian clock genes were rhythmically expressed in forskolin-synchronized GTCs. To mimic hypoxia, GTCs were treated with hypoxia-inducing reagents (CoCl2 or DMOG). Quantitative real-time PCR results demonstrated that hypoxia perturbed the mRNA expression of circadian clock genes and StAR. Notably, the increased expression of NR1D1 and the reduction of StAR expression in hypoxic GTCs were also detected by western blotting. In addition, progesterone secretion exhibited a notable decline in hypoxic GTCs. SR9009, an NR1D1 agonist, significantly decreased StAR expression at both the mRNA and protein levels and markedly inhibited progesterone secretion in GTCs. Moreover, SR8278, an NR1D1 antagonist, partially reversed the inhibitory effect of CoCl2 on mRNA and protein expression levels of StAR and progesterone synthesis in GTCs. Our results demonstrate that hypoxia reduces StAR expression via the activation of NR1D1 signaling in GTCs, thus inhibiting progesterone synthesis. These findings provide new insights into the NR1D1 regulation of progesterone synthesis in GTCs under hypoxic conditions.
Assuntos
Progesterona , Trofoblastos , Animais , Humanos , Trofoblastos/metabolismo , Cabras/genética , Hipóxia , RNA Mensageiro , Cobalto , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismoRESUMO
Recurrent pregnancy loss (RPL) is a common pathological problem during pregnancy, and its clinical etiology is complex and unclear. Dysfunction of trophoblasts may cause a series of pregnancy complications, including preeclampsia, fetal growth restriction, and RPL. Recently, lncRNAs have been found to be closely related to the occurrence and regulation of pregnancy-related diseases, but few studies have focused on their role in RPL. In this study, we identified a novel lncRNA BBOX1-AS1 that was significantly upregulated in villous tissues and serum of RPL patients. Functionally, BBOX1-AS1 inhibited proliferation, migration, invasion, tube formation and promoted apoptosis of trophoblast cells. Mechanistically, overexpression of BBOX1-AS1 activated the p38 and JNK MAPK signaling pathways by upregulating GADD45A expression. Further studies indicated that BBOX1-AS1 could increase the stability of GADD45A mRNA by binding hnRNPK and ultimately cause abnormal trophoblast function. Collectively, our study highlights that the BBOX1-AS1/hnRNPK/GADD45A axis plays an important role in trophoblast-induced RPL and that BBOX1-AS1 may serve as a potential target for the diagnosis of RPL.
Assuntos
MicroRNAs , Pré-Eclâmpsia , RNA Longo não Codificante , Feminino , Gravidez , Humanos , Trofoblastos/metabolismo , Proliferação de Células/genética , Sistema de Sinalização das MAP Quinases , Pré-Eclâmpsia/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Movimento Celular/genética , MicroRNAs/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Preeclampsia (PE) refers to a pregnancy-specific disease that begins with the placenta. Differentially expressed microRNAs (miRs) are a feature of PE. This study tried to elicit the functional mechanism of miR-515-5p in trophoblast cell behaviors in PE. First, HTR-8/SVneo cells were transfected with miR-515-5p mimic or miR-515-5p inhibitor. Then, relative expression levels of miR-515-5p and histone deacetylase 2 (HDAC2) in HTR-8/SVneo cells were determined by reverse transcription-quantitative polymerase chain reaction. The potential binding site of miR-515-5p and HDAC2 was predicted on Targetscan and their binding relationship was verified via dual-luciferase assay. Proliferation, apoptosis, invasion, and migration of HTR-8/SVneo cells were assessed via cell counting kit-8, flow cytometry, Transwell, and wound healing assays, respectively. Protein levels of Cleaved caspase-3, Bcl-2, and Bax were determined via Western blot. Overexpressed miR-515-5p impeded proliferation and stimulated apoptosis of HTR-8/SVneo cells, and decreased levels of Cleaved caspase-3 and Bax and elevated Bcl-2, whilst opposite results were observed after miR-515-5p inhibition. miR-515-5p targeted HDAC2. Knockdown of HDAC2 annulled the promotional action of miR-515-5p inhibition on proliferative, invasive, and migratory abilities and its antiapoptotic action on HTR-8/SVneo cells. In brief, miR-515-5p affected the proliferation, apoptosis, invasion, and migration of HTR-8/SVneo cells by targeting HDAC2.
Assuntos
MicroRNAs , Pré-Eclâmpsia , Gravidez , Feminino , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Trofoblastos/metabolismo , Caspase 3/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Linhagem Celular , Movimento Celular/genética , Apoptose/genética , Proliferação de Células/genéticaRESUMO
An increase of reactive oxygen species in the placenta and oxidative disbalance has been recognized as a significant factor contributing to pregnancy complications. Dietary intake of food rich in antioxidants during pregnancy could exert a protective role in the prevention of adverse outcomes such as preeclampsia, miscarriage, and others. Flavonoid taxifolin has shown numerous health-promoting effects in a large number of studies conducted on animals, as well as various human cell types in vitro. However, its effects on human placental cells-trophoblasts-have yet to be determined. Therefore, cytoprotective and genoprotective effects of taxifolin on trophoblast cell line HTR-8/SVneo under induced oxidative stress were explored in this study. Cytotoxicity of a range of taxifolin concentrations (1-150 µM) was evaluated using the MTT and crystal violet assays. A model of oxidative stress was achieved by exposing HTR-8/SVneo cells to H2O2. To determine cytoprotective and antigenotoxic effects, the cells were pre-incubated with three concentrations of taxifolin (10, 50, and 100 µM) and then exposed to H2O2. Taxifolin in concentrations of 1, 5, 10, 25, 50, and 100 µM showed no cytotoxic effects on HTR-8/SVneo cells, but 150 µM of taxifolin caused a significant decrease in adherent cell number, as detected by crystal violet assay. Pretreatment with the chosen concentrations of taxifolin showed a significant cytoprotective effect on H2O2-induced cytotoxicity, as determined by the MTT assay. Furthermore, taxifolin showed a significant reduction in H2O2-induced DNA damage, measured by comet assay. This study showed protective effects of taxifolin on human trophoblast cells exposed to oxidative damage. Further studies are needed to explore the underlying mechanisms.