Dysfunction Of Trophoblast Cells Research Articles

BMP5 promotes trophoblast functions upon N-glycosylation via the BMP5-SMAD1/5 signaling pathway in preeclampsia

IntroductionPreeclampsia (PE) is one of the most common pregnancy-related complications worldwide and currently lacks an effective treatment. While trophoblast cell dysfunction has been identified as the fundamental cause of PE, the underlying mechanisms remain unclear. Bone morphogenetic protein 5 (BMP5) is a secreted glycoprotein highly expressed in the placenta that is involved in cell proliferation, migration, and invasion. However, the role and mechanism of BMP5 glycosylation of trophoblast cell function remain unclear. MethodsThe expression of BMP5 and N-glycosylation in preeclamptic placental tissues was investigated. We predicted and validated the N-glycosylation sites of BMP5. Additionally, we evaluated the effect of BMP5 N-glycosylation on the proliferation, migration, invasion, and angiogenesis of human immortalized trophoblastic HTR-8/SVneo cells. Furthermore, the role of N-glycosylated BMP5 in activating the BMP5-SMAD1/5 signaling pathway and regulating trophoblastic cell functions was explored. ResultsOur study reveals that PHA-E+L (recognizing branching N-glycans) reactive N-glycans and BMP5 expression levels are lower in preeclamptic villous tissues compared to normal placental tissues. Additionally, we demonstrated that BMP5 is an N-glycosylation-modified protein. Furthermore, N-glycosylated BMP5 promoted the functional trophoblastic cells (HTR-8/SVneo). We also revealed that N-glycosylation of BMP5 regulates multiple cell functions through the BMP5-SMAD1/5 signaling pathway. ConclusionN-glycosylated BMP5 promotes trophoblast cell proliferation, migration, invasion, and angiogenesis. This study provides mechanistic insight as to how N-glycosylation of BMP5 in trophoblast cells can contribute to the pathogenesis of preeclampsia and provides a new basis for its diagnosis and treatment.

Cleistocalyx nervosum var. paniala mitigates oxidative stress and inflammation induced by PM10 soluble extract in trophoblast cells via miR-146a-5p

Air pollution poses a significant global concern, notably impacting pregnancy outcomes through mechanisms such as DNA damage, oxidative stress, inflammation, and altered miRNA expression, all of which can adversely affect trophoblast functions. Cleistocalyx nervosum var. paniala, known for its abundance of anthocyanins with diverse biological activities including anti-mutagenic, antioxidant, and anti-inflammatory properties, is the focus of this study examining its effect on Particulate Matter 10 (PM10) soluble extract-induced trophoblast cell dysfunction via miRNA expression. The study involved the extraction of C. nervosum fruit using 70% ethanol, followed by fractionation with hexane, dichloromethane, and ethyl acetate. Subsequent testing for total phenolics, flavonoids, anthocyanins, and antioxidant activity revealed the ethyl acetate fraction (CN-EtOAcF) as possessing the highest phenolic and anthocyanin content along with potent antioxidant activity, prompting its selection for further investigation. In vitro studies on HTR-8/SVneo cells demonstrated that 5–10 µg/mL PM10 soluble extract exposure inhibited cell proliferation, migration, invasion, and induced apoptosis. However, pretreatment with 20–80 µg/mL CN-EtOAcF followed by 5 µg/mL PM10 soluble extract exposure exhibited protective effects against PM10 soluble extract-induced damage, including inflammation inhibition and intracellular ROS suppression. Notably, CN-EtOAcF down-regulated PM10-induced miR-146a-5p expression, with SOX5 identified as a potential target. Overall, CN-EtOAcF demonstrated the potential to protect against PM10-induced harm in trophoblast cells, suggesting its possible application in future therapeutic approaches.

DOCK1 deficiency drives placental trophoblast cell dysfunction by influencing inflammation and oxidative stress, hallmarks of preeclampsia.

Preeclampsia (PE) is a globally prevalent obstetric disorder, pathologically characterized by abnormal placental development. Dysfunctions of angiogenesis, vasculogenesis and spiral artery remodeling are demonstrated to be involved in PE pathogenesis; however, the underlying mechanisms remain largely unknown. Here, we investigated the role of the dedicator of cytokinesis 1 (DOCK1), crucial molecule in various cellular processes, in PE progression using HTR-8 cells derived from first-trimester placental extravillous trophoblasts. Our analysis revealed an aberrant DOCK1 expression in the placental villi of PE patients and its impact on essential cellular functions for vascular network formation. A deficiency of DOCK1 in HTR-8 cells impaired the vascular network formation, exacerbated the expression of anti-angiogenic factor ENG, and reduced VEGF levels. Moreover, DOCK1 knockout amplified apoptosis, as indicated by an altered BCL2: BAX ratio and enhanced levels of cleaved PARP. DOCK1 depletion also boosted NF-κB activation and pro-inflammatory cytokine production (IL-6 and TNF-α). Furthermore, the mice treated with DOCK1 inhibitor, TBOPP, exhibited PE-like symptoms. These findings highlight the multifaceted roles of DOCK1 in the pathophysiology of PE, demonstrating that its deficiency can lead to placental dysfunction by orchestrating inflammatory responses and oxidative stress. These insights emphasize the pathogenic role of DOCK1 in PE development and suggest potential treatment strategies that require further exploration. In the graphical abstract, a split image of placental villi contrasts the effects of normal and reduced DOCK1 expression on preeclampsia. The left side illustrates adequate DOCK1 levels supporting healthy trophoblast function and effective spiral artery remodeling. The right side highlights the consequences of DOCK1 deficiency, leading to trophoblast dysfunction and impaired spiral artery remodeling, accompanied by angiogenic imbalance, increased inflammation, oxidative stress, and apoptosis, contributing to placental dysfunction and the development of preeclampsia.

Placental growth factor alleviates hyperglycemia-induced trophoblast pyroptosis by regulating mitophagy.

Hyperglycemia is closely related to trophoblast dysfunction during pregnancy and results in suppressed invasion, migration, and pro-inflammatory cell death of trophoblasts. Hyperglycemia is a dependent risk factor for gestational hypertension accompanied by decreased placental growth factor (PLGF), which is important for maternal and fetal development. However, there is currently a lack of evidence to support whether PLGF can alleviate trophoblast cell dysfunction caused by high blood sugar. Here, we aim to clarify the effect of hyperglycemia on trophoblast dysfunction and determine how PLGF affects this process. The changes in placental tissue histomorphology from gestational diabetes mellitus (GDM) patients were compared with those of normal placentas. HTR8/SVneo cells were cultured in different amounts of glucose to examine cellular pyroptosis, migration, and invasion as well as PLGF levels. Furthermore, the levels of pyroptosis-related proteins (NLRP3, pro-caspase1, caspase1, IL-1β, and Gasdermin D [GSDMD]) as well as autophagy-related proteins (LC3-II, Beclin1, and p62) were examined by Western blotting. The GFP-mRFP-LC3-II system and transmission electron microscopy were used to detect mitophagy levels, and small interfering RNAs targeting BCL2 Interacting Protein 3 (siBNIP3) and PTEN-induced kinase 1 (siPINK1) were used to determine the role of mitophagy in pyroptotic death of HTR-8/SVneo cells. Our results show that hyperglycemia upregulates NLRP3, pro-caspase1, caspase1, IL-1β at the protein level in GDM patients. High glucose (HG, 25 mM) inhibits viability, invasion, and migration of trophoblast cells while suppressing superoxide dismutase levels and promoting malondialdehyde production, thus leading to a senescence associated beta-gal-positive cell burst. PLGF levels in nucleus and the cytosol are also inhibited by HG, whereas PLGF treatment inhibited pyroptosis-related protein levels of NLRP3, pro-caspase1, caspase1, IL-1β, and GSDMD, Gasdermin D N-terminal domain (GSDMD-N). HG-induced mitochondrial dysfunction and BNIP3 and PINK1/Parkin expression. Knocking down BINP3 and PINK1 abolished the protective role of PLGF by preventing mitophagy. PLGF inhibited hyperglycemia, while PLGF reversed hyperglycemic injury by promoting mitophagy via the BNIP3/PINK1/Parkin pathway. Altogether, these results suggest that PLGF may protect against trophoblast dysfunction in diabetes.

Maternal exposure to beta-Cypermethrin disrupts placental development by dysfunction of trophoblast cells from oxidative stress

As a broad-spectrum and efficient insecticide, beta-Cypermethrin (β-CYP) poses a health risk to pregnancy. It matters the mechanisms of maternal exposure to β-CYP for impacting reproductive health. The placenta, a transient organ pivotal for maternal-fetal communication during pregnancy, plays a crucial role in embryonic development. The effect of β-CYP exposure on the placenta and its underlying molecular mechanisms remain obscure. The objective of this study was to investigate the effect of β-CYP exposure on placental development and the function of trophoblast, as well as the underlying mechanisms through CD-1 mouse model (1, 10, 20 mg/kg.bw) and in vitro HTR-8/SVneo cell model (12.5, 25, 50, 100 μM). We found slower weight gain and reduced uterine wet weight in pregnant mice with maternal exposure to β-CYP during pregnancy, as well as adverse pregnancy outcomes such as uterine bleeding and embryo resorption. The abnormal placental development in response to β-CYP was noticed, including imbalanced placental structure and disrupted labyrinthine vascular development. Trophoblasts, pivotal in placental development and vascular remodeling, displayed abnormal differentiation under β-CYP exposure. This aberration was characterized by thickened trophoblast layers in the labyrinthine zone, accompanied by mitochondrial and endoplasmic reticulum swelling within trophoblasts. Further researches on human chorionic trophoblast cell lines revealed that β-CYP exposure induced apoptosis in HTR-8/SVneo cells. This induction resulted in a notable decrease in migration and invasion abilities, coupled with oxidative stress and the inhibition of the Notch signaling pathway. N-acetylcysteine (an antioxidant) partially restored the impaired Notch signaling pathway in HTR-8/SVneo cells, and mitigated cellular functional damage attributed to β-CYP exposure. Collectively, exposure to β-CYP induced oxidative stress and then led to inhibition of the Notch signaling pathway and dysfunction of trophoblast cells, ultimately resulted in abnormal placenta and pregnancy. These findings indicate Reactive Oxygen Species as potential intervention targets to mitigate β-CYP toxicity. The comprehensive elucidation contributes to our understanding of β-CYP biosafety and offers an experimental basis for preventing and managing its reproductive toxicity.

HSPB8 binding to c-Myc alleviates hypoxia/reoxygenation-induced trophoblast cell dysfunction.

Preeclampsia (PE) is a pregnancy-specific syndrome with complex pathogenesis. The present study aimed to explore the role of heat shock protein B8 (HSPB8) and c-Myc in trophoblast cell dysfunction using a hypoxia/reoxygenation (H/R)-treated HTR8/SVneo cell model. HSPB8 expression in tissues of patients with PE was analyzed using the Gene Expression Omnibus database. Following detection of HSPB8 expression in H/R-stimulated HTR8/SVneo cells, HSPB8 was overexpressed by transfection of the gene with a HSPB8-specific plasmid. Cell Counting Kit-8, wound healing and Transwell assays were used to evaluate the proliferation, migration and invasion of HTR8/SVneo cells exposed to H/R conditions. Reactive oxygen species (ROS) were determined by 2,7-dichlorodihydrofluorescein diacetate staining. 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbo-cyanine iodide (JC-1) staining was applied to assess mitochondrial membrane potential. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were detected using the corresponding commercial kits. In addition, the induction of apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Moreover, the Biogrid database predicted that HSPB8 was bound to c-Myc, and a co-immunoprecipitation (Co-IP) assay was used to verify this interaction. Subsequently, c-Myc expression was silenced to conduct rescue experiments in HTR8/SVneo cells exposed to H/R conditions and upregulated HSPB8 expression. Notably, reduced HSPB8 expression was noted in PE tissues and H/R-stimulated HTR8/SVneo cells. HSPB8 enforced expression promoted the proliferation, migration and invasion of HTR8/SVneo cells. Moreover, H/R caused an increase in ROS and MDA levels as well as in TUNEL staining and a decrease in aggregated JC-1 fluorescence and SOD activity levels, which were restored following HSPB8 overexpression. Co-IP confirmed the interaction between HSPB8 and c-Myc. Moreover, knockdown of c-Myc expression compromised the effects of HSPB8 upregulation on trophoblast cell dysfunction following induction of H/R. Collectively, the data indicated that HSPB8 could improve mitochondrial oxidative stress by binding to c-Myc to alleviate trophoblast cell dysfunction. The findings may provide new insights into the pathogenesis of PE and highlight the role of HSPB8/c-Myc in the prevention and treatment of PE in the future.

ADAM9 deubiquitination induced by USP22 suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition of trophoblast cells in preeclampsia

IntroductionThe dysregulation of deubiquitination has been shown to affect the development of pre-eclampsia (PE). A disintegrin and metalloprotease 9 (ADAM9) plays roles in diverse physiological contexts, including PE. Here, this study aimed to investigate whether ADAM9 regulated trophoblast cell dysfunction through ubiquitin-specific protease 22 (USP22) deubiquitinase-mediated deubiquitination during PE. MethodsLevels of genes and proteins were tested via qRT-PCR and western blotting assays. Cell proliferation, migration, and invasion were detected using cell counting kit-8, 5-ethynyl-2′-deoxyuridine (EdU), flow cytometry, transwell and wound healing assays, respectively. Epithelial-mesenchymal transition related markers were assayed using western blotting. Proteins between USP22 and ADAM9 were identified by co-immunoprecipitation assay. ResultsADAM9 was highly expressed in PE patients, functionally, ADAM9 overexpression weakened the proliferation, migration, invasion, and EMT progression in trophoblast cells. Mechanistically, the deubiquitinase USP22 removed ubiquitination on ADAM9 and maintained its stability. Forced expression of USP22 also suppressed the proliferation and mobility in trophoblast cells. Moreover, the regulatory effects of USP22 on trophoblast cells were reversed by ADAM9 silencing. In addition, USP22 interacted with ADAM9 to regulate the activation of Wnt/β-catenin pathway. DiscussionADAM9 was deubiquitinated and stabilized by USP22 and then suppressed the proliferation, migration, invasion, and EMT progression in trophoblast cells, indicating a new pathway of USP10/RUNX1 axis in PE process.

FtMt reduces oxidative stress-induced trophoblast cell dysfunction via the HIF-1α/VEGF signaling pathway

BackgroundPreeclampsia (PE) is a complication of pregnancy that causes long-term adverse outcomes for the mother and fetus and may even lead to death. Oxidative stress caused by the imbalance of oxidants and antioxidants in the placenta has been considered as one of the key mechanisms of preeclampsia (together with inflammation, etc.), in which the placental mitochondria play an important role. The expression of hypoxia-inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) is known to be increased in patients with PE. Mitochondrial ferritin (FtMt) is known to protect the mitochondria from oxidative stress, although its specific role in PE remains unclear.MethodsWe used qRT-PCR and western blotting to detect the expression levels of FtMt, HIF-1α, and VEGF in placental tissues from patients with PE. Human chorionic trophoblast cells were also administered with hypoxia treatment, followed by the detection of cell proliferation, invasion and angiogenic capacity by CCK8, Transwell, and endothelial cell angiogenesis assays; we also detected the expression of HIF-1α and VEGF in these cells. Finally, overexpression or inhibitory FtMt lentiviral vectors, along with negative control vectors, were constructed and transfected into hypoxia-treated human chorionic trophoblast cells; this was followed by analyses of cell function.ResultsThe expression levels of FtMt, HIF-1α and VEGF in the PE group were higher than those in the control group (P < 0.05). Following hypoxia, there was an increase in the expression levels of HIF-1α and VEGF protein in trophoblast cells. There was also an increase in invasion ability and vascular formation ability along with a reduction in cell proliferation ability. These effects were reversed by transfecting cells with the knockout FtMt lentivirus vector. The differences were statistically significant.ConclusionAnalyses showed that FtMt plays a key role in the vascular regulation of PE trophoblast cells after hypoxia possibly acting via the HIF-1α/VEGF signaling pathway. These results provide us an enhanced understanding of the pathogenesis of PE and suggest that the HIF-1α/VEGF signaling pathway represents a new target for the treatment of PE.

Exposure to higher concentrations of exogenous ELABELA causes HTR-8/SVneo trophoblast cell dysfunction: A possible pathogenesis of pre-eclampsia.

Pre-eclampsia (PE) is a multi-system disorder of pregnancy which constitute one of the leading causes of maternal and perinatal mortality worldwide. Dysfunction of trophoblast cells have been play an a significant role in the development and progression of PE. ELABELA (ELA), an endogenous apelin receptor (APJ) ligand, is a hormone secreted by the placenta into circulation, we previously identified ELA and its cognate apelin receptor (APJ) as proteins that are differentially expressed in the placentas of humans with PE. However, a fact known to few about the potential regulatory effects of ELA on the functions of trophoblast cells. In this study, our experiments showed that exogenous ELA did not effectively affect the proliferation and apoptosis of HTR-8/SVneo cells in higher concentrations but inhibited the invasion and migration capabilities. Studies have shown that the epithelial-mesenchymal transition (EMT) event of trophoblast cells is abnormal through regulate the P13K/AKT pathway in PE. First we confirmed APJ expression in HTR-8/SVneo cells and ELA suppressed P13K/AKT signalling pathway-related factors (AKT, phospho (p)-AKT) phosphorylation in HTR-8/SVneo cells in a concentration-dependent manner. Next, we found that treatment with higher concentrations of ELA or the P13K inhibitor LY294002 significantly increased the expression of β-catenin which is the classical epithelial marker relative to the control group. Moreover, treatment with both LY294002 and ELA more strongly affected the β-catenin expression and the HTR8/SVneo cells invasion and migration. These results demonstrate that ELA inhibits trophoblast cell functions by altering the β-catenin expression get though the PI3K/AKT signalling pathway in PE. Thus, this result might provide a new therapeutic target for the treatment of PE.

Transcriptional Factor Forkhead Box D1 Upregulates Sirtuin3 by Activating the Wnt/β-Catenin Pathway to Alleviate HTR-8/Svneo Trophoblast Cell Dysfunction in Preeclampsia

The proliferation, invasion, migration and apoptosis of trophoblast cells in preeclampsia are closely related to the occurrence and development of preeclampsia. Transcription factors forkhead box D1 and Sirtuin3 are abnormally expressed in preeclampsia, and Sirtuin3 plays a regulatory role in cell proliferation, invasion and apoptosis in related diseases. However, the studies on forkhead box D1 and Sirtuin3 in preeclampsia and their specific mechanisms have not been reported so far. In this study, the expression of Sirtuin3 in Human chorionic trophoblast cells HTR-8/Svneo was inhibited by cell transfection, and then the effects of Sirtuin3 expression in interfering cells on cell invasion, migration and apoptosis were detected by MTT, TUNEL, Western blot, wound healing and Transwell techniques. Subsequently, the binding between forkhead box D1 and Sirtuin3 was predicted by JASPAR website and verified by luciferase assay and ChIP assay. Finally, cell invasion, migration and apoptosis were detected after overexpression of forkhead box D1 and interference with Sirtuin3, and the Wnt/β-catenin signaling pathway was detected to explore the mechanism. We found that interfering with Sirtuin3 induced apoptosis of HTR-8/Svneo cells and inhibited cell invasion and migration. Forkhead box D1 transcriptional activation of Sirtuin3 alleviated HTR-8/SVneo cell dysfunction through activation of the Wnt/β-catenin signaling pathway. Overall, transcriptional factor forkhead box D1 can upregulate Sirtuin3 by activating the Wnt/β-catenin pathway to alleviate HTR-8/Svneo trophoblast cell dysfunction in preeclampsia.

NINJ1 triggers extravillous trophoblast cell dysfunction through blocking the STAT3 signaling pathway.

Trophoblasts are the most important parts of the placenta in early pregnancy. Trophoblast cell dysfunction can induce embryo implantation insufficiency, thereby resulting in multiple diseases, including recurrent spontaneous abortion (RSA). A previous study indicates higher nerve injury-induced protein 1 (NINJ1) RNA levels in the villi tissues of RSA patients. This study aimed to investigate the effect of NINJ1 on trophoblast behaviors and pregnancy loss. Fresh villi tissues were obtained from with RSA patients and patients with artificial selective abortion for personal reasons, and NINJ1 expression in these tissues was detected. Extravillous trophoblast cell line HTR-8/SVneo was transfected with small-interfering RNA targeting NINJ1 or NINJ1 overexpression vector to perform loss-/gain-of-function experiments. Spontaneous abortion (SA) was induced by mating CBA/J females with DBA/2 males and the pregnant females were intraperitoneally injected with adenovirus vector carrying NINJ1 short hairpin RNA. NINJ1 mRNA and protein levels were higher in the villi tissues of RSA patients than those of artificial selective abortion patients. NINJ1 knockdown promoted trophoblast cell proliferation, migration and invasion but inhibited cell apoptosis. Moreover, conditioned medium from NINJ1-depleted trophoblasts promoted the angiogenesis of human umbilical vein endothelial cells. NINJ1 knockdown also promoted activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway in trophoblasts, and STAT3 inhibitor reversed NINJ1 knockdown-induced effects on trophoblast behaviors. Furthermore, pregnancy loss was attenuated by NINJ1 inhibition. NINJ1 contributes to the development of SA and triggers trophoblast cell dysfunction through inhibiting the STAT3 pathway.

IL10+/+ AND IL10-/- TROPHOBLAST CELLS PROLIFERATION, MIGRATION AND INVASION DURING HYPERGLYCEMIA AND EXPRESSION OF CENTRAL MOLECULES

Successful embryo implantation requires a functional communication between a blastocyst and a receptive endometrium during a brief period known as the window of implantation. Under certain inflammatory conditions, such as diabetes, the window of implantation can be affected preventing normal implantation which could lead to pregnancy loss. The aim of our current study was to evaluate the role of IL-10, TNFα, LIF, MMP-9, and NO in implantation and to establish their possible impact on the functional ability of trophoblast cells under hyperglycemic conditions. For this reason, the experiments were conducted on trophoblast cells isolated from placentas of IL-10+/+ (C57BL/6J) and IL-10 -/-(B6.129P2-Il10tm1Cgn/J) pregnant mice. The cells were stimulated by IL-10, TNF-alpha and LIF, and the effect of glucose concentration on their functional activity and expression ability of several markers were determined. The results of our investigation showed that during hyperglycemia anti-inflammatory cytokine IL-10 occurs as an inhibitor of TNFα production and decreases oxidative stress. IL-10 regulates LIF and MMP expressions and evolves in the control of invasion and proliferation processes of trophoblast cells. In conclusion, IL-10 and TNF-alpha play a central role in the processes associated with implantation during hyperglycemia. IL-10 is a suppressor of hyperglycemia-induced trophoblast cell dysfunction and LIF, MMP-9, and NO may be critical players in it.

Circ_0007611 stimulates IL-1 receptor accessory protein to inhibit trophoblast cell proliferation and induce cell apoptosis.

Preeclampsia (PE) is a common pregnancy disorder, and mounting evidence has revealed that circular RNA participates in PE development. However, the detailed molecular mechanism of circ_0007611 in PE progression remains unknown. RNA expressions of circ_0007611, microRNA-558 (miR-558), and IL-1 receptor accessory protein (IL1RAP) were detected by quantitative real-time polymerase chain reaction. Cell proliferation was investigated by clonogenicity, 5-Ethynyl-29-deoxyuridine, and DNA content quantitation assays. Cell apoptotic rate and angiogenesis were analyzed by cell apoptosis and tube formation assays, respectively. Protein expression was detected by western blot. The binding relationship between miR-558 and circ_0007611 or IL1RAP was identified by a dual-luciferase reporter or RNA immunoprecipitation assay. Circ_0007611 and IL1RAP expressions were significantly upregulated, while miR-558 was downregulated in the placental tissues of PE women in comparison with normal placental tissues. Functionally, circ_0007611 overexpression inhibited trophoblast cell proliferation and angiogenesis and induced cell apoptosis; however, circ_0007611 downregulation showed the opposite effects. Mechanistically, circ_0007611 acted as a miR-558 sponge, and miR-558 bound to IL1RAP. Besides, miR-558 overexpression or IL1RAP absence relieved circ_0007611-induced trophoblast cell dysfunction. Moreover, miR-558 contributed to cell proliferation and tube formation and inhibited cell apoptosis by reducing IL1RAP expression in trophoblast cells. Circ_0007611 aggravated trophoblast cell disorders by the miR-558/IL1RAP pathway in PE.

The Novel Peptide AEDPPE Alleviates Trophoblast Cell Dysfunction Associated With Preeclampsia by Regulating the NF-κB Signaling Pathway.

Background: Preeclampsia (PE) is a serious risk to the health of pregnant women and fetuses during pregnancy, and there is no effective treatment for this condition. Although many reports have confirmed the therapeutic effects of peptides in diseases, the role of peptides in PE remains poorly understood.Methods: A differentially expressed peptide in PE (AEDPPE) is derived from heat-shock protein beta-1 (HSPB1), amino acids 100 to 109 (DVNHFAPDEL), which we identified in a previous study. We synthesized AEDPPE and investigated its effect on HTR-8/SVneo cell function using a Cell Counting Kit-8, flow cytometric assay, and Transwell and wound-healing assays. Quantitative reverse transcription-PCR and ELISA were used to determine cytokine expression. Pull-down assay, mass spectrometry, Western blot analysis, and immunofluorescence were used to explore the potential targets and signaling pathways regulated by AEDPPE. Finally, we assessed the effect of AEDPPE in the lipopolysaccharide (LPS)-induced PE-like rat model.Results: AEDPPE significantly promoted the migration and invasion of HTR-8/SVneo cells, and it decreased the expression of interleukins 1 beta (IL-1β), interleukin 6 (IL-6), and interleukin 8 (IL-8). These functions performed by AEDPPE remained evident after injury to HTR-8/SVneo cells with tumor necrosis factor-alpha (TNF-α), and AEDPPE reversed the elevated sFlt-1/PlGF ratio induced by TNF-α. AEDPPE may exert these biological effects by binding to heat-shock protein 90β (HSP 90β) and, thus, affect the NF-κB signaling pathway. In an LPS-induced PE-like rat model, AEDPPE significantly improved PE symptoms and fetal rat outcomes.Conclusion: Our study showed that AEDPPE enhanced trophoblast migration and invasion and reduced inflammatory cytokine expression, and we hypothesized that these actions involved the NF-κB signaling pathway. The use of AEDPPE may thus develop into a novel modality in the treatment of PE.

Circ-PNPT1 contributes to gestational diabetes mellitus (GDM) by regulating the function of trophoblast cells through miR-889-3p/PAK1 axis

BackgroundGestational diabetes mellitus (GDM) is the most common medical complication of pregnancy. CircRNA polyribonucleotide nucleotidyltransferase 1 (circ-PNPT1) has been found to be abnormally expressed in GDM patients. However, function and mechanism of circ-PNPT1 in GDM remain largely undefined.MethodsLevels of circ-PNPT1, microRNA (miR)-889-3p and PAK1 (p21 (RAC1) activated kinase 1) were detected using quantitative real-time polymerase chain reaction and Western blot assays. Cell viability, apoptosis, migration and invasion were determined using cell counting kit-8 assay, flow cytometry, transwell and wound healing assays, respectively. The binding interaction between miR-889-3p and circ-PNPT1 or PAK1 was verified using dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays. Exosomes were obtained from culture media by the use of commercial kits and qualified by transmission electron microscopy (TEM).ResultsCirc-PNPT1 was highly expressed in the placental tissues of GDM and high glucose (HG)-induced trophoblast cells. Knockdown of circ-PNPT1 reversed HG-induced arrest of trophoblast cell viability, migration, invasion and the promotion of cell apoptosis. Mechanistically, we confirmed circ-PNPT1 could promote the expression of PAK1, the target of miR-889-3p, by directly sponging miR-889-3p, and circ-PNPT1 regulated HG-induced trophoblast cell dysfunction by miR-889-3p/PAK1 axis. Further studies showed circ-PNPT1 was packaged into exosomes and could be internalized by surrounding trophoblast cells.ConclusionCirc-PNPT1 promoted HG-induced trophoblast cell biological dysfunction through miR-889-3p/PAK1 axis. Meanwhile, it could be transferred from HG-induced trophoblast cells to surrounding untreated cells via exosomes.

Vitamin D alleviates hypoxia/reoxygenation-induced injury of human trophoblast HTR-8 cells by activating autophagy

IntroductionAttenuation of trophoblast cell dysfunction would be beneficial for retarding pre-eclampsia (PE). Vitamin D has been reported to improve trophoblast cell function in early PE, but the mechanism involved is not fully elucidated. This study is aimed to investigate whether vitamin D alleviates trophoblast cell dysfunction via regulating autophagy. MethodsHuman trophoblast HTR-8 cells were cultured in hypoxia/reoxygenation (H/R) condition to simulate the oxidative stress state of early PE in vitro. MTT, Transwell and tube formation assays were respectively applied to assess cell proliferation, invasion, and angiogenesis abilities. DCFH-DA staining was performed to detect cellular reactive oxygen species levels. GFP-RFP-LC3 plasmid transfection and transmission electron microscopy were subjected to monitor autophagy. Enzyme-linked immunosorbent assay and Western blot analysis were used to detect autophagy-related and pyroptosis-associated molecules. ResultsH/R led to severe impairments on the bio-function of HTR-8 cells, as evidenced by the deficiency of cell proliferation, invasion, and angiogenesis abilities, and the increase of cellular ROS production. Simultaneously, H/R inhibited autophagy and triggered pyroptosis. 1,25(OH)2D3, the hormonally active form of vitamin D, dramatically attenuated H/R-induced trophoblast dysfunction. Also, 1,25(OH)2D3 activated autophagy and inhibited pyroptosis. Additionally, autophagy-enhancer rapamycin exerted similar protective effect to that of 1,25(OH)2D3, whereas autophagy-inhibitor 3-methyladenine blocked the protective effect of 1,25(OH)2D3. DiscussionThe mechanism that vitamin D alleviates trophoblast cell dysfunction is associated with autophagy induction and pyroptosis inhibition.

Overexpressed Hsa_circ_0001326 Contributes to the Decreased Cell Viability in SWAN71 Cells by Regulating MiR-186-5p/p27 Kip1 Axis.

Preeclampsia (PE) is a severe pregnancy-specific complication responsible for a majority of maternal and fetal mortality. The dysfunction of trophoblast cells is known to be associated with the etiology of PE. Moreover, elevated expression of hsa_circ_0001326 was found in patients with PE without elucidating specific mechanisms. Thus, this study aimed to investigate the role of hsa_circ_0001326 in the dysfunction of trophoblast cells in vitro. Human trophoblast SWAN71 cells were used in this study. Cell proliferation, apoptosis and cell cycle were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining, cell counting kit-8 assay, Annexin V/propidium iodide (PI) staining and flow cytometry, respectively. Dual luciferase assay was performed to validate the predicted targets. Additionally, Western blot was conducted for protein detection. The results indicated overexpression (OE) of hsa_circ_0001326 remarkably decreased the viability and proliferation of SWAN71 cells. MiR-186-5p was identified as the target of hsa_circ_0001326. Meanwhile, p27 Kip1 was validated as the target of hsa_miR-186-5p. Moreover, the increased apoptosis and decreased migration induced by hsa_circ_0001326 OE were inhibited by p27 Kip1 knockdown. Hsa_circ_0001326 OE upregulated p27 Kip1 and cleaved caspase3 and downregulated CDK2 and cyclin E1 in cells, while these phenomena were reversed by p27 Kip1 knockdown. In addition, hsa_circ_0001326 OE induced G0/G1 cell cycle arrest was also attenuated in the presence of p27 Kip1 knockdown. Taken together, hsa_circ_0001326 OE contributed to the decreased viability of SWAN71 cells by targeting miR-186-5p via upregulation of p27 Kip1. Our findings were helpful to uncover the pathophysiological process of PE, as well as inspire the development of novel targeted therapy against PE.

Circular RNA PUM1 (CircPUM1) attenuates trophoblast cell dysfunction and inflammation in recurrent spontaneous abortion via the MicroRNA-30a-5p (miR-30a-5p)/JUNB axis

ABSTRACT Recurrent spontaneous abortion (RSA) is a threat to human reproductive health worldwide. CircPUM1 has been reported to participate in the pathogenesis of various diseases. However, there has been no report on its association with RSA yet. In this study, gene expressions were examined by RT-qPCR. Protein levels of JUNB and cleaved caspases-3 were detected by Western blotting. ELISA was used to detect TNF-α, IL-6, and IL-8 levels. Cell viability, migration, invasion, and apoapsis were analyzed using CCK-8, transwell, and flow cytometry assays. The association between miR-30a-5p and circPUM1 or JUNB was identified by bioinformatics analysis, dual-luciferase reporter assay, and RIP assay. Herein, we found circPUM1 was significantly downregulated in RSA placental samples. CircPUM1 knockdown induced decreased proliferation, migration, and invasion, but increased apoptosis, pro-apoptotic protein (cleaved caspases-3) level, and proinflammatory factor (TNF-α, IL-6, and IL-8) secretion in trophoblast cells. Furthermore, we confirmed that circPUM1 was a sponge for miR-30a-5p, and JUNB was directly targeted by miR-30a-5p. It was demonstrated that miR-30a-5p inhibition could reverse trophoblast cell dysfunction and inflammation induced by circPUM1 knockdown. In addition, we found that JUNB expression was negatively modulated by miR-30a-5p and positively regulated by circPUM1. Moreover, circPUM1 inhibition exacerbated dysfunction and inflammation in trophoblast cells via targeting JUNB. To sum up, our study indicated that circPUM1 could impair RSA occurrence and development by facilitating trophoblast cellular processes and protecting against inflammation via the miR-30a-5p/JUNB axis, providing a new target for the improvement of RSA diagnosis and treatment.

MiR-18a Contributes to Preeclampsia by Downregulating Smad2 (Full Length) and Reducing TGF-β Signaling

The study investigated the regulation of Smad2 by miR-18a and its role in preeclampsia (PE). Bioinformatics analysis showed that both Smad2 and Smad3 were the predicted targets for miR-18a. Mass spectrum analysis showed that two mature Smad2 isoforms existed in human placenta: full length, Smad2(FL), and that lacking exon3, Smad2(Δexon3). The protein level of Smad2(FL), but not Smad2(Δexon3) or Smad3, was significantly increased in severe PE (sPE) placenta, which was inversely correlated with the level of miR-18a. Elevated Smad2(FL) phosphorylation level appeared in sPE placenta, and Smad2 was colocalized with miR-18a in various subtypes of trophoblasts in human placenta. Smad2(FL) was validated as the direct target of miR-18a in HTR8/SVneo cells. miR-18a enhanced trophoblast cell invasion, which was blocked by the overexpression of Smad2(FL). Furthermore, overexpression of miR-18a repressed Smad2 activation and the inhibition of trophoblast cell invasion by transforming growth factor-β (TGF-β). In conclusion, our results suggest that miR-18a inhibits the expression of Smad2(FL), but not Smad2(Δexon3) or Smad3, which can reduce TGF-β signaling, leading to the enhancement of trophoblast cell invasion. A lack of miR-18a, which results in the upregulation of Smad2(FL), contributes to the development of PE.

Overexpression of tissue factor pathway inhibitor 2 attenuates trophoblast proliferation and invasion in preeclampsia.

Pre-eclampsia (PE) is a disorder of pregnancy characterized by proteinuria and high blood pressure, affecting 2-8% of pregnancies worldwide. Previous studies have shown that PE is closely associated with trophoblast cell dysfunction. Here, we investigated the role of tissue factor pathway inhibitor-2 (TFPI-2) in regulating the biological processes of trophoblast cells. The TFPI-2 levels in plasma samples and placental tissues were tested by ELISA, immunohistochemistry, qRT-PCR, and western blot. HTR8/Svneo cell line was used to simulate the primary trophoblast cells and H/R culture was applied to mimic the oxidative stress state of PE. MTT assay, Annexin V/propidium iodide (PI) apoptosis assay, and transwell assay were used to determine the cell proliferation, apoptosis, and invasion. The expression levels of matrix metalloproteinases (MMPs) were evaluated by western blot. The expression of TFPI-2 was remarkably up-regulated in both the serum and placenta of PE patients. Hypoxia/reoxygenation increased the expression of TFPI-2 in HTR-8/SVneo cell line. TFPI-2 promoted that cell proliferation and inhibited the cell apoptosis of HTR8/SVneo cells in H/R condition. In addition, downregulation of TFPI-2 increased the cell invasion and the expression of MMP2 and MMP9. This study reveals that TFPI-2 plays a crucial role in monitoring the biological function of trophoblast cells, which might provide theoretical basis and therapeutic targets for the treatment of PE.