Evaluating the role of DNA methyltransferases in trophoblast fusion and preeclampsia development: Insights from methylation-regulated genes.

The etiology of preeclampsia (PE), a complex and multifactorial condition, remains incompletely understood. DNA methylation, which is primarily regulated by three DNA methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B, plays a vital role in early embryonic development and trophectoderm differentiation. Yet, how DNMTs modulate trophoblast fusion and PE development remains unclear. In this study, we found that the DNMTs expression was downregulated during trophoblast cells fusion. Downregulation of DNMTs was observed during the reconstruction of the denuded syncytiotrophoblast (STB) layer of placental explants. Additionally, overexpression of DNMTs inhibited trophoblast fusion. Conversely, treatment with the DNA methylation inhibitor 5-aza-CdR decreased the expression of DNMTs and promoted trophoblast fusion. A combined analysis of DNA methylation data and gene transcriptome data obtained from the primary cytotrophoblasts (CTBs) fusion process identified 104 potential methylation-regulated differentially expressed genes (MeDEGs) with upregulated expression due to DNA demethylation, including CD59, TNFAIP3, SDC1, and CDK6. The transcription regulation region (TRR) of TNFAIP3 showed a hypomethylation with induction of 5-aza-CdR, which facilitated CREB recruitment and thereby participated in regulating trophoblast fusion. More importantly, clinical correlation analysis of PE showed that the abnormal increase in DNMTs may be involved in the development of PE. This study identified placental DNA methylation-regulated genes that may contribute to PE, offering a novel perspective on the role of epigenetics in trophoblast fusion and its implication in PE development.

HMGB1 regulates autophagy of placental trophoblast through ERK signaling pathway†.

OBJECTIVE: The purpose of this study is to investigate the role of high mobility group protein B1 (HMGB1) in placental development and fetal growth. METHODS: We employed the Cre-loxP recombination system to establish a placenta-specific HMGB1 knockout mouse model. Breeding HMGB1flox/flox mice with Elf5-Cre mice facilitated the knockout, leveraging Elf5 expression in extra-embryonic ectoderm, ectoplacental cone, and trophoblast giant cells at 12.5 days of embryonic development. The primary goal of this model was to elucidate the molecular mechanism of HMGB1 in placental development, assessing parameters such as placental weight, fetal weight, and bone development. Additionally, we utilized lentiviral interference and overexpression of HMGB1 in human trophoblast cells to further investigate HMGB1's functional role. RESULTS: Our findings indicate that the HMGB1flox/floxElf5cre/+ mouse displays fetal growth restriction, characterized by decreased placental and fetal weight and impaired bone development. The absence of HMGB1 inhibits autophagosome formation, impairs lysosomal degradation, and disrupts autophagic flux. Depletion of HMGB1 in human trophoblast cells also suppresses cell viability, proliferation, migration, and invasion by inhibiting the ERK signaling pathway. Overexpression of HMGB1 observed the opposite phenotypes. CONCLUSIONS: HMGB1 participates in the regulation of autophagy through the ERK signaling pathway and affects placental development.

Palmitic acid impairs human and mouse placental function by inhibiting trophoblast autophagy through induction of acyl-coenzyme A-binding protein (ACBP) upregulation.

STUDY QUESTION: Can exposure to palmitic acid (PA), a common saturated fatty acid, modulate autophagy in both human and mouse trophoblast cells through the regulation of acyl-coenzyme A-binding protein (ACBP)? SUMMARY ANSWER: PA exposure before and during pregnancy impairs placental development through mechanisms involving placental autophagy and ACBP expression. WHAT IS KNOWN ALREADY: High-fat diets, including PA, have been implicated in adverse effects on human placental and fetal development. Despite this recognition, the precise molecular mechanisms underlying these effects are not fully understood. STUDY DESIGN, SIZE, DURATION: Extravillous trophoblast (EVT) cell line HTR-8/SVneo and human trophoblast stem cell (hTSC)-derived EVT (hTSCs-EVT) were exposed to PA or vehicle control for 24 h. Female wild-type C57BL/6 mice were divided into PA and control groups (n = 10 per group) and subjected to a 12-week dietary intervention. Afterward, they were mated with male wild-type C57BL/6 mice and euthanized on Day 14 of gestation. Female ACBPflox/flox mice were also randomly assigned to control and PA-exposed groups (each with 10 mice), undergoing the same dietary intervention and mating with ACBPflox/floxELF5-Cre male mice, followed by euthanasia on Day 14 of gestation. The study assessed the effects of PA on mouse embryonic development and placental autophagy. Additionally, the role of ACBP in the pathogenesis of PA-induced placental toxicity was investigated. PARTICIPANTS/MATERIALS, SETTING, METHODS: The findings were validated using real-time PCR, Western blot, immunofluorescence, transmission electron microscopy, and shRNA knockdown approaches. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to PA-upregulated ACBP expression in both human HTR-8/SVneo cells and hTSCs-EVT, as well as in mouse placenta. PA exposure also induced autophagic dysfunction in HTR-8/SVneo cells, hTSCs-EVT, and mouse placenta. Through studies on ACBP placental conditional knockout mice and ACBP knockdown human trophoblast cells, it was revealed that reduced ACBP expression led to trophoblast malfunction and affected the expression of autophagy-related proteins LC3B-II and P62, thereby impacting embryonic development. Conversely, ACBP knockdown partially mitigated PA-induced impairment of placental trophoblast autophagy, observed both in vitro in human trophoblast cells and in vivo in mice. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Primary EVT cells from early pregnancy are fragile, limiting research use. Maintaining their viability is tough, affecting data reliability. The study lacks depth to explore PA diet cessation effects after 12 weeks. Without follow-up, understanding postdiet impacts on pregnancy stages is incomplete. Placental abnormalities linked to elevated PA diet in embryos lack confirmation due to absence of control groups. Clarifying if issues stem solely from PA exposure is difficult without proper controls. WIDER IMPLICATIONS OF THE FINDINGS: Consuming a high-fat diet before and during pregnancy may result in complications or challenges in successfully carrying the pregnancy to term. It suggests that such dietary habits can have detrimental effects on the health of both the mother and the developing fetus. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the National Natural Science Foundation of China (82171664, 82301909) and the Natural Science Foundation of Chongqing Municipality of China (CSTB2022NS·CQ-LZX0062, cstc2019jcyj-msxmX0749, and cstc2021jcyj-msxmX0236). The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

miR-526b-5p/c-Myc/Foxp1 participates in recurrent spontaneous abortion by regulating the proliferation, migration, and invasion of trophoblasts.

PURPOSE: As a member of the C19MC family, miR-526b-5p is mainly expressed in the placental tissue and is a well-known tumor suppressor microRNA. However, its effect on the function of trophoblasts and its role in the development of recurrent spontaneous abortion (RSA) remains unclear. METHODS: Transcriptome sequencing, quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, 5-ethynyl-2'-deoxyuridine (Edu) proliferation analysis, cell counting kit-8 (CCK8) assay, Transwell assays, and wound healing were used to detect the proliferation, migration, and invasion capacity of trophoblasts. Target genes of miR-526b-5p were obtained by the dual luciferase reporter system. The promoter-reporter system and ChIP-qPCR were used to prove that c-Myc positively regulated the expression of Foxp1 RESULTS: The miR-526b-5p levels were significantly higher in patients with RSA than in controls. High expression of miR-526b-5p inhibited the proliferation, migration, and invasion of trophoblast cell line. By contrast, low expression of miR-526b-5p promoted the proliferation and migration of trophoblast cell line. Target genes of miR-526b-5p were c-Myc and Foxp1. c-Myc positively regulated the expression of Foxp1 by binding to the Foxp1 promoter location -146/-135. Finally, miR-526b-5p impeded the proliferation, migration, and invasion of trophoblasts by negatively regulating c-Myc by rescue experiments. CONCLUSION: Thus, miR-526b-5p affected the proliferation, migration, and invasion of trophoblasts by targeting c-Myc and Foxp1. Low expression of c-Myc further deactivated the positive transcriptional regulation of c-Myc on Foxp1, which may be the mechanism of RSA. This study provides potential therapeutic targets and clues for the diagnosis and treatment of RSA.

The roles of ADAMDEC1 in trophoblast differentiation during normal pregnancy and preeclampsia.

Human cytotrophoblast (CTB) differentiation into syncytiotrophoblast (STB) is essential for placental formation and function. Understanding the molecular mechanisms involved in trophoblast differentiation is necessary as it would help in the development of novel therapeutic agents to treat placentation-mediated pregnancy complications. In this study, we found a common upregulated gene, ADAM-like Decysin-1 (ADAMDEC1), from five published microarray and RNA-sequencing datasets. Interference to ADAMDEC1 impaired forskolin-induced BeWo cells differentiation, while ADAMDEC1 overexpression promoted BeWo cells and 3D JEG-3 spheroids differentiation. Interestingly, ADAMDEC1 may inhibit Thrombospondin 1 rather than E-cadherin to trigger the activation of the cAMP signal pathway during CTB differentiation into STB. More importantly, a decreasing in ADAMDEC1 might be involved in the development of preeclampsia. Therefore, ADAMDEC1 is expected to become a new target for prediction of and intervention in placenta-derived pregnancy diseases.

[HADHA Inhibits the Migration and Invasion of HTR-8/SVneo Cells by Regulating PI3K/AKT Signaling Pathway].

Objective: To explore the effects of hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) on the migration and invasion of HTR-8/SVneo cells, a human trophoblast cell line, and its potential mechanism of action. Methods: Immunofluorescence staining was done to evaluate the expression levels of HADHA in samples of normal villi and recurrent spontaneous abortion (RSA) villi at 6-8 weeks. Lentiviral infection system was used to construct stable HTR-8/SVneo cell lines with HADHA overexpression and knockdown. Western blot, qRT-PCR, Wound-healing assay, and Transwell assay were used to determine the effect of HADHA on the migration and invasion of HTR-8/SVneo cells and the expression of relevant genes. Transcriptome sequencing and bioinformatics analysis were done to screen for the potential target genes and signaling pathways regulated by HADHA. The specific molecular mechanism of how HADHA regulates the migration and invasion of HTR-8/SVneo cells was examined by adding the inhibitor of protein kinase B (PKB/AKT). Results: HADHA was highly expressed in extravillous trophoblasts (EVT) of RSA villus samples as compared with samples from the normal control group. In HTR-8/SVneo cells overexpressing HADHA, the expression levels of migration and invasion-related genes, including HLA-G, MMP2, MMP9, and NCAD, were decreased (P<0.01,P<0.05), and the migration and invasion abilities of HTR-8/SVneo cells were weakened (P<0.05). HADHA knockdown increased the expression levels of HLA-G, MMP2, MMP9, and NCAD (P<0.01, P<0.05), and promoted the migration and invasion of HTR-8/SVneo cells (P<0.05). In addition, HADHA overexpression decreased the phosphorylation levels of PI3K and AKT (P<0.05) and inhibited the PI3K/AKT signaling pathway. HADHA knockdown activated the PI3K/AKT signaling pathway. When MK-2206, an AKT inhibitor, was added to stable HTR-8/SVneo cell lines with HADHA knockdown, the migration and invasion of the cells were significantly reduced. Conclusion: HADHA inhibits the migration and invasion of HTR-8/SVneo cells by inhibiting the PI3K/AKT signaling pathway.

Molecular network of miR-1343 regulates the pluripotency of porcine pluripotent stem cells via repressing OTX2 expression.

Porcine OTX2 was found to be highly activated in porcine iPS cells (piPSCs) that were reported by different laboratories worldwide. To reveal the regulatory function of OTX2 in porcine reprogrammed cells, we screened porcine miRNA-seq databases and found two miRNAs, miR-1343 and miR-545, that could specifically bind to 3'UTR of OTX2 and suppress endogenous OTX2 expression in piPSCs. Knockdown of OTX2 by miR-1343 and miR-545 could significantly increase the expression of SOX2 and ESRRB, but did not alter the expressions of OCT4 and KLF4, and improve the pluripotency of piPSCs. The promoter-based assays showed that OTX2 potentially bound to the promoter region of SOX2 and ESRRB and suppressed their expression. On the other hand, SOX2 could interact with OTX2 promoter. Ectopic expression of SOX2 could significantly decrease OTX2 promoter activity, showing that there is a negative feedback loop between SOX2 and OTX2. Additionally, SOX2 and ESRRB significantly stimulated miR-1343 expression in piPSCs, but OTX2 down regulated the expression of miR-1343 in either direct or indirect manners. In summary, this study demonstrates that there is a regulatory network mediated by miR-1343, in which downregulation of OTX2 by miR-1343 can elevate the expression of pluripotent genes that were then sustain the pluripotency of piPSCs.

Suppression of GATA3 promotes epithelial-mesenchymal transition and simultaneous cellular senescence in human extravillous trophoblasts.

The regulatory mechanism of the transcription factor GATA3 in the differentiation and maturation process of extravillous trophoblasts (EVT) in early pregnancy placenta, as well as its relevance to the occurrence of pregnancy disorders, remains poorly understood. This study leveraged single-cell RNA sequencing data from placental organoid models and placental tissue to explore the dynamic changes in GATA3 expression during EVT maturation. The expression pattern exhibited an initial upregulation followed by subsequent downregulation, with aberrant GATA3 localization observed in cases of recurrent miscarriage (RM). By identifying global targets regulated by GATA3 in primary placental EVT cells, JEG3, and HTR8/SVneo cell lines, this study offered insights into its regulatory mechanisms across different EVT cell models. Shared regulatory targets among these cell types and activation of trophoblast cell marker genes emphasized the importance of GATA3 in EVT differentiation and maturation. Knockdown of GATA3 in JEG3 cells led to repression of GATA3-induced epithelial-mesenchymal transition (EMT), as evidenced by changes in marker gene expression levels and enhanced migration ability. Additionally, interference with GATA3 accelerated cellular senescence, as indicated by reduced proliferation rates and increased activity levels for senescence-associated ß-galactosidase enzyme, along with elevated expression levels for senescence-associated genes. This study provides comprehensive insights into the dual role of GATA3 in regulating EMT and cellular senescence during EVT differentiation, shedding light on the dynamic changes in GATA3 expression in normal and pathological placental conditions.

TBX3 reciprocally controls key trophoblast lineage decisions in villi during human placenta development in the first trimester.

Human trophoblastic lineage development is intertwined with placental development and pregnancy outcomes, but the regulatory mechanisms underpinning this process remain inadequately understood. In this study, based on single-nuclei RNA sequencing (snRNA-seq) analysis of the human early maternal-fetal interface, we compared the gene expression pattern of trophoblast at different developmental stages. Our findings reveal a predominant upregulation of TBX3 during the transition from villous cytotrophoblast (VCT) to syncytiotrophoblast (SCT), but downregulation of TBX3 as VCT progresses into extravillous trophoblast cells (EVT). Immunofluorescence analysis verified the primary expression of TBX3 in SCT, partial expression in MKi67-positive VCT, and absence in HLA-G-positive EVT, consistent with our snRNA-seq results. Using immortalized trophoblastic cell lines (BeWo and HTR8/SVneo) and human primary trophoblast stem cells (hTSCs), we observed that TBX3 knockdown impedes SCT formation through RAS-MAPK signaling, while TBX3 overexpression disrupts the cytoskeleton structure of EVT and hinders EVT differentiation by suppressing FAK signaling. In conclusion, our study suggests that the spatiotemporal expression of TBX3 plays a critical role in regulating trophoblastic lineage development via distinct signaling pathways. This underscores TBX3 as a key determinant during hemochorial placental development.

CDEMI: Characterizing differences in microbial composition and function in microbiome data.

Microbial communities influence host phenotypes through microbiota-derived metabolites and interactions between exogenous active substances (EASs) and the microbiota. Owing to the high dynamics of microbial community composition and difficulty in microbial functional analysis, the identification of mechanistic links between individual microbes and host phenotypes is complex. Thus, it is important to characterize variations in microbial composition across various conditions (for example, topographical locations, times, physiological and pathological conditions, and populations of different ethnicities) in microbiome studies. However, no web server is currently available to facilitate such characterization. Moreover, accurately annotating the functions of microbes and investigating the possible factors that shape microbial function are critical for discovering links between microbes and host phenotypes. Herein, an online tool, CDEMI, is introduced to discover microbial composition variations across different conditions, and five types of microbe libraries are provided to comprehensively characterize the functionality of microbes from different perspectives. These collective microbe libraries include (1) microbial functional pathways, (2) disease associations with microbes, (3) EASs associations with microbes, (4) bioactive microbial metabolites, and (5) human body habitats. In summary, CDEMI is unique in that it can reveal microbial patterns in distributions/compositions across different conditions and facilitate biological interpretations based on diverse microbe libraries. CDEMI is accessible at http://rdblab.cn/cdemi/.

Poly(N-Isopropylacrylamide) Based Electrically Conductive Hydrogels and Their Applications.

Poly(N-isopropylacrylamide) (PNIPAM) based electrically conductive hydrogels (PNIPAM-ECHs) have been extensively studied in recent decades due to their thermal-responsive (leading to the volume change of hydrogels) and electrically conductive performance. The incorporation of conductive components into the PNIPAM hydrogel network makes it become conductive hydrogel, and as a result, the PNIPAM hydrogel could become sensitive to an electrical signal, greatly expanding its application. In addition, conductive components usually bring new stimuli-responsive properties of PNIPAM-based hydrogels, such as near-infrared light and stress/strain responsive properties. PNIPAM-ECHs display a wide range of applications in human motion detection, actuators, controlled drug release, wound dressings, etc. To summarize recent research advances and achievements related to PNIPAM-ECHs, this manuscript first reviews the design and structure of representative PNIPAM-ECHs according to their conductive components. Then, the applications of PNIPAM-ECHs have been classified and discussed. Finally, the remaining problems related to PNIPAM-ECHs have been summarized and a future research direction is proposed which is to fabricate PNIPAM-ECHs with integrated multifunctionality.

Suppressing self-discharge of Li-B/CoS2 thermal batteries by using a carbon-coated CoS2 cathode.

Thermal batteries with molten salt electrolytes are used for many military applications, primarily as power sources for guided missiles. The Li-B/CoS2 couple is designed for high-power, high-voltage thermal batteries. However, their capacity and safe properties are influenced by acute self-discharge that results from the dissolved lithium anode in molten salt electrolytes. To solve those problems, in this paper, carbon coated CoS2 was prepared by pyrolysis reaction of sucrose at 400 °C. The carbon coating as a physical barrier can protect CoS2 particles from damage by dissolved lithium and reduce the self-discharge reaction. Therefore, both the discharge efficiency and safety of Li-B/CoS2 thermal batteries are increased remarkably. Discharge results show that the specific capacity of the first discharge plateau of carbon-coated CoS2 is 243 mA h g-1 which is 50 mA h g-1 higher than that of pristine CoS2 at a current density of 100 mA cm-2. The specific capacity of the first discharge plateau at 500 mA cm-2 for carbon-coated CoS2 and pristine CoS2 are 283 mA h g-1 and 258 mA h g-1 respectively. The characterizations by XRD and DSC indicate that the carbonization process has no noticeable influence on the intrinsic crystal structure and thermal stability of pristine CoS2.

The oncogene Etv5 promotes MET in somatic reprogramming and orchestrates epiblast/primitive endoderm specification during mESCs differentiation.

Unipotent spermatogonial stem cells (SSCs) can be efficiently reprogrammed into pluripotent stem cells only by manipulating the culture condition, without introducing exogenous reprogramming factors. This phenotype raises the hypothesis that the endogenous transcription factors (TFs) in SSCs may facilitate reprogramming to acquire pluripotency. In this study, we screened a pool of SSCs TFs (Bcl6b, Lhx1, Foxo1, Plzf, Id4, Taf4b, and Etv5), and found that oncogene Etv5 could dramatically increase the efficiency of induced pluripotent stem cells (iPSCs) generation when combined with Yamanaka factors. We also demonstrated that Etv5 could promote mesenchymal-epithelial transition (MET) at the early stage of reprogramming by regulating Tet2-miR200s-Zeb1 axis. In addition, Etv5 knockdown in mouse embryonic stem cells (mESCs) could decrease the genomic 5hmC level by downregulating Tet2. Furthermore, the embryoid body assay revealed that Etv5 could positively regulate primitive endoderm specification through regulating Gata6 and negatively regulate epiblast specification by inhibiting Fgf5 expression. In summary, our findings provide insights into understanding the regulation mechanisms of Etv5 under the context of somatic reprogramming, mESCs maintenance, and differentiation.

Common microRNA-mRNA interactions exist among distinct porcine iPSC lines independent of their metastable pluripotent states.

Previous evidences have proved that porcine-induced pluripotent stem cells (piPSCs) could be induced to distinctive metastable pluripotent states. This raises the issue of whether there is a common transcriptomic profile existing among the piPSC lines at distinctive state. In this study, we performed conjoint analysis of small RNA-seq and mRNA-seq for three piPSC lines which represent LIF dependence, FGF2 dependence and LFB2i dependence, respectively. Interestingly, we found there are 16 common microRNAs which potentially target 13 common mRNAs among the three piPSC lines. Dual-luciferase reporter assay validated that miR-370, one of the 16 common microRNAs, could directly target the 3'UTR of LIN28A. When the differentiation occurred, miR-370 could be activated in piPSCs and switched off the expression of LIN28A. Ectopic expression of miR-370 in piPSCs could reduce LIN28A expression, decrease the alkaline phosphatase activity, slow down the proliferation, and further cause the downregulation of downstream pluripotent genes (OCT4, SOX2, NANOG, SALL4 and ESRRB) and upregulation of differentiation relevant genes (SOX9, JARID2 and JMJD4). Moreover, these phenotypes caused by miR-370 could be rescued by overexpressing LIN28A. Collectively, our findings suggest that a set of common miRNA-mRNA interactions exist among the distinct piPSC lines, which orchestrate the self-renewal and differentiation of piPSCs independent of their metastable pluripotent states.

OTX2 impedes self-renewal of porcine iPS cells through downregulation of NANOG expression.

The transcription factor Otx2 acts as a negative switch in the regulation of transition from naive to primed pluripotency in mouse pluripotent stem cells. However, the molecular features and function of porcine OTX2 have not been well elucidated in porcine-induced pluripotent stem cells (piPSCs). By studying high-throughput transcriptome sequencing and interfering endogenous OTX2 expression, we demonstrate that OTX2 is able to downgrade the self-renewal of piPSCs. OTX2 is highly expressed in porcine brain, reproductive tissues, and preimplantation embryos, but is undetectable in fibroblasts and most somatic tissues. However, the known piPSC lines reported previously produced different levels of OTX2 depending on the induction procedures and culture conditions. Overexpression of porcine OTX2 can reduce the percentage of alkaline phosphatase-positive colonies and downregulate NANOG and OCT4 expression. In contrast, knockdown of OTX2 can significantly increase endogenous expressions of NANOG, OCT4, and ESRRB, and stabilize the pluripotent state of piPSCs. On the other hand, NANOG can directly bind to the OTX2 promoter as shown in ChIP-seq data and repress OTX2 promoter activity in a dose-dependent manner. These observations indicate that OTX2 and NANOG can form a negative feedback circuitry to regulate the pluripotency of porcine iPS cells.