FOXO1 modulates the biofunctions of trophoblast cells in preeclampsia via the DUSP9/p38/JNK signaling pathway

FOXO1 modulates the biofunctions of trophoblast cells in preeclampsia via the DUSP9/p38/JNK signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article FOXO1 modulates the biofunctions of trophoblast cells in preeclampsia via the DUSP9/p38/JNK signaling pathway Zhendong Qin, Xiaotao Bian, Jinfeng Li, Yanhong Yi, Junli Lu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5649547/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Preeclampsia (PE) is currently one of the major causes threatening the health and leading to death of pregnant women and fetuses. The onset of PE is attributed to cellular biological dysfunction resulting from the disruption of the molecular regulatory network in the trophoblast cells. We discovered that FOXO1 was downregulated in the placenta of preeclampsia. Methods In order to delve deeper into the involvement of FOXO1 in the development of preeclampsia, trophoblast cell lines were generated with manipulated levels of FOXO1, either through overexpression or knockdown, to elucidate its biological function and underlying mechanisms. Results The expression level of FOXO1 is positively correlated with the invasive, migratory, and proliferative abilities of trophoblast cells. Transcriptome sequencing analysis revealed DUSP9 as a potential target gene of FOXO1. The suppression of DUSP9 expression has been shown to markedly diminish the invasive, migratory, and proliferative abilities of trophoblast cells. Silencing DUSP9 in trophoblast cells that exhibit elevated levels of FOXO1 can attenuate their physiological functions. We found that overexpression/inhibition of FOXO1 can correspondingly suppress/activate the p38/JNK signaling pathway. Notably, the inhibition of DUSP9 in the context of FOXO1 overexpression can activate the p38/JNK signaling pathway. Conclusions FOXO1 modulates the biofunctions of trophoblast cells in preeclampsia via the DUSP9/p38/JNK signaling pathway. Preeclampsia FOXO1 DUSP9 p38/JNK signaling pathway biofunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Preeclampsia is a common and severe complication during pregnancy, characterized by the onset of high blood pressure and proteinuria after 20 weeks of gestation( 1 ). It can lead to multiple organ dysfunction in the mother and an increased risk of fetal mortality( 2 ). Apart from limited symptomatic treatment and termination of pregnancy, there are currently no more effective treatments for this condition available. The pathogenesis of this disease has not been fully elucidated so far; however, it is generally believed to be placenta-derived. Research has demonstrated that FOXO1 plays a pivotal role in embryonic implantation and early development. FoxO1, as a cell-specific core transcription factor, primarily exhibits its significance during embryonic implantation and endometrial remodeling( 3 ). As an emerging marker of decidualization and endometrial receptivity, FoxO1 contributes to our comprehension of the biology of the endometrium( 3 – 5 ). Nevertheless, its involvement in placental development and placenta-related disorders remains elusive. The FoxO family encompasses transcription factors, with FoxO1 being a member of the "O" subclass within the forkhead box protein family( 6 , 7 ). These Fox proteins possess a conserved DNA binding domain known as the "forkhead box"( 6 ). Within the realm of mammalian tissues, there exist four distinct members of the FoxO transcription factor group: FoxO1, FoxO3, FoxO4, and FoxO6; each exhibiting unique expression patterns( 6 , 8 , 9 ). In response to growth factor deprivation, FoxOs undergo nuclear translocation and subsequently upregulate an array of target genes involved in governing cellular resistance and metabolism, cell cycle progression, oxidative stress response, as well as apoptosis( 9 ). Dual Specificity Phosphatase 9 (DUSP9) is a member of the dual-specificity phosphatase family and functions as a critical regulator of the mitogen-activated protein kinase (MAPK) signaling pathways. The MAPK pathways include a group of proteins involved in relaying biochemical signals from the cell surface to the DNA in the cell nucleus. These pathways control a variety of cellular processes, such as proliferation, differentiation, and apoptosis( 10 ). In recent studies, DUSP9 has been revealed to participate in inflammatory responses in the development of nonalcoholic fatty liver disease and the process of parturition via p38 and JNK signaling pathways( 11 , 12 ). DUSP9 is dispensable for mammalian embryonic development, but essential for placental function, and its deletion can cause embryonic death( 13 ). Moreover, DUSP9 was reported to be associated with pregnancy complications, such as gestational diabetes mellitus and preeclampsia( 14 , 15 ). p38 MAPKs, a subclass of MAPK, are typically activated by stress stimuli such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock. The p38 MAPK pathway, once activated, can influence the expression of inflammatory cytokines, which play a role in the body's immune response. c-Jun N-terminal kinases (JNKs), another subclass of MAPKs, are also activated by stress and have been implicated in the processes of inflammation, apoptosis, and cellular proliferation. These kinases are activated by a variety of extracellular stresses, including UV irradiation, inflammatory cytokines, and osmotic stress, and they, in turn, regulate the activity of numerous transcription factors, such as c-Jun and activating transcription factor-2 (ATF-2). DUSP9 is known to inactivate both p38 and JNK by dephosphorylating them, thereby affecting their signaling capabilities( 16 ). The regulatory functions of DUSP9 are therefore crucial for maintaining cellular homeostasis. Improper regulation of these kinases by DUSP9 has been implicated in various diseases, including preeclampsia( 17 ). Preeclampsia involves an abnormal maternal inflammatory response, and dysregulation of p38 and JNK pathways can exacerbate this by increasing the expression of pro-inflammatory cytokines like TNF-alpha and IL-6. An overactive p38 MAPK pathway can lead to increased vascular permeability and endothelial dysfunction, both of which are hallmark features of preeclampsia. Endothelial dysfunction involves the impaired function of the endothelial layer lining the blood vessels, causing systemic vascular resistance and contributing significantly to the hypertension observed in preeclamptic patients. The study objectives are to delineate the functional role of FOXO1 in trophoblast cells and to elucidate how the DUSP9/p38/JNK signaling pathway modulates its activity. This will involve examining the expression and activation patterns of FOXO1 in trophoblast cells under normal and preeclamptic conditions. Additionally, the study aims to characterize the effects of manipulating DUSP9 and the p38/JNK pathway on FOXO1 activity and trophoblast cell function. This could include using specific inhibitors or gene knockdown approaches to modulate pathway components and assessing the resultant effects on cell survival, proliferation, differentiation, and stress response. Results The expression level of FOXO1 was decreased in preeclampsia placenta In order to examine the expression profile of FOXO1 in preeclampsia placenta, placental specimens were collected from pregnant individuals undergoing cesarean section at Beijing Chaoyang Hospital. Initially, immunofluorescence analysis was performed to determine the localization of FOXO1 expression in placental tissue. The results indicated a predominant localization of FOXO1 within syncytiotrophoblast cells (Fig. 1 A). Due to the heterogeneous structure of placental tissue, samples were obtained from both the amniotic and chorionic regions of the placenta for evaluating FOXO1 expression levels. A reduction in FOXO1 mRNA expression levels was observed in both the amniotic and chorionic surfaces of pre-eclamptic placental tissue compared to normal placental tissue (Fig. 1 B). Alterations in protein levels were also noted, with a significant decrease in FOXO1 expression observed in preeclamptic placental tissues compared to the control group (Fig. 1 C, D). Generation of stable transfected cell lines with altered levels of FOXO1 expression We chose to utilize the frequently employed trophoblast cell lines HTR8/SVneo cells and JEG3 cells for in vitro experiments. Initially, we employed the lentiviral transduction technique to achieve stable overexpression or knockdown of FOXO1 in both HTR8 and JEG3 cells. Three short hairpin RNAs (shRNAs: 438i, 439i 440i) specific to FOXO1 and whole sequence of FOXO1 were designed and utilized to conduct lentiviruses for the purpose of knocking down and overexpressing FOXO1. Based on the findings from RT-PCR and western blot analyses, it was concluded that Lv-438i demonstrates a knockdown efficiency of 90%, whereas LV-ov exhibits an overexpression efficiency exceeding 200% (Fig. 2 A.B.C.D). FOXO1 regulates the invasive, migratory, and proliferative capacities of trophoblast cells The transwell assay was employed to assess the regulatory influence of FOXO1 on the invasive and migratory capacities of trophoblast cells. The downregulation of FOXO1 has been shown to result in reduced invasive and migratory capacities in JEG3 (Fig. 3 A, B) and HTR8 cells (Fig. 3 D, E), while upregulation of FOXO1 has been demonstrated to markedly augment the invasiveness and migratory potential of trophoblast cells (Fig. 3 A, B, D, E). The CCK8 assay is utilized to assess cellular proliferative capacity. In comparison to the control group, suppression of FOXO1 expression impedes the proliferation of HTR8 and JEG3 cells, whereas upregulation of FOXO1 markedly enhances trophoblast cell proliferation (Fig. 3 C, F). DUSP9 may be a target gene of FOXO1 To elucidate the regulatory mechanism of FOXO1 in trophoblast cells, transcriptome sequencing analysis was conducted on cells with stable knockdown of FOXO1. We identified a total of 403 genes that were differentially expressed, comprising 265 genes that were downregulated and 138 genes that were upregulated (Fig. 4 A). DUSP9 may be a potential target gene of FOXO1. The expression pattern of DUSP9 was investigated in the preeclampsia placenta. A notable reduction in protein expression levels of DUSP9 was observed on both the amniotic and chorionic membrane surfaces in preeclampsia compared to the control group (Fig. 4 B, C, D). FOXO1 modulates the biofunction of trophoblast cells via DUSP9 The expression level of DUSP9 in trophoblast cells was manipulated through transfection with specific siRNA targeting DUSP9. SiRNA interference technology was employed to identify an effective siRNA sequence that downregulated DUSP9 among three potential candidates, for further investigation (Fig. 5 A, B). We further investigated the impact of knocking down DUSP9 on the function of trophoblast cells based on overexpression of FOXO1. The reduction of DUSP9 expression markedly impeded the enhancement of invasion and migration capabilities in trophoblast cells by FOXO1 (Fig. 5 C, D, E, F). Furthermore, we assessed the impact of DUSP9 knockdown on cell proliferation. Our findings revealed that the inhibition of DUSP9 could effectively counteract the stimulatory effect of FOXO1 on trophoblast cell proliferation (Fig. 5 G, H). FOXO1 regulating the p38/JNK signaling pathway through DUSP9 in trophoblast cells To clarify the mechanism of FOXO1 in trophoblast cells, we conducted dusp9 interference in trophoblast cells that were overexpressing FOXO1 to explore changes in signaling pathways. According to the data we obtained, overexpression of FOXO1 significantly suppresses the p38/JNK signaling pathway(Fig. 6 A-B). In comparison to the group with FOXO1 overexpression, depletion of DUSP9 expression leads to elevated phosphorylation levels of p38/JNK(Fig. 6 C-D). Hence, it can be concluded that FOXO1 directly regulates the p38/JNK signaling pathway through DUSP9. Discussion The main findings of the study will be summarized, highlighting the role of FOXO1 and the DUSP9/p38/JNK signaling pathway in trophoblast cells in the context of preeclampsia. Preeclampsia represents a significant obstetric complication characterized by high blood pressure and often accompanied by damage to organs such as the liver and kidneys. This condition primarily surfaces after the 20th week of pregnancy and affects both mother and fetus, resulting in elevated risks during pregnancy and delivery( 18 ). Understanding the molecular mechanisms underlying preeclampsia can provide insights that pave the way for novel therapeutic approaches. One of the critical discoveries from the study involves the transcription factor FOXO1, a member of the Forkhead box O (FOXO) family. FOXO1 has been recognized for its diverse roles in cellular processes, including metabolism, cell cycle regulation, and apoptosis( 19 ). Within trophoblast cells, which play an integral role in placental development and function, FOXO1 reportedly exerts substantial influence. Under normal physiological conditions, proper trophoblast function is imperative for the adequate formation and maintenance of the placenta, ensuring optimal nutrient and gas exchange between the mother and fetus( 20 , 21 ). The study's findings illuminate how aberrations in FOXO1 activity contribute to the pathogenesis of preeclampsia. Researchers observed that FOXO1 expression and activity levels are markedly altered in trophoblast cells derived from preeclamptic pregnancies. Specifically, there is a noted downregulation of FOXO1 in these cells, correlating with abnormal cellular behaviors that underpin the disease state( 22 ). These aberrations can include impaired cell proliferation, defective migration and invasion capabilities, and increased apoptotic activity. The misregulation of these processes can compromise the structural integrity and functionality of the placenta, thereby contributing to the adverse maternal and fetal outcomes associated with preeclampsia( 23 , 24 ). The role of FOXO1 in trophoblast cells, which are essential for the development and function of the placenta, is particularly significant when considering the pathophysiology of preeclampsia, a severe pregnancy complication characterized by high blood pressure and damage to other organs. Existing literature highlights the pivotal role of trophoblast cells in mediating the invasion of maternal blood vessels, a process crucial for establishing adequate maternal-fetal circulation. This vascular remodeling is essential for ensuring sufficient oxygen and nutrient supply to the developing fetus. Dysregulation in trophoblast function can lead to impaired placentation, which is a hallmark of preeclampsia. Studies have demonstrated that FOXO1 plays a significant role in regulating the genes involved in trophoblast invasion and differentiation( 25 ). Research findings suggest that FOXO1's regulatory effects on trophoblast cells are mediated through several pathways. For instance, FOXO1 can upregulate the expression of matrix metalloproteinases (MMPs)( 26 – 28 ), enzymes that degrade extracellular matrix components, thereby facilitating trophoblast invasion into the maternal decidua. Additionally, FOXO1 has been shown to influence the production of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis during placental development. VEGF promotes the formation of new blood vessels, which is indispensable for proper placental function( 27 ). An impairment in these FOXO1-mediated processes could contribute to the poor placentation observed in preeclampsia. Furthermore, FOXO1 is involved in the oxidative stress response within trophoblast cells. Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms( 29 , 30 ), is a prominent feature in the placentas of preeclamptic pregnancies. Another aspect of FOXO1's significance in trophoblast cells relates to its role in apoptotic pathways. FOXO1 can induce the expression of pro-apoptotic genes such as BCL2-like 11 (BCL2L11) and Fas ligand (FASLG), which orchestrate the programmed cell death process( 31 ). Regulation of trophoblast cell turnover through controlled apoptosis is essential for placental development and function. In preeclampsia, aberrant apoptosis of trophoblast cells has been documented, potentially leading to placental insufficiency. Therefore, the modulation of apoptosis by FOXO1 is a critical factor in ensuring trophoblast cell viability and function. Overall, the regulatory role of FOXO1 in trophoblast cell functions such as invasion, differentiation, oxidative stress response, and apoptosis underscores its significance in the context of preeclampsia. The disruption of FOXO1-mediated pathways can potentially contribute to the impaired placentation and heightened oxidative stress characteristic of preeclamptic pregnancies. A deeper understanding of FOXO1's mechanisms of action in trophoblasts could pave the way for novel therapeutic strategies aimed at alleviating the adverse outcomes associated with preeclampsia. As research continues to uncover the intricacies of FOXO1 regulation and function, its potential as a therapeutic target in preeclampsia becomes increasingly apparent. In trophoblast cells associated with preeclamptic conditions, the study identifies aberrant activity in the DUSP9/p38/JNK axis. A significant finding is the reduced expression of DUSP9, which results in the sustained activation of both p38 and JNK kinases. This hyperactivation triggers a cascade of downstream signaling events that exacerbate the pathological cellular activities observed in preeclampsia. For instance, p38 and JNK activation are linked to heightened inflammatory responses, increased oxidative stress levels, and the promotion of apoptosis. These activities collectively contribute to the dysfunctional state of the trophoblast cells, further compromising placental development and exacerbating the symptoms of preeclampsia. The interplay between FOXO1 and the DUSP9/p38/JNK pathway is a critical nexus in the molecular landscape of preeclampsia. The study posits that the reduced activity of FOXO1 might be both a cause and consequence of the altered signaling through the DUSP9/p38/JNK pathway. For instance, decreased FOXO1 activity could lead to the upregulation of pro-apoptotic genes and inflammatory mediators, which are potentiated by the concurrent hyperactivation of p38 and JNK due to diminished DUSP9 activity. Conversely, the imbalanced signaling through the p38 and JNK pathways could enhance FOXO1 activity, creating a feedback loop that perpetuates the pathogenic state in trophoblast cells. Future investigations into the mechanistic intricacies of the DUSP9/p38/JNK/FOXO1 pathway, particularly focusing on how environmental and genetic factors influence its components and interactions, might pave the way for novel predictive markers and targeted treatments for preeclampsia, enhancing maternal and fetal health outcomes. Understanding these signaling dynamics in greater detail can significantly advance our knowledge of trophoblast biology and the pathophysiology of preeclampsia, leading to better diagnostic, preventive, and therapeutic strategies. In summary, while the current study provides a foundational understanding of the involvement of FOXO1 and the DUSP9/p38/JNK pathway in preeclampsia, it is essential to address its limitations and pursue future research avenues that enrich our understanding of the disease. Expanding the scope of study populations, improving experimental models, and incorporating temporal and multifaceted analyses will significantly advance our knowledge and potentially lead to effective therapeutics and interventions for preeclampsia. Subjects, Materials and Methods Cell culture JEG3 and HTR8/SVneo cells are purchased from ATCC. JEG3 and HTR8/SVneo cells were cultured in DMEM medium containing 10% FBS, 1% penicillin/streptomycin in T25cm2 culture flasks in an incubator at 37℃ with 5% CO 2 . With the approval of the Research Ethics Committee at Beijing Chaoyang Hospital, a total of 20 cases involving postpartum placental tissue from pregnant women diagnosed with preeclampsia and those with normal pregnancies were collected from the obstetrics and gynecology department between March and December 2023. The pregnancies were terminated for non-medical reasons, and all normal pregnancies were validated through ultrasound examinations and blood tests. The collected samples were categorized into two groups: the Preeclampsia (PE) group and the Control (CTRL) group. For experimental purposes, placental tissues were sectioned to dimensions of 3 cm x 2 cm x 1 cm and preserved in a 50 ml solution tube containing 4% paraformaldehyde for subsequent paraffin embedding LentiVirus Lentiviral infection and the construction of stably transformed cell lines: In 6-well plates, JEG3 and HTR8/SVneo cells were infected with lentiviral interference plasmids FOXO1-OV, FOXO1-i (438i; 439i; 440i), and the control vector plasmid when the fusion of JEG3 and HTR8/SVneo cells reached 70%-80%.Stable cell lines were screened by culturing with medium containing genistein (200ng/ul) and puromycin (100ng/ul) for 3d.Then, the cells were expanded and the expression of FOXO1 was verified by Western blot and PCR. Plasmid transient:In 6-well plates, lentivirus-infected FOXO1-OV JEG3 and HTR8/SVneo cells were transfected using Lipo2000 and si-Dusp9 (ctrl-003-004-006) plasmids when lentivirus-infected JEG3 and HTR8/SVneo cell fusion reached 70%-80%. 250ul opti-MEM and 5ul lipo2000 were added to a 1.5ml centrifuge tube in a 1.5ml centrifuge tube and blown to mix (step1);250ul opti-MEM and 500ng plasmid were taken in a 1.5ml centrifuge tube and blown to mix (step2); The step1, 2 liquids were mixed, blown and left to stand for 20 minutes. Subsequently, 500ul of opti-MEM was added to each well, followed by 500ul of step1, 2 mixture and transferred to the incubator. After 4–6 h, DMEM was supplemented to 2 ml and incubated in the incubator for 2 d. Then, Dusp9 expression under FOXO1-OV was verified by Western blot and PCR. RNA extraction, reverse transcription and real time PCR First, total RNA was extracted from trophoblast cells by operating according to the instructions of TRizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration of total RNA was determined by a Thermo Fisher Ultra-Micro Spectrophotometer.The cDNA was obtained by reverse transcription using the RR036A,TaKaRa Reverse Transcription Kit in a 10ul, 1ug RNA system at 37°C for 15min; 85°C for 5; and 4°C later. Subsequently, RT-PCR experiments were performed using the TB Green Premix Ex Taq II (RR820A, TaKaRa) kit and Applied Biosystems 7500.cDNA amplification was performed in the following cycles:95°C for 30 s, followed by 40 cycles at 95°C for 5 s, 60°C for 34 s. With GAPDH as the endogenous reference gene, the relative quantitative method of ΔΔCT was used to calculate the expression changes of target genes.Each sample was analyzed three times. Western blot The cell lysate is prepared in a RIPA buffer containing a mixture of protease inhibitors and phosphatase inhibitors.The total proteins were extracted from placental tissue and trophoblast cells on ice and separated by centrifugation at 4℃.The concentration of extracted protein was determined by BCA method, and the same amount of protein was transferred to nitrocellulosing membrane (PVDF) by SDS-PAGE gel treatment. Then seal with 5% BSA for 1–2 hours. Add monoclonal FOXO1 (1:1000), phosphorylation (p-)JNK(1:1000),JNK (1:1000), phosphorylation (p-)p38(1:1000), p38(1:1000), Dusp-9 (1:1000) and GAPDH (1:1000), Place at 4℃ overnight. After PVDF membrane was washed with PBST, goat anti-rabbit IgG-HRP (1:20000) was added and incubated at room temperature for 2 h. The proteins were displayed with ECL luminescence kit and gel imaging system. The absorbance values were analyzed using ImageJ. Transwell Insert Invasion and migration Assay Migration: In a 24-well plate with 8mm pore size transwell chambers (Costar, ME, USA), 6*10^4 cells were resuspended with 100ul of DMEM medium containing 0.1% DMEM, followed by inoculation of the cells into the transwell chambers, and the lower wells were filled with 700ul of DMEM medium containing 10% FBS to form a concentration difference. Chemical priming was performed. After 24 h, the chambers were removed and the cells were fixed by submerging the chambers in 75% anhydrous ethanol for 15 min; subsequently, they were stained with 1X crystal violet. Then, the cells were wiped with a cotton swab from the chamber wall on the membrane, and the infiltrated cells were observed by inverted microscope (Leica, Solms, Germany). Invasion: Cell invasion ability was assayed in 24-well plates using transwell chambers (Costar, ME, USA) with 8mm pore size and the bottom of the chambers evenly coated with Matrigel (Corning, NY, USA). 8*10^4 cells were resuspended with 100ul of DMEM medium containing 0.1% DMEM, and subsequently the cells were inoculated into transwell chambers, and the lower wells were filled with 700ul of DMEM medium containing 10% FBS to form a concentration difference for chemo priming.After 24 h, the chambers were removed, and the cells were fixed by submerging the chambers in 75% anhydrous ethanol for 15 min; subsequently, stained with 1X crystal violet. Then, the cells were wiped with a cotton swab from the chamber wall on the membrane, and the infiltrated cells were observed by inverted microscope (Leica, Solms, Germany). Cell Proliferation Assay The cells were evenly spread into 96-well plates (2*103 cells/100ul/well), and 4 groups of time gradients were set 0h,24h,48h,72h with 8 sub-wells in each group. Then, 10ul of cell counting kit (CCK-8) (KGI Bio, Jiangsu) was added to each well at different time points and incubated at 37℃ for 2 h. Subsequently, the absorbance was measured at 450 nm. Immunofluorescence Paraffin sections of human placenta were prepared and preheated in an incubator for 30 minutes. Subsequently, the sections were dewaxed using xylene followed by a gradient ethanol series. Tissue repair was performed with citric acid solution, and the samples were washed three times with PBS. Hydrogen peroxide was utilized to inactivate catalase within the tissue; thereafter, a sealing solution containing 5% BSA was added and incubated for one hour. Primary antibodies against FOXO1 and CK8 (diluted 1:100) were then applied, and the slides were placed in a humidified chamber at 4°C overnight. The following day, the samples underwent three washes with PBS before being incubated with fluorescent secondary antibodies for one hour. DAPI was subsequently applied to each slide at a volume of 100 µL per piece for ten minutes, followed by three washes with PBS lasting five minutes each. Finally, an anti-fluorescence quencher was used to seal the slides prior to observation under a fluorescence microscope. Statistical analyses SPSS 22.0 (IBM, SPSS, Chicago, US) was used. Tests were conducted with one-way analysis of variance followed by Tukey’s post hoc test for multiple groups and Student’s t-test for two groups. P < 0.05 was statistically significant. Declarations Ethics approval and consent to participate This experimental study was conducted with the approval of the Ethics Committee of Beijing Chaoyang Hospital, Capital Medical University(2024-S-457). All participating patients signed an informed consent document. All experiments were conducted in strict accordance with the Declaration of Helsinki. Consent for publication All authors agree to the publication of the research findings presented in this manuscript entitled " FOXO1 modulates the biofunctions of trophoblast cells in preeclampsia via the DUSP9/p38/JNK signaling pathway" in the Biological Research. We have carefully read and understood the submission guidelines and requirements of the journal. We certify that the data, figures, and images presented in this manuscript are original or have been authorized and licensed legally. I guarantee that ethical standards have been followed in human experiments, and informed consent has been obtained from the subjects. We hereby declare that we have made every effort to avoid errors and misconduct in this research and that the results presented in this manuscript are truthful and reliable. I also declare that I will not be held responsible for any negative consequences resulting from the publication of this manuscript. Availability of data and materials Data will be made available from the corresponding author upon reasonable request. Clinical trial number Not applicable. Consent for publication Not Applicable Competing interests There is no conflicts of interest in this work. Funding This work was supported by the Beijing Natural Science Foundation (7232069), National Natural Science Foundation of China (82201863), Natural Science Foundation of Hebei Province（H2023103009）and National Key Research and Development Program of China (2021YFC2700705). Authors' contributions Zhendong Qin: Methodology, writing-review and editing. Xiaotao Bian: Data curation, formal analysis, writing review and editing. Jinfeng Li: Conceptualization, resources, supervision. Yanhong Yi: Resources, data curation. Junli Lu: resources, supervision, methodology. Guangming Cao: Conceptualization, Acquisition of data, writing-review and editing. Acknowledgements None Authors' information Guangming Cao, Junli Lu, Zhendong Qin, Jinfeng Li, Yanhong Yi: Department of Obstetrics and Gynecology, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China. Xiaotao Bian: Department of Pathology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Beijing Maternal and Child Health Care Hospital, Beijing 100026, China. References ACOG Practice Bulletin No. 202: Gestational Hypertension and Preeclampsia. Obstet Gynecol. 2019;133(1):1. Pittara T, Vyrides A, Lamnisos D, Giannakou K. Pre-eclampsia and long-term health outcomes for mother and infant: an umbrella review. BJOG. 2021;128(9):1421–30. Kajihara T, Brosens JJ, Ishihara O. The role of FOXO1 in the decidual transformation of the endometrium and early pregnancy. Med Mol Morphol. 2013;46(2):61–8. 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FoxO1 is a cell-specific core transcription factor for endometrial remodeling and homeostasis during menstrual cycle and early pregnancy. Hum Reprod Update. 2021;27(3):570–83. Macchi R, Sotelo AD, Parrado AC, Salaverry LS, Blanco GA, Castro MS, Rey-Roldán EB, Canellada AM. Losartan impairs HTR-8/SVneo trophoblast migration through inhibition of angiotensin II-induced pro-inflammatory profile in human endometrial stromal cells. Toxicol Appl Pharmcol. 2023;461. Sissaoui S, Egginton S, Ting L, Ahmed A, Hewett PW. Hyperglycaemia up-regulates placental growth factor (PlGF) expression and secretion in endothelial cells via suppression of PI3 kinase-Akt signalling and activation of FOXO1. Sci Rep. 2021;11(1). Liu F, Zhu XT, Li Y, Wang CJ, Fu JL, Hui J, Xiao Y, Liu L, Yan R, Li XF, Liu Y. Magnesium demethylcantharidate inhibits hepatocellular carcinoma cell invasion and metastasis via activation transcription factor FOXO1. Eur J Pharmacol. 2021;911. Zhang WY, Wu FJ. Linoleic acid induces human ovarian granulosa cell inflammation and apoptosis through the ER-FOXO1-ROS-NFκB pathway. Sci Rep. 2024;14(1). Sun WY, Xu T, Lin HJ, Yin YL, Xu SW. BPA and low-Se exacerbate apoptosis and autophagy in the chicken bursa of Fabricius by regulating the ROS/AKT/FOXO1 pathway. Sci Total Environ. 2024;908. Kizil HE, Caglayan C, Darendelioglu E, Ayna A, Gür C, Kandemir FM, Küçükler S. Morin ameliorates methotrexate-induced hepatotoxicity via targeting Nrf2/HO-1 and Bax/Bcl2/Caspase-3 signaling pathways. Mol Biol Rep. 2023;50(4):3479–88. Additional Declarations No competing interests reported. Supplementary Files supplementaryfile.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5649547","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392462550,"identity":"95815be8-1794-420c-83dc-1c7720c23dd6","order_by":0,"name":"Zhendong Qin","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhendong","middleName":"","lastName":"Qin","suffix":""},{"id":392462551,"identity":"4a2c1f50-fe0b-4908-aa69-f5cabfe8558c","order_by":1,"name":"Xiaotao Bian","email":"","orcid":"","institution":"Beijing Obstetrics and Gynecology Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaotao","middleName":"","lastName":"Bian","suffix":""},{"id":392462552,"identity":"d78bd5d5-c7ae-47d0-9dd0-2e289f2fcad6","order_by":2,"name":"Jinfeng Li","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jinfeng","middleName":"","lastName":"Li","suffix":""},{"id":392462553,"identity":"6c2ca70b-91a3-40e7-8839-a3857d3f412b","order_by":3,"name":"Yanhong Yi","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanhong","middleName":"","lastName":"Yi","suffix":""},{"id":392462554,"identity":"d6da9344-7328-4d92-9c7a-e1bcc0afc305","order_by":4,"name":"Junli Lu","email":"","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Junli","middleName":"","lastName":"Lu","suffix":""},{"id":392462555,"identity":"2da350db-3bf3-423e-a87b-e82aa9bd798f","order_by":5,"name":"Guangming Cao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIiWNgGAWjYFADZhBRIcHDz97Y+OAD8VrO2MhJ9hxuNpxBtE2MbWnGBjfS26Q58Cjibz98+DVPzR27te28h18wsB1O3HDzYYM0A4OdnG4Ddi0SZ9LSrHmOPUvedpgvzYKB53DizNuJDcYFDMnGZgewazFgyDEz5mE7nGx2mMfMgEHicGIfUEvyDIYDidtwaeF/A9TyD6bF4HBiw82DDYd58GmRyDF+zNt22A6oxfgBQ0KascANxsZmfFokbjxLY5zbdzgBZAsDwwFQICc2M84wwO0X/v7kwx/efDtsb3b+jPEHxn+gqDz+/MeHCjs5XFqAgE0CSCQ2ABnSf5CCBR9gBqUOexhjFIyCUTAKRgEGAAArZGPfdYxCBwAAAABJRU5ErkJggg==","orcid":"","institution":"Beijing Chao-Yang Hospital","correspondingAuthor":true,"prefix":"","firstName":"Guangming","middleName":"","lastName":"Cao","suffix":""}],"badges":[],"createdAt":"2024-12-16 00:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5649547/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5649547/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72181845,"identity":"8f5c8448-e6d1-4bf7-aa68-634f134c7066","added_by":"auto","created_at":"2024-12-23 12:51:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":329109,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression level of FOXO1 was decreased in preeclampsia placenta. A. immunofluorescent staining of FOXO1 in placenta from preeclampsia and control group. FOXO1 staining (green immunofluorescence), CK8 staining (red immunofluorescence) and DAPI staining (Blue immunofluorescence). B. Comparison of FOXO1 expression level in placenta basal and chorionic placenta from preeclampsia and control group by RT-PCR. C-D. Western blot analysis of FOXO1 in preeclampsia and control placenta. Means ± SD are indicated. * indicates P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5649547/v1/b057e4697d9090f31ae5592e.png"},{"id":72181846,"identity":"ed27821b-9eeb-4d32-91c4-e91cc518b30c","added_by":"auto","created_at":"2024-12-23 12:51:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113501,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration of stable transfected cell lines with altered levels of FOXO1 expression in trophoblast cell lines. RT-PCR and western blot analysis of FOXO1 expression in JEG3 cell(A, B) and HTR8/SVneo cells(C, D). Three short hairpin RNAs (shRNAs: 438i, 439i 440i) specific to FOXO1 and whole sequence of FOXO1 were designed and utilized to conduct lentiviruses for the purpose of knocking down and overexpressing FOXO1. Means ± SD are indicated. * indicates P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5649547/v1/db14173be8054f321c31f350.png"},{"id":72181847,"identity":"6c69d3c7-c463-464c-93e3-9fe25bc29cd9","added_by":"auto","created_at":"2024-12-23 12:51:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":938331,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of FOXO1 on the migration, invasion and proliferation of trophablast cells. Transwell experiment showing that silencing FOXO1 inhibits the migratory and invasive ability (NCi vs. 438i) and overexpression of FOXO1 promote the migratory ability (OVNC vs. OV) both in JEG3 cells (A, B) and HTR8/SVneo cells (D, E). B. CCK8 proliferation experiment demonstrating the ability of silencing FOXO1 to inhibit the cell proliferation and overexpressing FOXO1 promote cell proliferation both in JEG3 cells (C) and HTR8/SVneo cells (F). *p \u0026lt; 0.05; *p \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5649547/v1/77e7066902ac7fffd40e0fea.png"},{"id":72181848,"identity":"78c890ea-eded-4096-8dda-2c4625cb8f81","added_by":"auto","created_at":"2024-12-23 12:51:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":89335,"visible":true,"origin":"","legend":"\u003cp\u003eDUSP9 may be a target gene of FOXO1. A. The differential expressed genes of FOXO1-siRNA(438i) vs. control(NCi) were shown as volcano plot. B. Comparison of DUSP9 expression level in placenta basal and chorionic placenta from preeclampsia and control group by RT-PCR. C-D. Western blot analysis of DUSP9 in preeclampsia and control placenta. Means ± SD are indicated. * indicates P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5649547/v1/e5f43370f6005beaf44cac2c.png"},{"id":72181853,"identity":"59fe0040-035a-4918-9fd6-1598e2b8f9cc","added_by":"auto","created_at":"2024-12-23 12:51:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":317978,"visible":true,"origin":"","legend":"\u003cp\u003eFOXO1 modulates the biofunction of trophoblast cells via DUSP9. A-B. Three short siRNAs (D003i, D004i, D005i) specific to Dusp9 were designed and utilized for the purpose of knocking down. C-F. Transwell experiment showing that the reduction of DUSP9 expression markedly impeded the enhancement of invasion and migration capabilities in trophoblast cells by FOXO1. G-H. CCK8 proliferation experiment demonstrating that the inhibition of DUSP9 could effectively counteract the stimulatory effect of FOXO1 on trophoblast cell proliferation.\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5649547/v1/5f249b9f8ddfd520d8eb99af.png"},{"id":72181852,"identity":"7471a456-fe3f-4eae-a96c-bd6e2e710d97","added_by":"auto","created_at":"2024-12-23 12:51:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":153525,"visible":true,"origin":"","legend":"\u003cp\u003eFOXO1 regulating the p38/JNK signaling pathway through DUSP9 in trophoblast cells. A-B. Western blot bands showed that FOXO1 knockdown or overexpress regulate p38/JNK signaling pathway in HTR8 and Jeg3 cells. C-D. In comparison to the group with FOXO1 overexpression, depletion of DUSP9 expression leads to elevated phosphorylation levels of p38/JNK. Means ± SD are indicated. * indicates P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5649547/v1/347c2723037a89a764d5f2e2.png"},{"id":72256421,"identity":"5068ef57-4fbe-462d-83c0-3d635d42d5d3","added_by":"auto","created_at":"2024-12-24 09:47:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3030837,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5649547/v1/e2928ff3-0fb4-49cb-a0b0-dd7f6c601d2a.pdf"},{"id":72181866,"identity":"c93e400a-d605-4287-bb66-86cd1bb28600","added_by":"auto","created_at":"2024-12-23 12:51:09","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":67946234,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-5649547/v1/942a26913f35e77d90117076.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"FOXO1 modulates the biofunctions of trophoblast cells in preeclampsia via the DUSP9/p38/JNK signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreeclampsia is a common and severe complication during pregnancy, characterized by the onset of high blood pressure and proteinuria after 20 weeks of gestation(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It can lead to multiple organ dysfunction in the mother and an increased risk of fetal mortality(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Apart from limited symptomatic treatment and termination of pregnancy, there are currently no more effective treatments for this condition available. The pathogenesis of this disease has not been fully elucidated so far; however, it is generally believed to be placenta-derived.\u003c/p\u003e \u003cp\u003eResearch has demonstrated that FOXO1 plays a pivotal role in embryonic implantation and early development. FoxO1, as a cell-specific core transcription factor, primarily exhibits its significance during embryonic implantation and endometrial remodeling(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). As an emerging marker of decidualization and endometrial receptivity, FoxO1 contributes to our comprehension of the biology of the endometrium(\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Nevertheless, its involvement in placental development and placenta-related disorders remains elusive.\u003c/p\u003e \u003cp\u003eThe FoxO family encompasses transcription factors, with FoxO1 being a member of the \"O\" subclass within the forkhead box protein family(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). These Fox proteins possess a conserved DNA binding domain known as the \"forkhead box\"(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Within the realm of mammalian tissues, there exist four distinct members of the FoxO transcription factor group: FoxO1, FoxO3, FoxO4, and FoxO6; each exhibiting unique expression patterns(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In response to growth factor deprivation, FoxOs undergo nuclear translocation and subsequently upregulate an array of target genes involved in governing cellular resistance and metabolism, cell cycle progression, oxidative stress response, as well as apoptosis(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDual Specificity Phosphatase 9 (DUSP9) is a member of the dual-specificity phosphatase family and functions as a critical regulator of the mitogen-activated protein kinase (MAPK) signaling pathways. The MAPK pathways include a group of proteins involved in relaying biochemical signals from the cell surface to the DNA in the cell nucleus. These pathways control a variety of cellular processes, such as proliferation, differentiation, and apoptosis(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In recent studies, DUSP9 has been revealed to participate in inflammatory responses in the development of nonalcoholic fatty liver disease and the process of parturition via p38 and JNK signaling pathways(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). DUSP9 is dispensable for mammalian embryonic development, but essential for placental function, and its deletion can cause embryonic death(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Moreover, DUSP9 was reported to be associated with pregnancy complications, such as gestational diabetes mellitus and preeclampsia(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ep38 MAPKs, a subclass of MAPK, are typically activated by stress stimuli such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock. The p38 MAPK pathway, once activated, can influence the expression of inflammatory cytokines, which play a role in the body's immune response. c-Jun N-terminal kinases (JNKs), another subclass of MAPKs, are also activated by stress and have been implicated in the processes of inflammation, apoptosis, and cellular proliferation. These kinases are activated by a variety of extracellular stresses, including UV irradiation, inflammatory cytokines, and osmotic stress, and they, in turn, regulate the activity of numerous transcription factors, such as c-Jun and activating transcription factor-2 (ATF-2). DUSP9 is known to inactivate both p38 and JNK by dephosphorylating them, thereby affecting their signaling capabilities(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The regulatory functions of DUSP9 are therefore crucial for maintaining cellular homeostasis. Improper regulation of these kinases by DUSP9 has been implicated in various diseases, including preeclampsia(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Preeclampsia involves an abnormal maternal inflammatory response, and dysregulation of p38 and JNK pathways can exacerbate this by increasing the expression of pro-inflammatory cytokines like TNF-alpha and IL-6. An overactive p38 MAPK pathway can lead to increased vascular permeability and endothelial dysfunction, both of which are hallmark features of preeclampsia. Endothelial dysfunction involves the impaired function of the endothelial layer lining the blood vessels, causing systemic vascular resistance and contributing significantly to the hypertension observed in preeclamptic patients.\u003c/p\u003e \u003cp\u003eThe study objectives are to delineate the functional role of FOXO1 in trophoblast cells and to elucidate how the DUSP9/p38/JNK signaling pathway modulates its activity. This will involve examining the expression and activation patterns of FOXO1 in trophoblast cells under normal and preeclamptic conditions. Additionally, the study aims to characterize the effects of manipulating DUSP9 and the p38/JNK pathway on FOXO1 activity and trophoblast cell function. This could include using specific inhibitors or gene knockdown approaches to modulate pathway components and assessing the resultant effects on cell survival, proliferation, differentiation, and stress response.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe expression level of FOXO1 was decreased in preeclampsia placenta\u003c/h2\u003e \u003cp\u003eIn order to examine the expression profile of FOXO1 in preeclampsia placenta, placental specimens were collected from pregnant individuals undergoing cesarean section at Beijing Chaoyang Hospital. Initially, immunofluorescence analysis was performed to determine the localization of FOXO1 expression in placental tissue. The results indicated a predominant localization of FOXO1 within syncytiotrophoblast cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Due to the heterogeneous structure of placental tissue, samples were obtained from both the amniotic and chorionic regions of the placenta for evaluating FOXO1 expression levels. A reduction in FOXO1 mRNA expression levels was observed in both the amniotic and chorionic surfaces of pre-eclamptic placental tissue compared to normal placental tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Alterations in protein levels were also noted, with a significant decrease in FOXO1 expression observed in preeclamptic placental tissues compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneration of stable transfected cell lines with altered levels of FOXO1 expression\u003c/h3\u003e\n\u003cp\u003eWe chose to utilize the frequently employed trophoblast cell lines HTR8/SVneo cells and JEG3 cells for \u003cem\u003ein vitro\u003c/em\u003e experiments. Initially, we employed the lentiviral transduction technique to achieve stable overexpression or knockdown of FOXO1 in both HTR8 and JEG3 cells. Three short hairpin RNAs (shRNAs: 438i, 439i 440i) specific to FOXO1 and whole sequence of FOXO1 were designed and utilized to conduct lentiviruses for the purpose of knocking down and overexpressing FOXO1. Based on the findings from RT-PCR and western blot analyses, it was concluded that Lv-438i demonstrates a knockdown efficiency of 90%, whereas LV-ov exhibits an overexpression efficiency exceeding 200% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA.B.C.D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eFOXO1 regulates the invasive, migratory, and proliferative capacities of trophoblast cells\u003c/h3\u003e\n\u003cp\u003eThe transwell assay was employed to assess the regulatory influence of FOXO1 on the invasive and migratory capacities of trophoblast cells. The downregulation of FOXO1 has been shown to result in reduced invasive and migratory capacities in JEG3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B) and HTR8 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E), while upregulation of FOXO1 has been demonstrated to markedly augment the invasiveness and migratory potential of trophoblast cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, D, E). The CCK8 assay is utilized to assess cellular proliferative capacity. In comparison to the control group, suppression of FOXO1 expression impedes the proliferation of HTR8 and JEG3 cells, whereas upregulation of FOXO1 markedly enhances trophoblast cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDUSP9 may be a target gene of FOXO1\u003c/h3\u003e\n\u003cp\u003eTo elucidate the regulatory mechanism of FOXO1 in trophoblast cells, transcriptome sequencing analysis was conducted on cells with stable knockdown of FOXO1. We identified a total of 403 genes that were differentially expressed, comprising 265 genes that were downregulated and 138 genes that were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). DUSP9 may be a potential target gene of FOXO1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression pattern of DUSP9 was investigated in the preeclampsia placenta. A notable reduction in protein expression levels of DUSP9 was observed on both the amniotic and chorionic membrane surfaces in preeclampsia compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C, D).\u003c/p\u003e\n\u003ch3\u003eFOXO1 modulates the biofunction of trophoblast cells via DUSP9\u003c/h3\u003e\n\u003cp\u003eThe expression level of DUSP9 in trophoblast cells was manipulated through transfection with specific siRNA targeting DUSP9. SiRNA interference technology was employed to identify an effective siRNA sequence that downregulated DUSP9 among three potential candidates, for further investigation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). We further investigated the impact of knocking down DUSP9 on the function of trophoblast cells based on overexpression of FOXO1. The reduction of DUSP9 expression markedly impeded the enhancement of invasion and migration capabilities in trophoblast cells by FOXO1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D, E, F). Furthermore, we assessed the impact of DUSP9 knockdown on cell proliferation. Our findings revealed that the inhibition of DUSP9 could effectively counteract the stimulatory effect of FOXO1 on trophoblast cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFOXO1 regulating the p38/JNK signaling pathway through DUSP9 in trophoblast cells\u003c/h2\u003e \u003cp\u003eTo clarify the mechanism of FOXO1 in trophoblast cells, we conducted dusp9 interference in trophoblast cells that were overexpressing FOXO1 to explore changes in signaling pathways. According to the data we obtained, overexpression of FOXO1 significantly suppresses the p38/JNK signaling pathway(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). In comparison to the group with FOXO1 overexpression, depletion of DUSP9 expression leads to elevated phosphorylation levels of p38/JNK(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D). Hence, it can be concluded that FOXO1 directly regulates the p38/JNK signaling pathway through DUSP9.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main findings of the study will be summarized, highlighting the role of FOXO1 and the DUSP9/p38/JNK signaling pathway in trophoblast cells in the context of preeclampsia. Preeclampsia represents a significant obstetric complication characterized by high blood pressure and often accompanied by damage to organs such as the liver and kidneys. This condition primarily surfaces after the 20th week of pregnancy and affects both mother and fetus, resulting in elevated risks during pregnancy and delivery(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Understanding the molecular mechanisms underlying preeclampsia can provide insights that pave the way for novel therapeutic approaches.\u003c/p\u003e \u003cp\u003eOne of the critical discoveries from the study involves the transcription factor FOXO1, a member of the Forkhead box O (FOXO) family. FOXO1 has been recognized for its diverse roles in cellular processes, including metabolism, cell cycle regulation, and apoptosis(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Within trophoblast cells, which play an integral role in placental development and function, FOXO1 reportedly exerts substantial influence. Under normal physiological conditions, proper trophoblast function is imperative for the adequate formation and maintenance of the placenta, ensuring optimal nutrient and gas exchange between the mother and fetus(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe study's findings illuminate how aberrations in FOXO1 activity contribute to the pathogenesis of preeclampsia. Researchers observed that FOXO1 expression and activity levels are markedly altered in trophoblast cells derived from preeclamptic pregnancies. Specifically, there is a noted downregulation of FOXO1 in these cells, correlating with abnormal cellular behaviors that underpin the disease state(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). These aberrations can include impaired cell proliferation, defective migration and invasion capabilities, and increased apoptotic activity. The misregulation of these processes can compromise the structural integrity and functionality of the placenta, thereby contributing to the adverse maternal and fetal outcomes associated with preeclampsia(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe role of FOXO1 in trophoblast cells, which are essential for the development and function of the placenta, is particularly significant when considering the pathophysiology of preeclampsia, a severe pregnancy complication characterized by high blood pressure and damage to other organs. Existing literature highlights the pivotal role of trophoblast cells in mediating the invasion of maternal blood vessels, a process crucial for establishing adequate maternal-fetal circulation. This vascular remodeling is essential for ensuring sufficient oxygen and nutrient supply to the developing fetus. Dysregulation in trophoblast function can lead to impaired placentation, which is a hallmark of preeclampsia. Studies have demonstrated that FOXO1 plays a significant role in regulating the genes involved in trophoblast invasion and differentiation(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearch findings suggest that FOXO1's regulatory effects on trophoblast cells are mediated through several pathways. For instance, FOXO1 can upregulate the expression of matrix metalloproteinases (MMPs)(\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), enzymes that degrade extracellular matrix components, thereby facilitating trophoblast invasion into the maternal decidua. Additionally, FOXO1 has been shown to influence the production of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis during placental development. VEGF promotes the formation of new blood vessels, which is indispensable for proper placental function(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). An impairment in these FOXO1-mediated processes could contribute to the poor placentation observed in preeclampsia.\u003c/p\u003e \u003cp\u003eFurthermore, FOXO1 is involved in the oxidative stress response within trophoblast cells. Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), is a prominent feature in the placentas of preeclamptic pregnancies. Another aspect of FOXO1's significance in trophoblast cells relates to its role in apoptotic pathways. FOXO1 can induce the expression of pro-apoptotic genes such as BCL2-like 11 (BCL2L11) and Fas ligand (FASLG), which orchestrate the programmed cell death process(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Regulation of trophoblast cell turnover through controlled apoptosis is essential for placental development and function. In preeclampsia, aberrant apoptosis of trophoblast cells has been documented, potentially leading to placental insufficiency. Therefore, the modulation of apoptosis by FOXO1 is a critical factor in ensuring trophoblast cell viability and function.\u003c/p\u003e \u003cp\u003eOverall, the regulatory role of FOXO1 in trophoblast cell functions such as invasion, differentiation, oxidative stress response, and apoptosis underscores its significance in the context of preeclampsia. The disruption of FOXO1-mediated pathways can potentially contribute to the impaired placentation and heightened oxidative stress characteristic of preeclamptic pregnancies. A deeper understanding of FOXO1's mechanisms of action in trophoblasts could pave the way for novel therapeutic strategies aimed at alleviating the adverse outcomes associated with preeclampsia. As research continues to uncover the intricacies of FOXO1 regulation and function, its potential as a therapeutic target in preeclampsia becomes increasingly apparent.\u003c/p\u003e \u003cp\u003eIn trophoblast cells associated with preeclamptic conditions, the study identifies aberrant activity in the DUSP9/p38/JNK axis. A significant finding is the reduced expression of DUSP9, which results in the sustained activation of both p38 and JNK kinases. This hyperactivation triggers a cascade of downstream signaling events that exacerbate the pathological cellular activities observed in preeclampsia. For instance, p38 and JNK activation are linked to heightened inflammatory responses, increased oxidative stress levels, and the promotion of apoptosis. These activities collectively contribute to the dysfunctional state of the trophoblast cells, further compromising placental development and exacerbating the symptoms of preeclampsia.\u003c/p\u003e \u003cp\u003eThe interplay between FOXO1 and the DUSP9/p38/JNK pathway is a critical nexus in the molecular landscape of preeclampsia. The study posits that the reduced activity of FOXO1 might be both a cause and consequence of the altered signaling through the DUSP9/p38/JNK pathway. For instance, decreased FOXO1 activity could lead to the upregulation of pro-apoptotic genes and inflammatory mediators, which are potentiated by the concurrent hyperactivation of p38 and JNK due to diminished DUSP9 activity. Conversely, the imbalanced signaling through the p38 and JNK pathways could enhance FOXO1 activity, creating a feedback loop that perpetuates the pathogenic state in trophoblast cells.\u003c/p\u003e \u003cp\u003eFuture investigations into the mechanistic intricacies of the DUSP9/p38/JNK/FOXO1 pathway, particularly focusing on how environmental and genetic factors influence its components and interactions, might pave the way for novel predictive markers and targeted treatments for preeclampsia, enhancing maternal and fetal health outcomes. Understanding these signaling dynamics in greater detail can significantly advance our knowledge of trophoblast biology and the pathophysiology of preeclampsia, leading to better diagnostic, preventive, and therapeutic strategies.\u003c/p\u003e \u003cp\u003eIn summary, while the current study provides a foundational understanding of the involvement of FOXO1 and the DUSP9/p38/JNK pathway in preeclampsia, it is essential to address its limitations and pursue future research avenues that enrich our understanding of the disease. Expanding the scope of study populations, improving experimental models, and incorporating temporal and multifaceted analyses will significantly advance our knowledge and potentially lead to effective therapeutics and interventions for preeclampsia.\u003c/p\u003e"},{"header":"Subjects, Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eJEG3 and HTR8/SVneo cells are purchased from ATCC. JEG3 and HTR8/SVneo cells were cultured in DMEM medium containing 10% FBS, 1% penicillin/streptomycin in T25cm2 culture flasks in an incubator at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e With the approval of the Research Ethics Committee at Beijing Chaoyang Hospital, a total of 20 cases involving postpartum placental tissue from pregnant women diagnosed with preeclampsia and those with normal pregnancies were collected from the obstetrics and gynecology department between March and December 2023. The pregnancies were terminated for non-medical reasons, and all normal pregnancies were validated through ultrasound examinations and blood tests. The collected samples were categorized into two groups: the Preeclampsia (PE) group and the Control (CTRL) group. For experimental purposes, placental tissues were sectioned to dimensions of 3 cm x 2 cm x 1 cm and preserved in a 50 ml solution tube containing 4% paraformaldehyde for subsequent paraffin embedding\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLentiVirus\u003c/h2\u003e \u003cp\u003eLentiviral infection and the construction of stably transformed cell lines: In 6-well plates, JEG3 and HTR8/SVneo cells were infected with lentiviral interference plasmids FOXO1-OV, FOXO1-i (438i; 439i; 440i), and the control vector plasmid when the fusion of JEG3 and HTR8/SVneo cells reached 70%-80%.Stable cell lines were screened by culturing with medium containing genistein (200ng/ul) and puromycin (100ng/ul) for 3d.Then, the cells were expanded and the expression of FOXO1 was verified by Western blot and PCR.\u003c/p\u003e \u003cp\u003ePlasmid transient:In 6-well plates, lentivirus-infected FOXO1-OV JEG3 and HTR8/SVneo cells were transfected using Lipo2000 and si-Dusp9 (ctrl-003-004-006) plasmids when lentivirus-infected JEG3 and HTR8/SVneo cell fusion reached 70%-80%. 250ul opti-MEM and 5ul lipo2000 were added to a 1.5ml centrifuge tube in a 1.5ml centrifuge tube and blown to mix (step1);250ul opti-MEM and 500ng plasmid were taken in a 1.5ml centrifuge tube and blown to mix (step2); The step1, 2 liquids were mixed, blown and left to stand for 20 minutes. Subsequently, 500ul of opti-MEM was added to each well, followed by 500ul of step1, 2 mixture and transferred to the incubator. After 4\u0026ndash;6 h, DMEM was supplemented to 2 ml and incubated in the incubator for 2 d. Then, Dusp9 expression under FOXO1-OV was verified by Western blot and PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, reverse transcription and real time PCR\u003c/h2\u003e \u003cp\u003eFirst, total RNA was extracted from trophoblast cells by operating according to the instructions of TRizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration of total RNA was determined by a Thermo Fisher Ultra-Micro Spectrophotometer.The cDNA was obtained by reverse transcription using the RR036A,TaKaRa Reverse Transcription Kit in a 10ul, 1ug RNA system at 37\u0026deg;C for 15min; 85\u0026deg;C for 5; and 4\u0026deg;C later.\u003c/p\u003e \u003cp\u003eSubsequently, RT-PCR experiments were performed using the TB Green Premix Ex Taq II (RR820A, TaKaRa) kit and Applied Biosystems 7500.cDNA amplification was performed in the following cycles:95\u0026deg;C for 30 s, followed by 40 cycles at 95\u0026deg;C for 5 s, 60\u0026deg;C for 34 s. With GAPDH as the endogenous reference gene, the relative quantitative method of ΔΔCT was used to calculate the expression changes of target genes.Each sample was analyzed three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eThe cell lysate is prepared in a RIPA buffer containing a mixture of protease inhibitors and phosphatase inhibitors.The total proteins were extracted from placental tissue and trophoblast cells on ice and separated by centrifugation at 4℃.The concentration of extracted protein was determined by BCA method, and the same amount of protein was transferred to nitrocellulosing membrane (PVDF) by SDS-PAGE gel treatment. Then seal with 5% BSA for 1\u0026ndash;2 hours. Add monoclonal FOXO1 (1:1000), phosphorylation (p-)JNK(1:1000),JNK (1:1000), phosphorylation (p-)p38(1:1000), p38(1:1000), Dusp-9 (1:1000) and GAPDH (1:1000), Place at 4℃ overnight. After PVDF membrane was washed with PBST, goat anti-rabbit IgG-HRP (1:20000) was added and incubated at room temperature for 2 h. The proteins were displayed with ECL luminescence kit and gel imaging system. The absorbance values were analyzed using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTranswell Insert Invasion and migration Assay\u003c/h2\u003e \u003cp\u003eMigration: In a 24-well plate with 8mm pore size transwell chambers (Costar, ME, USA), 6*10^4 cells were resuspended with 100ul of DMEM medium containing 0.1% DMEM, followed by inoculation of the cells into the transwell chambers, and the lower wells were filled with 700ul of DMEM medium containing 10% FBS to form a concentration difference. Chemical priming was performed. After 24 h, the chambers were removed and the cells were fixed by submerging the chambers in 75% anhydrous ethanol for 15 min; subsequently, they were stained with 1X crystal violet. Then, the cells were wiped with a cotton swab from the chamber wall on the membrane, and the infiltrated cells were observed by inverted microscope (Leica, Solms, Germany).\u003c/p\u003e \u003cp\u003eInvasion: Cell invasion ability was assayed in 24-well plates using transwell chambers (Costar, ME, USA) with 8mm pore size and the bottom of the chambers evenly coated with Matrigel (Corning, NY, USA). 8*10^4 cells were resuspended with 100ul of DMEM medium containing 0.1% DMEM, and subsequently the cells were inoculated into transwell chambers, and the lower wells were filled with 700ul of DMEM medium containing 10% FBS to form a concentration difference for chemo priming.After 24 h, the chambers were removed, and the cells were fixed by submerging the chambers in 75% anhydrous ethanol for 15 min; subsequently, stained with 1X crystal violet. Then, the cells were wiped with a cotton swab from the chamber wall on the membrane, and the infiltrated cells were observed by inverted microscope (Leica, Solms, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell Proliferation Assay\u003c/h2\u003e \u003cp\u003eThe cells were evenly spread into 96-well plates (2*103 cells/100ul/well), and 4 groups of time gradients were set 0h,24h,48h,72h with 8 sub-wells in each group. Then, 10ul of cell counting kit (CCK-8) (KGI Bio, Jiangsu) was added to each well at different time points and incubated at 37℃ for 2 h. Subsequently, the absorbance was measured at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eParaffin sections of human placenta were prepared and preheated in an incubator for 30 minutes. Subsequently, the sections were dewaxed using xylene followed by a gradient ethanol series. Tissue repair was performed with citric acid solution, and the samples were washed three times with PBS. Hydrogen peroxide was utilized to inactivate catalase within the tissue; thereafter, a sealing solution containing 5% BSA was added and incubated for one hour. Primary antibodies against FOXO1 and CK8 (diluted 1:100) were then applied, and the slides were placed in a humidified chamber at 4\u0026deg;C overnight. The following day, the samples underwent three washes with PBS before being incubated with fluorescent secondary antibodies for one hour. DAPI was subsequently applied to each slide at a volume of 100 \u0026micro;L per piece for ten minutes, followed by three washes with PBS lasting five minutes each. Finally, an anti-fluorescence quencher was used to seal the slides prior to observation under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eSPSS 22.0 (IBM, SPSS, Chicago, US) was used. Tests were conducted with one-way analysis of variance followed by Tukey\u0026rsquo;s post hoc test for multiple groups and Student\u0026rsquo;s t-test for two groups. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experimental study was conducted with the approval of the Ethics Committee of Beijing Chaoyang Hospital, Capital Medical University(2024-S-457). All participating patients signed an informed consent document. All experiments were conducted in strict accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to the publication of the research findings presented in this manuscript entitled \u0026quot; FOXO1 modulates the biofunctions of trophoblast cells in preeclampsia via the DUSP9/p38/JNK signaling pathway\u0026quot; in the Biological Research. We have carefully read and understood the submission guidelines and requirements of the journal. We certify that the data, figures, and images presented in this manuscript are original or have been authorized and licensed legally. I guarantee that ethical standards have been followed in human experiments, and informed consent has been obtained from the subjects. We hereby declare that we have made every effort to avoid errors and misconduct in this research and that the results presented in this manuscript are truthful and reliable. I also declare that I will not be held responsible for any negative consequences resulting from the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no conflicts of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Beijing Natural Science Foundation (7232069), National Natural Science Foundation of China (82201863), Natural Science Foundation of Hebei Province（H2023103009）and National Key Research and Development Program of China (2021YFC2700705).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhendong Qin:\u0026nbsp;Methodology, writing-review and editing. Xiaotao Bian: Data curation, formal analysis, writing review and editing. Jinfeng Li:\u0026nbsp;Conceptualization, resources, supervision. Yanhong Yi: Resources, data curation. Junli Lu:\u0026nbsp;resources, supervision, methodology. Guangming Cao:\u0026nbsp;Conceptualization,\u0026nbsp;Acquisition of data, writing-review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuangming Cao, Junli Lu, Zhendong Qin, Jinfeng Li, Yanhong Yi: Department of Obstetrics and Gynecology, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXiaotao Bian: Department of Pathology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Beijing Maternal and Child Health Care Hospital, Beijing 100026, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eACOG Practice Bulletin No. 202: Gestational Hypertension and Preeclampsia. Obstet Gynecol. 2019;133(1):1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePittara T, Vyrides A, Lamnisos D, Giannakou K. Pre-eclampsia and long-term health outcomes for mother and infant: an umbrella review. BJOG. 2021;128(9):1421\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKajihara T, Brosens JJ, Ishihara O. The role of FOXO1 in the decidual transformation of the endometrium and early pregnancy. Med Mol Morphol. 2013;46(2):61\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVasquez YM, Mazur EC, Li X, Kommagani R, Jiang L, Chen R, Lanz RB, Kovanci E, Gibbons WE, DeMayo FJ. 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Mol Biol Rep. 2023;50(4):3479\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Preeclampsia, FOXO1, DUSP9, p38/JNK signaling pathway, biofunction","lastPublishedDoi":"10.21203/rs.3.rs-5649547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5649547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePreeclampsia (PE) is currently one of the major causes threatening the health and leading to death of pregnant women and fetuses. The onset of PE is attributed to cellular biological dysfunction resulting from the disruption of the molecular regulatory network in the trophoblast cells. We discovered that FOXO1 was downregulated in the placenta of preeclampsia.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn order to delve deeper into the involvement of FOXO1 in the development of preeclampsia, trophoblast cell lines were generated with manipulated levels of FOXO1, either through overexpression or knockdown, to elucidate its biological function and underlying mechanisms.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe expression level of FOXO1 is positively correlated with the invasive, migratory, and proliferative abilities of trophoblast cells. Transcriptome sequencing analysis revealed DUSP9 as a potential target gene of FOXO1. The suppression of DUSP9 expression has been shown to markedly diminish the invasive, migratory, and proliferative abilities of trophoblast cells. Silencing DUSP9 in trophoblast cells that exhibit elevated levels of FOXO1 can attenuate their physiological functions. We found that overexpression/inhibition of FOXO1 can correspondingly suppress/activate the p38/JNK signaling pathway. Notably, the inhibition of DUSP9 in the context of FOXO1 overexpression can activate the p38/JNK signaling pathway.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eFOXO1 modulates the biofunctions of trophoblast cells in preeclampsia via the DUSP9/p38/JNK signaling pathway.\u003c/p\u003e","manuscriptTitle":"FOXO1 modulates the biofunctions of trophoblast cells in preeclampsia via the DUSP9/p38/JNK signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 12:51:03","doi":"10.21203/rs.3.rs-5649547/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"73e1f1a3-9b6a-4cab-a4c5-9be48b613f53","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-24T09:38:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-23 12:51:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5649547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5649547","identity":"rs-5649547","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}