MELTF-AS1 Regulates DUSP5 and Trophoblast Function via EZH2 to Inhibit Preeclampsia

MELTF-AS1 Regulates DUSP5 and Trophoblast Function via EZH2 to Inhibit Preeclampsia | 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 Article MELTF-AS1 Regulates DUSP5 and Trophoblast Function via EZH2 to Inhibit Preeclampsia Lingling Jiang, yi shen, Meilikang Li, Haili Kai, Xinyi Kang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8076487/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Objective: To explore the mechanism by which lncRNA MELTF-AS1 binds EZH2 to downregulate target gene DUSP5, thereby promoting trophoblast function and inhibiting the occurrence of preeclampsia (PE). Methods: Bioinformatic re-analysis of dataset GSE183466 identified the lncRNA MELTF-AS1 as a key dysregulated transcript in preeclampsia. Transcriptome sequencing after MELTF-AS1 knockdown identified DUSP5. CCK-8, colony formation, and Transwell assays evaluated effects on HTR-8/SVneo cell proliferation/invasion. Nuclear-cytoplasmic separation localizedthe action site of MELTF-AS1. RIP-qPCR/CHIP-qPCR clarified binding mechanisms of MELTF-AS1/DUSP5, with reduced uterine perfusion pressure mice verifying conclusions. Results: In this study, Bioinformatic analysis identified that MELTF-AS1 was significantly downregulated in the PE group. Transcriptome sequencing after MELTF-AS1 knockdown showed that DUSP5 was significantly upregulated. MELTF-AS1 enhanced cell proliferation/invasion, while DUSP5 inhibited them. Nuclear-cytoplasmic separation assay revealed that MELTF-AS1 was expressed in both the nucleus and cytoplasm. Further RIP-qPCR and CHIP-qPCR showed that MELTF-AS1 could bind to EZH2, promoting the enrichment of EZH2 at the DUSP5 promoter region and increasing the level of H3K27me3 modification, thereby reducing DUSP5 transcriptional level. Conclusion: This study reveals the regulatory mechanism mediated by MELTF-AS1, and the MELTF-AS1/DUSP5 regulatory pathway may provide new predictive and therapeutic intervention strategies for PE. Biological sciences/Cell biology Biological sciences/Molecular biology Preeclampsia MELTF-AS1 EZH2 DUSP5 trophoblast cell function Figures Figure 1 Figure 2 Figure 3 Introduction Preeclampsia (PE), a pregnancy-specific syndrome characterized by hypertension and proteinuria after 20 weeks of gestation, remains a leading cause of maternal and perinatal morbidity and mortality worldwide [ 1 ] . Despite decades of research, the pathogenic mechanisms underlying PE remain incompletely understood, with growing evidence highlighting that impaired trophoblast cell function—including inadequate proliferation, invasion, and differentiation—plays a pivotal role in the development of PE [ 2 , 3 ] . Trophoblast cells are essential for placental implantation and spiral artery remodeling [ 4 ] ; dysfunction of these cells leads to inadequate placental perfusion, triggering systemic inflammation and endothelial dysfunction, which are hallmarks of PE [ 5 – 7 ] .​ Long non-coding RNAs (lncRNAs), a class of non-protein-coding RNAs longer than 200 nucleotides, have emerged as key regulators of gene expression in various physiological and pathological processes, including placental development and PE. Accumulating studies have demonstrated that aberrant lncRNA expression is closely associated with trophoblast cell dysfunction in PE [ 8 , 9 ] . For instance, lncRNA H19 and MALAT1 have been reported to modulate trophoblast proliferation and invasion by targeting specific signaling pathways, thereby participating in PE pathogenesis [ 10 – 14 ] . However, the majority of lncRNAs involved in PE remain uncharacterized, and their precise regulatory mechanisms in trophoblast cell function and PE progression require further exploration.​ Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), mediates gene silencing through trimethylation of histone H3 at lysine 27 (H3K27me3) [ 15 ] . EZH2 has been shown to regulate trophoblast cell proliferation, invasion, and apoptosis, and its dysregulation is implicated in PE development [ 16 , 17 ] . Dual-specificity phosphatase 5 (DUSP5), a member of the DUSP family that dephosphorylates mitogen-activated protein kinases (MAPKs), has been reported to inhibit cell proliferation and invasion in multiple cancers [ 18 – 20 ] . However, the expression pattern and functional role of DUSP5 in PE, as well as its upstream regulatory mechanisms, remain unclear. MELTF-AS1 is a newly identified lncRNA whose biological function has not been fully elucidated [ 21 , 22 ] . Bioinformatic re-analysis of an NCBI lncRNA dataset (GSE183466) from preeclampsia placentas revealed that MELTF-AS1 was significantly down-regulated, suggesting its potential involvement in PE pathogenesis. Based on these findings, we hypothesized that MELTF-AS1 may regulate trophoblast cell function and PE development through interaction with EZH2 and subsequent modulation of DUSP5 expression.​ In the present study, we aimed to: (1) confirm the differential expression of MELTF-AS1 in PE placental tissues; (2) investigate the effects of MELTF-AS1 and DUSP5 on trophoblast cell proliferation and invasion; (3) clarify the subcellular localization of MELTF-AS1 and its binding interaction with EZH2; (4) explore whether MELTF-AS1 regulates DUSP5 expression via EZH2-mediated H3K27me3 modification; and (5) verify the regulatory pathway in a mouse model of PE. The results of this study may provide novel insights into the molecular mechanisms of PE and identify potential therapeutic targets for this devastating disease. Results MELTF-AS1 promotes proliferation and invasion of trophoblast cells Emerging evidence indicates that placental dysfunction is the cornerstone of the pathogenesis of PE, in which defective extravillous trophoblast (EVT) invasion leads to insufficient spiral artery remodeling and subsequent placental ischemia. Numerous studies have shown that aberrant lncRNA expression is closely associated with trophoblast dysfunction in PE. In order to explore the relationship between lncRNAs and PE, the research team obtained the lncRNA dataset of PE from NCBI (GSE183466) and conducted a re-analysis of bioinformatics to identify the relevant target long non-coding RNAs. The results revealed that compared with the control group, there were 68 downregulated lncRNAs and 76 upregulated lncRNAs in the PE group (Fig. 1 A). Specifically, MELTF-AS1 was significantly downregulated in the PE group. Therefore, we modulated the expression of MELTF-AS1 in the HTR-8/SVneo cell line and verified the effect of MELTF-AS1 on trophoblast function using the CCK-8 assay, colony formation assay, and Transwell assay. These experiments demonstrated that MELTF-AS1 promoted the invasion and proliferation capabilities of trophoblast cells(Fig. 1 B-D).. Additionally, nuclear-cytoplasmic separation assay showed that MELTF-AS1, as a key lincRNA, is present in both the cytoplasm and nucleus(Fig. 1 E). MELTF-AS1 Involves in the Pathogenesis of Preeclampsia by Epigenetic Regulation of DUSP5 Binding to EZH2 In order to understand the molecular mechanism of MELTF-AS1 resistance to PE, we performed transcriptome sequencing on HTR-8/SVneo cells in MELTF-AS1 silencing and control groups. RNA sequencing analysis indicated that following the depletion of MELTF-AS1, the transcript levels of 147 genes were upregulated by at least 2-fold, while the expression levels of 107 mRNAs were downregulated by 2-fold or more(Fig. 2 A).MELTF-AS1-related signaling pathways predicted by the GO database suggest changes in cell migration and invasion in trophoblast cells where MELTF-AS1 is up/down regulated(Fig. 2 B). Among the enriched genes, there are well-known genes related to proliferation and invasion, such as PTEN, p53, CXCR4, etc (data not shown). Compared with other genes, the change of DUSP5 was the most significant after changing the expression of MELTF-AS1.After down-regulating MELTF-AS1, the expression of DUSP5 increased; after up-regulating MELTF-AS1, the expression of DUSP5 decreased (Fig. 2 D-E). So, we speculate that MELTF-AS1 affects trophoblast function by regulating DUSP5. Consequently, we conducted CCK8, Transwell and Colony Formation assays using the HTR-8/SVneo cell lines, respectively. These experiments indicated that DUSP5 may also impede trophoblast cell invasion and proliferation (Fig. 2 F-G). To verify whether MELTF-AS1 and DUSP5 can bind directly to exert their effects, we performed RIP-qPCR. The results showed that MELTF-AS1 and DUSP5 could not bind directly. Subsequently, through literature review, we found that MELTF-AS1 can increase the level of H3K27me3 modification by binding to EZH2, thereby affecting the transcription of downstream factors. Therefore, using RIP-qPCR and CHIP-qPCR assays, we demonstrated that MELTF-AS1 can bind to EZH2, which in turn promotes the enrichment of EZH2 in the promoter region of DUSP5 and increases the level of H3K27me3 modification, ultimately resulting in the transcriptional silencing of DUSP5 (Fig. 2 H-I). Aberrant DUSP5 expression in PE mice models and human placentas To verify the key role of the MELTF-AS1/DUSP5 axis in the pathological process of PE in vivo, we successfully established a mouse model with reduced uterine perfusion pressure (RUPP) via surgery, which is a classic in vivo model for PE research (Fig. 3 A). First, we confirmed that the RUPP model successfully simulated the maternal cardiovascular symptoms of PE. We continuously monitored the systolic blood pressure (SBP) of pregnant mice. The results showed that compared with the control group, mice in the RUPP group exhibited a significant increase in SBP starting from mid-gestation (around embryonic day 14, E14), which persisted until late gestation and met the diagnostic criteria for hypertension. This data clearly indicated that the RUPP surgery successfully induced gestational hypertension—a core clinical feature of PE.Second, we evaluated the impact of the RUPP model on pregnancy outcomes and observed typical placental and fetal developmental abnormalities. We weighed the placentas and embryos of mice in late gestation. Statistical analysis revealed that both placental weight and embryonic weight in the RUPP group were significantly lower than those in the control group. Reduced placental weight directly reflects placental hypoplasia and dysfunction, while the significant decrease in embryonic weight serves as direct evidence of fetal growth restriction (FGR) caused by placental insufficiency. Together, these two indicators confirmed that the RUPP model can effectively recapitulate the adverse pregnancy outcomes of PE (Fig. 3 B-C). Immunohistochemistry (IHC) and Western blot analyses confirmed that compared with the control group, the expression of DUSP5 in the RUPP group was significantly increased(Fig. 3 D-E). These findings were consistent with our in vitro results. The same conclusion was also obtained in human placental tissues(Fig. 3 F-G).In conclusion, this part of the in vivo experiment not only verified the successful construction of the RUPP model, but also correlated the expression changes of DUSP5 with the three core phenotypes of preeclampsia - gestational hypertension, placental insufficiency, and fetal growth restriction. These results provide key in vivo experimental evidence for elucidating the specific role of DUSP5 in the pathogenesis of preeclampsiaTherefore, the study proposes the mechanism depicted in (Fig. 3 H) . Discussion This study systematically explored the role and molecular mechanism of lncRNA MELTF-AS1 in preeclampsia (PE) pathogenesis, with three core discoveries: (1) Clinical correlation: MELTF-AS1 is significantly downregulated in placental tissues of PE patients, while its downstream target gene DUSP5 is notably upregulated; (2) In vitro functional validation: MELTF-AS1 promotes proliferation and invasion of HTR-8/SVneo trophoblast cells, whereas DUSP5 exerts the opposite inhibitory effect, and the two form a regulatory axis controlling trophoblast function; (3) Molecular mechanism: MELTF-AS1 does not directly bind to DUSP5, but instead interacts with EZH2 (the catalytic subunit of PRC2) to enhance EZH2 enrichment at the DUSP5 promoter region, increase H3K27me3 modification, and ultimately silence DUSP5 transcription; (4) In vivo verification: The RUPP mouse model (a classic PE-like model) recapitulates the "MELTF-AS1 downregulation/DUSP5 upregulation" pattern, and this expression imbalance is associated with PE core phenotypes—gestational hypertension, placental hypoplasia, and fetal growth restriction (FGR)—which is consistent with observations in human PE placentas. These results collectively confirm that the MELTF-AS1/EZH2/DUSP5 epigenetic regulatory axis is a key mediator of PE development. PE is fundamentally driven by placental dysfunction, and defective extravillous trophoblast (EVT) invasion—resulting in insufficient spiral artery remodeling and placental ischemia—is widely recognized as its initiating event [ 23 – 25 ] . Our findings link lncRNA-mediated epigenetic regulation to this core pathological process:First, trophoblast function regulation: CCK-8, colony formation, and Transwell assays demonstrated that MELTF-AS1 enhances trophoblast proliferation and invasion—two critical capabilities for successful placental implantation. Conversely, DUSP5 (a MAPK phosphatase previously reported to inhibit cell motility in cancers) suppresses these functions. In PE, reduced MELTF-AS1 expression weakens this "pro-function" signal, while elevated DUSP5 further amplifies trophoblast dysfunction, creating a "double negative" effect that impairs spiral artery remodeling and placental perfusion.Second, epigenetic mechanism innovation: Unlike many lncRNAs that directly bind target mRNAs to regulate translation,MELTF-AS1 adopts an EZH2-dependent epigenetic silencing strategy. RIP-qPCR confirmed the MELTF-AS1-EZH2 interaction, and CHIP-qPCR validated that this complex specifically targets the DUSP5 promoter to induce H3K27me3—a well-characterized repressive histone modification. This mechanism explains why MELTF-AS1 (detected in both nucleus and cytoplasm via nuclear-cytoplasmic separation) exerts transcriptional regulation: its nuclear localization enables collaboration with EZH2 to modulate chromatin state, while cytoplasmic localization may imply additional uncharacterized roles (e.g., competing with other RNAs), providing a basis for future functional exploration. Numerous studies have implicated lncRNAs in PE [ 26 , 27 ] . For example, lncRNA H19 promotes trophoblast invasion by sponging miR-675 [ 14 , 28 ] , and MALAT1 regulates trophoblast function via the PI3K/Akt pathway [ 13 ] . However, these studies primarily focus on "lncRNA-miRNA-mRNA" ceRNA networks or signaling pathway modulation, whereas our work identifies a novel lncRNA-protein-histone modification axis (MELTF-AS1/EZH2/H3K27me3/DUSP5) in PE. This expands the understanding of lncRNA-mediated epigenetic regulation in placental development and PE.Regarding DUSP5, prior research has focused on its role in cancer (e.g., inhibiting breast cancer cell proliferation) and cardiovascular diseases [ 18 , 29 ] , but its involvement in PE was unknown. Our study is the first to demonstrate that DUSP5 is a key downstream effector of MELTF-AS1, with its upregulation contributing to trophoblast dysfunction and PE phenotypes. This fills the gap in knowledge about DUSP5’s physiological function in pregnancy and pathological role in PE.In terms of animal models, the RUPP model is widely used to simulate PE due to its ability to replicate placental ischemia and gestational hypertension [ 30 ] . Our validation that RUPP mice exhibit "MELTF-AS1 down/DUSP5 up"—consistent with human PE placentas—strengthens the clinical relevance of the identified regulatory axis, supporting its potential as a conserved mechanism across species. This study identifies a novel MELTF-AS1/EZH2/DUSP5 epigenetic regulatory axis that controls trophoblast function and PE development. The findings not only deepen our understanding of PE’s molecular mechanisms but also provide potential diagnostic biomarkers and therapeutic targets for this life-threatening pregnancy complication. Addressing the current limitations and expanding on these discoveries will be critical for translating this basic research into clinical practice. Materials and methods Sample collection and Ethics statement This study was approved by the Nantong First People’s Hospital and was conducted in strict accordance with the Declaration of Helsinki. All study participants were duly informed, and human tissue samples were collected with their consent through signed informed consent forms. Inclusion and exclusion criteria for tissue collection were strictly adhered to as per the latest International Federation of Obstetricians guidelines. Plasmid construction. The plasmid and bacterial suspension were constructed by Ribobro (China). The bacterial suspension was added to 1 ml centrifuge tubes containing liquid LB medium (with AMP added) and incubated at 37°C on a shaking incubator for 12–16 hours. After 12 hours, 1 ml of the bacterial suspension was transferred from the centrifuge tube to a 50 ml culture bottle and incubated at 37°C on a shaking incubator for 12–16 hours. The Endo-free Plasmid Mini Kit II (Omage, D6950-02B) was used to extract the plasmid and use a nanodrop ultraviolet spectrophotometer to determine the DNA content and purity of the plasmid. The plasmid DNA was stored at -20°C. Construction of Reduced Uterine Perfusion Pressure mouse model At 12.5 days of mouse pregnancy, a surgery to reduce uterine perfusion pressure was performed and suturing was carried out, while the control group only performed the operation of opening the abdominal cavity and suturing for repositioning.After 18.5 days of gestation, the placenta and embryonic tissue were retrieved. Model and Sample Preparation of Mice RUPP mice (6–9 weeks old, weighing 180–200 g) were obtained from Animal Experiment Center of Nanjing Medical University (Nanjing, China) for all animal experiments. Animal experiments were approved by the Nantong First People’s Hospital Animal Experimentation Ethics Committee, and the experiments were conducted following the guidelines of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mice were categorized into respective groups and provided with nesting materials and sufficient food. After 18.5 days of gestation, pregnant mice were anesthetized using isoflurane and euthanized by intraperitoneal injection of an overdose of pentobarbital at a dose of 150 mg/kg of body weight.Then the placenta and embryonic tissue were retrieved. Cell culture HTR-8/SVneo cells were obtained from American Type Culture Collection (ATCC) for all cell experiments. The culture conditions for HTR-8/SVneo cells were: RPMI-1640 culture medium containing 10% FBS and 1% antibiotics, cultured in a humidified incubator at 37°C with 5% CO2 and 90% humidity. The complete culture medium was replaced every 2–3 days based on the cell growth condition, and the cells are passaged and used for subsequent experiments when the cell density is between 80% and 90%. Cell transfection After three passages, the cells were digested when the cell density was about 80%. The cells were counted and 2×10 5 cells/well were plated into 6-well plates for culture. When the cell density is between 80% and 90%, siRNA and pDNA were transfected with Lipofectamine 3000 and returned to the incubator after transfection. After 6–8 hours, the complete culture medium was replaced. The cells were collected 24–48 hours after transfection and the total RNA and total protein were extracted for qPCR or Western Blot to verify the transfection efficiency or for other experiments. Chromatin immunoprecipitation assay (ChIP-qPCR) The experiment used Merckmillipore EZ-Magna ChIP A/G (Merck, 17-10086). a. Formaldehyde treated cells, cross-linked fixation: target protein-DNA complex; b. Cell lysis and ultrasonic interruption: 200-1000BP chromatin; c. Antibody binds to the target protein-DNA complex: antibody-target protein-DNA complex; d. Protein A/G binding antibody-target protein-DNA complex: e. Precipitate the complex and wash it to remove non-specific binding proteins; f. Elution to obtain enriched target protein-DNA complex; g. Decrosslinking and purification of enriched DNA fragments; h. qPCR analysis was verified. The relative primer sequences are presented in File S1. RNA immunoprecipitation quantitative PCR (RIP-qPCR) Collect HTR-8/SVneo cells and lyse them with RIP lysis buffer containing RNase inhibitors to preserve RNA-protein complexes. Centrifuge to remove cell debris, retaining the supernatant (total lysate). Aliquot the lysate into tubes. Add specific antibody against the target protein (e.g., EZH2) to the experimental group, IgG antibody (negative control) to the control group, and set an "Input" group (untreated lysate, for normalization). Incubate overnight at 4°C with rotation.Add Protein A/G magnetic beads to each tube, incubate for 2h at 4°C to capture antibody-protein-RNA complexes. Wash the beads thoroughly with buffer to remove non-specifically bound components.Treat the captured complexes with DNase I to eliminate DNA contamination, then extract RNA using phenol-chloroform method. Reverse-transcribe the RNA into cDNA. Perform qPCR using primers specific to the target RNA (e.g., MELTF-AS1). Calculate the enrichment of target RNA in the experimental group relative to IgG control (normalized to Input) to verify RNA-protein interaction. RNA isolation and qPCR analyses Total RNA was extracted from placental tissues or cultured cells using FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme Biotech Co., Ltd; (Chian) RC112). RNA was reverse transcribed to cDNA in a 10 µL reaction volume using the HiScript II Q RT SuperMix (Vazyme Biotech Co., Ltd; (Chian) R222). Quantitative real-time PCR was performed using ChamQ SYBR qPCR Master Mix (High ROX Premixed) (Vazyme Biotech Co., Ltd(Chian); Q341) on a CFX 96 system. The expression of MELTF-AS1 was normalized to the housekeeping gene U6, and the expression of DUSP5 was normalized to the housekeeping gene GAPDH. The relative primer sequences are presented in File S1. Immunohistochemistry (IHC) Immunohistochemistry (IHC) was performed on paraffin sections using the EnVision G2 Double stain System (DAKO, K5361, DAB+/Permanent Red). Briefly, slides were dewaxed, rehydrated, and subjected to antigen retrieval. After blocking endogenous enzymes, primary antibodies (DUSP5, 1:50, Proteintech #30256-1-AP) or negative control IgG were applied. Subsequent steps included incubation with polymer/HRP, DAB solution, and secondary antibody(DD13,Xiamen Talent Biomedical Technology Co.,Ltd.) Slides were counterstained with hematoxylin, dehydrated, and mounted. IHC images were captured by using confocal microscopy (Leica SP5). Western Blot analyses For Western Blot analysis, cell samples or placental tissue samples (5–10 ug) were homogenized in 2x SDS-sample buffer. Samples were heated, further homogenized, and loaded onto 4%–15% gradient or 10% SDS gels. The intensity of protein bands was quantified using Quantity One software (Bio-Rad, Hercules, CA). Protein bands were quantified using ImageJ or Quantity One software (Tanon, China). GAPDH served as an internal control in the assays. The antibodies used in this experiment and the conditions of Western Blot are shown in Table S2. The uncropped Western Blot images are shown in File S3. CCK8 Assay Seed HTR-8/SVneo cells into 96-well plates at an appropriate density with 100µL culture medium per well, and set 3–5 replicate wells for each group. Include blank control wells (only culture medium without cells) to eliminate background interference. Incubate the plate at 37°C with 5% CO 2 until cells adhere (usually 24h), then perform specific treatments (e.g., transfection with MELTF-AS1 overexpression/knockdown vectors).After the treatment period, add 10µL CCK-8 reagent to each well (avoid generating bubbles), and incubate the plate for another 1–4 h at 37°C.Measure the absorbance (OD value) of each well at 450 nm using a microplate reader. The OD value is positively correlated with the number of viable cells, reflecting cell proliferation activity. Colony Formation Assay Cells were dispersed into a single-cell suspension at 48h after transfected. The colony-forming assay was carried out by incubating 1×10 3 cells at 37°C for two weeks in a culture dish containing 10% FBS medium. The cells were then stabilized, and dyed using 0.1% crystal violet, and the colonies counted manually. Experiments were repeated three times for all groups. Transwell assays Transwell chambers (Millipore, Darmstadt, Germany) used for cell invasion assays were first pretreated with 50 µL of a 1:9 Matrigel/DMEM solution (BD, New Jersey, USA). Subsequently, 1 × 10 5 cells were dispersed in DMEM without FBS (1 mL) and 200 µL of cellular solution was placed in the upper chamber. Following this, DMEM containing 10% FBS (600 µL) was placed into the lower chamber to act as a chemotactic agent. 48h later, residual cells in the upper chamber were scraped off, while invading cells were immobilized in 4% paraformaldehyde and then dyed in 2% crystal violet. The invading cells were counted by light microscopy (D-35578, Leica, Weztlar, Germany). RNA-seq analysis After total RNA was extracted from the sample, mRNA was enriched with magnetic beads with Oligo (dT). A fragmentation Buffer was added to the obtained mRNA to make the fragments into short fragments. The first cDNA strand was synthesized using random hexamers based on the fragment mRNA as a template. The second strand of cDNA was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I, which was purified and eluted with EB buffer solution through terminal repair, base A and sequencing joint, then recovered by agarose gel electrophoresis and amplified by PCR to complete the entire library preparation. The constructed library was sequenced by Illumina HiSeq2000. The experiment was commissioned by Sangon Biotech Co., Ltd.(China).The sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive under the accession number : [SRR36422258], [SRR36422257], [SRR36422255], [SRR36422256], [SRR36422254], [SRR36422253]. Nuclear-cytoplasmic separation assay Collect target cells (e.g., HTR-8/SVneo) in logarithmic growth phase, wash 2–3 times with pre-cooled PBS to remove culture medium residues, and centrifuge at 800×g for 5min at 4°C to obtain cell pellets.Resuspend cell pellets in ice-cold cytoplasmic lysis buffer (containing NP-40, RNase inhibitor, and protease inhibitor) to disrupt the cell membrane without damaging the nuclear envelope. Incubate on ice for 5–10 min, then centrifuge at 3,000×g for 10min at 4°C. Transfer the supernatant (cytoplasmic fraction) to a new pre-cooled tube and store on ice.Resuspend the remaining pellet (crude nuclei) in ice-cold nuclear wash buffer (with protease inhibitor) to remove residual cytoplasm. Centrifuge at 3,000×g for 5min at 4°C, discard the supernatant, and repeat washing 2times. Resuspend the purified nuclear pellet in nuclear lysis buffer (containing SDS or RIPA, RNase/protease inhibitors), incubate on ice for 15–20 min to lyse the nuclear membrane, and centrifuge at 12,000×g for 15min at 4°C. Collect the supernatant (nuclear fraction).Detect the expression of cytoplasmic marker and nuclear marker in both fractions by Western blot or qPCR to confirm effective separation. Statistical analysis Quantitative data are expressed as means ± standard error of the mean (SEM). All statistical analyses were performed using a two-tailed Student’s t-test or ANOVA. Correlation analyses between variables were performed using the Pearson rank correlation test. p < 0.05 was considered significant, and the level of significance was assigned as * if p < 0.05 and as ** if p ≤ 0.01, respectively. Declarations Author Contributions L.J., Y.S. and X.D. designed and conducted experiments, collected and analyzed the data, and wrote the original manuscript. L.J., Y.S. and M.L. carried out experiments and collected and analyzed the data. L.J., Y.S.,M.L., H.K. and X.K. agnelli, performed the experiments and collected the data. D.C. and L.C, provided knowledge insights and critical discussion on this project. All authors have made critical revisions to the important knowledge content in the manuscript and have read and approved the final version. Data Availability The sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive under the accession number: [SRR36422258], [SRR36422257], [SRR36422255], [SRR36422256], [SRR36422254], [SRR36422253]. All data generated or analysed during this study are included in this article. Statement This study has been reported in accordance with the ARRIVE 2.0 guidelines. All experimental protocols have been approved by [The Ethics Committee of Nantong First People's Hospital] (approval number: [2023KT158]) Additional information Declaration of interests The authors declare this study was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. References Say, L., et al., Global causes of maternal death: a WHO systematic analysis. Lancet Glob Health, 2014. 2 (6): p. e323-33. Fan, C., et al., TET1 modulates trophoblast function by regulation of ODC1 in preeclampsia. 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Curr Issues Mol Biol, 2025. 47 (7). Wang, H., et al., G protein-coupled estrogen receptor promotes human extravillous trophoblast invasion via YAP-Snail-mediated CYR61 expression. Cell Signal, 2025. 135 : p. 112033. Jiang, S., et al., Preeclampsia-Associated lncRNA INHBA-AS1 Regulates the Proliferation, Invasion, and Migration of Placental Trophoblast Cells. Mol Ther Nucleic Acids, 2020. 22 : p. 684-695. Song, X., et al., Dysregulation of LncRNAs in Placenta and Pathogenesis of Preeclampsia. Curr Drug Targets, 2017. 18 (10): p. 1165-1170. Ogoyama, M., et al., LncRNA H19-Derived miR-675-5p Accelerates the Invasion of Extravillous Trophoblast Cells by Inhibiting GATA2 and Subsequently Activating Matrix Metalloproteinases. Int J Mol Sci, 2021. 22 (3). Tang, C., et al., Impact of knockout of dual-specificity protein phosphatase 5 on structural and mechanical properties of rat middle cerebral arteries: implications for vascular aging. Geroscience, 2024. 46 (3): p. 3135-3147. van Kammen, C.M., et al., Reduced uterine perfusion pressure as a model for preeclampsia and fetal growth restriction in murine: a systematic review and meta-analysis. Am J Physiol Heart Circ Physiol, 2024. 327 (1): p. H89-h107. Additional Declarations No competing interests reported. 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10:16:13","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95535,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8076487/v1/89dff8358860c172adaf819f.html"},{"id":100773407,"identity":"5bf9173e-31fc-4cdb-9870-c957236fa6bf","added_by":"auto","created_at":"2026-01-21 10:16:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":466019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMELTF-AS1 promotes proliferation and invasion of trophoblast cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)We performed a re-analysis of one publicly available preeclampsia-associated lncRNA dataset (GSE183466). Differently expressed LncRNA was shown.\u003c/p\u003e\n\u003cp\u003e(B)Proliferation of HTR-8/SVneo cells following transfection with siRNAs (siMELTF-AS1) or a plasmid DNA expressing human MELTF-AS1(pMELTF-AS1), as determined using colony-forming assay.\u003c/p\u003e\n\u003cp\u003e(C)Proliferation of HTR-8/SVneo cells following transfection with siRNAs (siMELTF-AS1) or a plasmid DNA expressing human (MELTF-AS1), as determined using CCK8 assay.\u003c/p\u003e\n\u003cp\u003e(D)Invasion of HTR-8/SVneo cells following transfection with siMELTF-AS1 or pMELTF-AS1, as determined by transwell assay.\u003c/p\u003e\n\u003cp\u003e(E)Nucleoplasmic separation experiment.\u003c/p\u003e\n\u003cp\u003eError bars are mean with SEM of technical replicates (n = 3). *p \u0026lt; 0.05 and **p \u0026lt; 0.01; significance by Student’s t test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8076487/v1/ae81cd5670747f6398e70464.png"},{"id":100773581,"identity":"f9aab4de-3f4e-478c-bd1e-85e65db84ad7","added_by":"auto","created_at":"2026-01-21 10:19:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":423505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMELTF-AS1 Involves in the Pathogenesis of Preeclampsia by Epigenetic Regulation of DUSP5 Binding to EZH2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Transcriptome sequencing was performed to profile gene expression in HTR-8/SVneo cells MELTF-AS1-knockdown. Genes that are differentially expressed are shown.\u003c/p\u003e\n\u003cp\u003e(B and C)The GO analysis revealed that there was enrichment of biological pathways (BP), cellular compartments (CC) and molecular functions (MF) in all genes with altered expression from the HTR-8/SVneo cells in vitro.\u003c/p\u003e\n\u003cp\u003e(D) DUSP5 expression in HTR-8/SVneo cells transfected with a specific siRNAs (siMELTF-AS1) or a plasmid DNA expressing human MELTF-AS1 (pMELTF-AS1) as measured by qRT-PCR.\u003c/p\u003e\n\u003cp\u003e(E) Western Blot of MELTF-AS1 from HTR-8/SVneo cells transfected with a specific siRNAs (siMELTF-AS1) or a plasmid DNA expressing human MELTF-AS1 (pMELTF-AS1). Proteins were isolated at 48 h post-transfection.\u003c/p\u003e\n\u003cp\u003e(F) Proliferation of HTR-8/SVneo cells following transfection with siRNAs (siDUSP5) or a plasmid DNA expressing human DUSP5 (pDUSP5), as determined using colony-forming assay.\u003c/p\u003e\n\u003cp\u003e(G) Invasion of HTR-8/SVneo cells following transfection with siDUSP5 or pDUSP5, as determined by transwell assay.\u003c/p\u003e\n\u003cp\u003e(H) RIP-qPCR shows that MELTF-AS1 is highly bound to EZH2.\u003c/p\u003e\n\u003cp\u003e(I) LncRNA MELTF-AS1 participates in the epigenetic silencing of target genes by recruiting EZH2 and promoting its binding on chromatin, thereby catalyzing H3K27me3 modification.\u003c/p\u003e\n\u003cp\u003eError bars are mean with SEM of technical replicates (n = 3). *p \u0026lt; 0.05 and **p \u0026lt; 0.01; significance by Student’s t test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8076487/v1/5ed9b8d17df4fd1592139d29.png"},{"id":100773587,"identity":"d4253fec-b7c4-4418-88d4-f3e104ecd1dc","added_by":"auto","created_at":"2026-01-21 10:19:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":429528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAberrant DUSP5 expression in PE mice models and human placentas\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Differences in placenta and fetus among RUPP mice and control mice.\u003c/p\u003e\n\u003cp\u003e(B) The blood pressure of RUPP mice was higher than that of normal pregnant mice.\u003c/p\u003e\n\u003cp\u003e(C) Statistical analysis reveals significant differences in the weights of the placenta and fetus among RUPPmice and normal counterparts.\u003c/p\u003e\n\u003cp\u003e(D) The protein expression of DUSP5 was measured by Western Blot in RUPP mice and normal mice.\u003c/p\u003e\n\u003cp\u003e(E) Immunohistochemistry (IHC) analysis of placental specimens from RUPP mice and normal mice.\u003c/p\u003e\n\u003cp\u003e(F) The protein expression of DUSP5 was measured by Western Blot in PE placenta and normal placenta.\u003c/p\u003e\n\u003cp\u003e(G) Immunohistochemistry (IHC) analysis of placental specimens from PE placenta and normal placenta.\u003c/p\u003e\n\u003cp\u003e(H) Mechanism diagram\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8076487/v1/4d872cffd14bd5bd226cca1d.png"},{"id":100776343,"identity":"63700b2f-5135-4606-af71-cd48682aef7e","added_by":"auto","created_at":"2026-01-21 11:02:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2199948,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8076487/v1/f9b89854-f34c-4d03-9203-a0bcd9fea059.pdf"},{"id":100773133,"identity":"987e5668-9097-4502-9a54-701737c1a918","added_by":"auto","created_at":"2026-01-21 10:14:07","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":349907,"visible":true,"origin":"","legend":"","description":"","filename":"supplymentaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8076487/v1/cf283a65bda3826dedfa3273.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"MELTF-AS1 Regulates DUSP5 and Trophoblast Function via EZH2 to Inhibit Preeclampsia","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreeclampsia (PE), a pregnancy-specific syndrome characterized by hypertension and proteinuria after 20 weeks of gestation, remains a leading cause of maternal and perinatal morbidity and mortality worldwide\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Despite decades of research, the pathogenic mechanisms underlying PE remain incompletely understood, with growing evidence highlighting that impaired trophoblast cell function\u0026mdash;including inadequate proliferation, invasion, and differentiation\u0026mdash;plays a pivotal role in the development of PE\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Trophoblast cells are essential for placental implantation and spiral artery remodeling\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e; dysfunction of these cells leads to inadequate placental perfusion, triggering systemic inflammation and endothelial dysfunction, which are hallmarks of PE\u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.​\u003c/p\u003e \u003cp\u003eLong non-coding RNAs (lncRNAs), a class of non-protein-coding RNAs longer than 200 nucleotides, have emerged as key regulators of gene expression in various physiological and pathological processes, including placental development and PE. Accumulating studies have demonstrated that aberrant lncRNA expression is closely associated with trophoblast cell dysfunction in PE\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. For instance, lncRNA H19 and MALAT1 have been reported to modulate trophoblast proliferation and invasion by targeting specific signaling pathways, thereby participating in PE pathogenesis\u003csup\u003e[\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. However, the majority of lncRNAs involved in PE remain uncharacterized, and their precise regulatory mechanisms in trophoblast cell function and PE progression require further exploration.​\u003c/p\u003e \u003cp\u003eEnhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), mediates gene silencing through trimethylation of histone H3 at lysine 27 (H3K27me3)\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. EZH2 has been shown to regulate trophoblast cell proliferation, invasion, and apoptosis, and its dysregulation is implicated in PE development\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Dual-specificity phosphatase 5 (DUSP5), a member of the DUSP family that dephosphorylates mitogen-activated protein kinases (MAPKs), has been reported to inhibit cell proliferation and invasion in multiple cancers\u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. However, the expression pattern and functional role of DUSP5 in PE, as well as its upstream regulatory mechanisms, remain unclear.\u003c/p\u003e \u003cp\u003eMELTF-AS1 is a newly identified lncRNA whose biological function has not been fully elucidated\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Bioinformatic re-analysis of an NCBI lncRNA dataset (GSE183466) from preeclampsia placentas revealed that MELTF-AS1 was significantly down-regulated, suggesting its potential involvement in PE pathogenesis. Based on these findings, we hypothesized that MELTF-AS1 may regulate trophoblast cell function and PE development through interaction with EZH2 and subsequent modulation of DUSP5 expression.​\u003c/p\u003e \u003cp\u003eIn the present study, we aimed to: (1) confirm the differential expression of MELTF-AS1 in PE placental tissues; (2) investigate the effects of MELTF-AS1 and DUSP5 on trophoblast cell proliferation and invasion; (3) clarify the subcellular localization of MELTF-AS1 and its binding interaction with EZH2; (4) explore whether MELTF-AS1 regulates DUSP5 expression via EZH2-mediated H3K27me3 modification; and (5) verify the regulatory pathway in a mouse model of PE. The results of this study may provide novel insights into the molecular mechanisms of PE and identify potential therapeutic targets for this devastating disease.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMELTF-AS1 promotes proliferation and invasion of trophoblast cells\u003c/h2\u003e \u003cp\u003eEmerging evidence indicates that placental dysfunction is the cornerstone of the pathogenesis of PE, in which defective extravillous trophoblast (EVT) invasion leads to insufficient spiral artery remodeling and subsequent placental ischemia. Numerous studies have shown that aberrant lncRNA expression is closely associated with trophoblast dysfunction in PE. In order to explore the relationship between lncRNAs and PE, the research team obtained the lncRNA dataset of PE from NCBI (GSE183466) and conducted a re-analysis of bioinformatics to identify the relevant target long non-coding RNAs. The results revealed that compared with the control group, there were 68 downregulated lncRNAs and 76 upregulated lncRNAs in the PE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSpecifically, MELTF-AS1 was significantly downregulated in the PE group. Therefore, we modulated the expression of MELTF-AS1 in the HTR-8/SVneo cell line and verified the effect of MELTF-AS1 on trophoblast function using the CCK-8 assay, colony formation assay, and Transwell assay. These experiments demonstrated that MELTF-AS1 promoted the invasion and proliferation capabilities of trophoblast cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D).. Additionally, nuclear-cytoplasmic separation assay showed that MELTF-AS1, as a key lincRNA, is present in both the cytoplasm and nucleus(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMELTF-AS1 Involves in the Pathogenesis of Preeclampsia by Epigenetic Regulation of DUSP5 Binding to EZH2\u003c/h3\u003e\n\u003cp\u003eIn order to understand the molecular mechanism of MELTF-AS1 resistance to PE, we performed transcriptome sequencing on HTR-8/SVneo cells in MELTF-AS1 silencing and control groups. RNA sequencing analysis indicated that following the depletion of MELTF-AS1, the transcript levels of 147 genes were upregulated by at least 2-fold, while the expression levels of 107 mRNAs were downregulated by 2-fold or more(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).MELTF-AS1-related signaling pathways predicted by the GO database suggest changes in cell migration and invasion in trophoblast cells where MELTF-AS1 is up/down regulated(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Among the enriched genes, there are well-known genes related to proliferation and invasion, such as PTEN, p53, CXCR4, etc (data not shown). Compared with other genes, the change of DUSP5 was the most significant after changing the expression of MELTF-AS1.After down-regulating MELTF-AS1, the expression of DUSP5 increased; after up-regulating MELTF-AS1, the expression of DUSP5 decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSo, we speculate that MELTF-AS1 affects trophoblast function by regulating DUSP5. Consequently, we conducted CCK8, Transwell and Colony Formation assays using the HTR-8/SVneo cell lines, respectively. These experiments indicated that DUSP5 may also impede trophoblast cell invasion and proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G).\u003c/p\u003e \u003cp\u003eTo verify whether MELTF-AS1 and DUSP5 can bind directly to exert their effects, we performed RIP-qPCR. The results showed that MELTF-AS1 and DUSP5 could not bind directly. Subsequently, through literature review, we found that MELTF-AS1 can increase the level of H3K27me3 modification by binding to EZH2, thereby affecting the transcription of downstream factors. Therefore, using RIP-qPCR and CHIP-qPCR assays, we demonstrated that MELTF-AS1 can bind to EZH2, which in turn promotes the enrichment of EZH2 in the promoter region of DUSP5 and increases the level of H3K27me3 modification, ultimately resulting in the transcriptional silencing of DUSP5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I).\u003c/p\u003e\n\u003ch3\u003eAberrant DUSP5 expression in PE mice models and human placentas\u003c/h3\u003e\n\u003cp\u003eTo verify the key role of the MELTF-AS1/DUSP5 axis in the pathological process of PE in vivo, we successfully established a mouse model with reduced uterine perfusion pressure (RUPP) via surgery, which is a classic in vivo model for PE research (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). First, we confirmed that the RUPP model successfully simulated the maternal cardiovascular symptoms of PE. We continuously monitored the systolic blood pressure (SBP) of pregnant mice. The results showed that compared with the control group, mice in the RUPP group exhibited a significant increase in SBP starting from mid-gestation (around embryonic day 14, E14), which persisted until late gestation and met the diagnostic criteria for hypertension. This data clearly indicated that the RUPP surgery successfully induced gestational hypertension\u0026mdash;a core clinical feature of PE.Second, we evaluated the impact of the RUPP model on pregnancy outcomes and observed typical placental and fetal developmental abnormalities. We weighed the placentas and embryos of mice in late gestation. Statistical analysis revealed that both placental weight and embryonic weight in the RUPP group were significantly lower than those in the control group. Reduced placental weight directly reflects placental hypoplasia and dysfunction, while the significant decrease in embryonic weight serves as direct evidence of fetal growth restriction (FGR) caused by placental insufficiency. Together, these two indicators confirmed that the RUPP model can effectively recapitulate the adverse pregnancy outcomes of PE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunohistochemistry (IHC) and Western blot analyses confirmed that compared with the control group, the expression of DUSP5 in the RUPP group was significantly increased(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). These findings were consistent with our in vitro results. The same conclusion was also obtained in human placental tissues(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G).In conclusion, this part of the in vivo experiment not only verified the successful construction of the RUPP model, but also correlated the expression changes of DUSP5 with the three core phenotypes of preeclampsia - gestational hypertension, placental insufficiency, and fetal growth restriction. These results provide key in vivo experimental evidence for elucidating the specific role of DUSP5 in the pathogenesis of preeclampsiaTherefore, the study proposes the mechanism depicted in (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) .\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study systematically explored the role and molecular mechanism of lncRNA MELTF-AS1 in preeclampsia (PE) pathogenesis, with three core discoveries: (1) Clinical correlation: MELTF-AS1 is significantly downregulated in placental tissues of PE patients, while its downstream target gene DUSP5 is notably upregulated; (2) In vitro functional validation: MELTF-AS1 promotes proliferation and invasion of HTR-8/SVneo trophoblast cells, whereas DUSP5 exerts the opposite inhibitory effect, and the two form a regulatory axis controlling trophoblast function; (3) Molecular mechanism: MELTF-AS1 does not directly bind to DUSP5, but instead interacts with EZH2 (the catalytic subunit of PRC2) to enhance EZH2 enrichment at the DUSP5 promoter region, increase H3K27me3 modification, and ultimately silence DUSP5 transcription; (4) In vivo verification: The RUPP mouse model (a classic PE-like model) recapitulates the \"MELTF-AS1 downregulation/DUSP5 upregulation\" pattern, and this expression imbalance is associated with PE core phenotypes\u0026mdash;gestational hypertension, placental hypoplasia, and fetal growth restriction (FGR)\u0026mdash;which is consistent with observations in human PE placentas. These results collectively confirm that the MELTF-AS1/EZH2/DUSP5 epigenetic regulatory axis is a key mediator of PE development.\u003c/p\u003e \u003cp\u003ePE is fundamentally driven by placental dysfunction, and defective extravillous trophoblast (EVT) invasion\u0026mdash;resulting in insufficient spiral artery remodeling and placental ischemia\u0026mdash;is widely recognized as its initiating event\u003csup\u003e[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Our findings link lncRNA-mediated epigenetic regulation to this core pathological process:First, trophoblast function regulation: CCK-8, colony formation, and Transwell assays demonstrated that MELTF-AS1 enhances trophoblast proliferation and invasion\u0026mdash;two critical capabilities for successful placental implantation. Conversely, DUSP5 (a MAPK phosphatase previously reported to inhibit cell motility in cancers) suppresses these functions. In PE, reduced MELTF-AS1 expression weakens this \"pro-function\" signal, while elevated DUSP5 further amplifies trophoblast dysfunction, creating a \"double negative\" effect that impairs spiral artery remodeling and placental perfusion.Second, epigenetic mechanism innovation: Unlike many lncRNAs that directly bind target mRNAs to regulate translation,MELTF-AS1 adopts an EZH2-dependent epigenetic silencing strategy. RIP-qPCR confirmed the MELTF-AS1-EZH2 interaction, and CHIP-qPCR validated that this complex specifically targets the DUSP5 promoter to induce H3K27me3\u0026mdash;a well-characterized repressive histone modification. This mechanism explains why MELTF-AS1 (detected in both nucleus and cytoplasm via nuclear-cytoplasmic separation) exerts transcriptional regulation: its nuclear localization enables collaboration with EZH2 to modulate chromatin state, while cytoplasmic localization may imply additional uncharacterized roles (e.g., competing with other RNAs), providing a basis for future functional exploration.\u003c/p\u003e \u003cp\u003eNumerous studies have implicated lncRNAs in PE\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. For example, lncRNA H19 promotes trophoblast invasion by sponging miR-675\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, and MALAT1 regulates trophoblast function via the PI3K/Akt pathway\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, these studies primarily focus on \"lncRNA-miRNA-mRNA\" ceRNA networks or signaling pathway modulation, whereas our work identifies a novel lncRNA-protein-histone modification axis (MELTF-AS1/EZH2/H3K27me3/DUSP5) in PE. This expands the understanding of lncRNA-mediated epigenetic regulation in placental development and PE.Regarding DUSP5, prior research has focused on its role in cancer (e.g., inhibiting breast cancer cell proliferation) and cardiovascular diseases\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, but its involvement in PE was unknown. Our study is the first to demonstrate that DUSP5 is a key downstream effector of MELTF-AS1, with its upregulation contributing to trophoblast dysfunction and PE phenotypes. This fills the gap in knowledge about DUSP5\u0026rsquo;s physiological function in pregnancy and pathological role in PE.In terms of animal models, the RUPP model is widely used to simulate PE due to its ability to replicate placental ischemia and gestational hypertension\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Our validation that RUPP mice exhibit \"MELTF-AS1 down/DUSP5 up\"\u0026mdash;consistent with human PE placentas\u0026mdash;strengthens the clinical relevance of the identified regulatory axis, supporting its potential as a conserved mechanism across species.\u003c/p\u003e \u003cp\u003eThis study identifies a novel MELTF-AS1/EZH2/DUSP5 epigenetic regulatory axis that controls trophoblast function and PE development. The findings not only deepen our understanding of PE\u0026rsquo;s molecular mechanisms but also provide potential diagnostic biomarkers and therapeutic targets for this life-threatening pregnancy complication. Addressing the current limitations and expanding on these discoveries will be critical for translating this basic research into clinical practice.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and Ethics statement\u003c/h2\u003e \u003cp\u003e This study was approved by the Nantong First People\u0026rsquo;s Hospital and was conducted in strict accordance with the Declaration of Helsinki. All study participants were duly informed, and human tissue samples were collected with their consent through signed informed consent forms. Inclusion and exclusion criteria for tissue collection were strictly adhered to as per the latest International Federation of Obstetricians guidelines.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmid construction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe plasmid and bacterial suspension were constructed by Ribobro (China). The bacterial suspension was added to 1 ml centrifuge tubes containing liquid LB medium (with AMP added) and incubated at 37\u0026deg;C on a shaking incubator for 12\u0026ndash;16 hours. After 12 hours, 1 ml of the bacterial suspension was transferred from the centrifuge tube to a 50 ml culture bottle and incubated at 37\u0026deg;C on a shaking incubator for 12\u0026ndash;16 hours. The Endo-free Plasmid Mini Kit II (Omage, D6950-02B) was used to extract the plasmid and use a nanodrop ultraviolet spectrophotometer to determine the DNA content and purity of the plasmid. The plasmid DNA was stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConstruction of Reduced Uterine Perfusion Pressure mouse model\u003c/h3\u003e\n\u003cp\u003eAt 12.5 days of mouse pregnancy, a surgery to reduce uterine perfusion pressure was performed and suturing was carried out, while the control group only performed the operation of opening the abdominal cavity and suturing for repositioning.After 18.5 days of gestation, the placenta and embryonic tissue were retrieved.\u003c/p\u003e\n\u003ch3\u003eModel and Sample Preparation of Mice\u003c/h3\u003e\n\u003cp\u003eRUPP mice (6\u0026ndash;9 weeks old, weighing 180\u0026ndash;200 g) were obtained from Animal Experiment Center of Nanjing Medical University (Nanjing, China) for all animal experiments. Animal experiments were approved by the Nantong First People\u0026rsquo;s Hospital Animal Experimentation Ethics Committee, and the experiments were conducted following the guidelines of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mice were categorized into respective groups and provided with nesting materials and sufficient food. After 18.5 days of gestation, pregnant mice were anesthetized using isoflurane and euthanized by intraperitoneal injection of an overdose of pentobarbital at a dose of 150 mg/kg of body weight.Then the placenta and embryonic tissue were retrieved.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHTR-8/SVneo cells were obtained from American Type Culture Collection (ATCC) for all cell experiments. The culture conditions for HTR-8/SVneo cells were: RPMI-1640 culture medium containing 10% FBS and 1% antibiotics, cultured in a humidified incubator at 37\u0026deg;C with 5% CO2 and 90% humidity. The complete culture medium was replaced every 2\u0026ndash;3 days based on the cell growth condition, and the cells are passaged and used for subsequent experiments when the cell density is between 80% and 90%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003eAfter three passages, the cells were digested when the cell density was about 80%. The cells were counted and 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well were plated into 6-well plates for culture. When the cell density is between 80% and 90%, siRNA and pDNA were transfected with Lipofectamine 3000 and returned to the incubator after transfection. After 6\u0026ndash;8 hours, the complete culture medium was replaced. The cells were collected 24\u0026ndash;48 hours after transfection and the total RNA and total protein were extracted for qPCR or Western Blot to verify the transfection efficiency or for other experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation assay (ChIP-qPCR)\u003c/h2\u003e \u003cp\u003eThe experiment used Merckmillipore EZ-Magna ChIP A/G (Merck, 17-10086). a. Formaldehyde treated cells, cross-linked fixation: target protein-DNA complex; b. Cell lysis and ultrasonic interruption: 200-1000BP chromatin; c. Antibody binds to the target protein-DNA complex: antibody-target protein-DNA complex; d. Protein A/G binding antibody-target protein-DNA complex: e. Precipitate the complex and wash it to remove non-specific binding proteins; f. Elution to obtain enriched target protein-DNA complex; g. Decrosslinking and purification of enriched DNA fragments; h. qPCR analysis was verified. The relative primer sequences are presented in File S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation quantitative PCR (RIP-qPCR)\u003c/h2\u003e \u003cp\u003eCollect HTR-8/SVneo cells and lyse them with RIP lysis buffer containing RNase inhibitors to preserve RNA-protein complexes. Centrifuge to remove cell debris, retaining the supernatant (total lysate). Aliquot the lysate into tubes. Add specific antibody against the target protein (e.g., EZH2) to the experimental group, IgG antibody (negative control) to the control group, and set an \"Input\" group (untreated lysate, for normalization). Incubate overnight at 4\u0026deg;C with rotation.Add Protein A/G magnetic beads to each tube, incubate for 2h at 4\u0026deg;C to capture antibody-protein-RNA complexes. Wash the beads thoroughly with buffer to remove non-specifically bound components.Treat the captured complexes with DNase I to eliminate DNA contamination, then extract RNA using phenol-chloroform method. Reverse-transcribe the RNA into cDNA. Perform qPCR using primers specific to the target RNA (e.g., MELTF-AS1). Calculate the enrichment of target RNA in the experimental group relative to IgG control (normalized to Input) to verify RNA-protein interaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and qPCR analyses\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from placental tissues or cultured cells using FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme Biotech Co., Ltd; (Chian) RC112). RNA was reverse transcribed to cDNA in a 10 \u0026micro;L reaction volume using the HiScript II Q RT SuperMix (Vazyme Biotech Co., Ltd; (Chian) R222). Quantitative real-time PCR was performed using ChamQ SYBR qPCR Master Mix (High ROX Premixed) (Vazyme Biotech Co., Ltd(Chian); Q341) on a CFX 96 system. The expression of MELTF-AS1 was normalized to the housekeeping gene U6, and the expression of DUSP5 was normalized to the housekeeping gene GAPDH. The relative primer sequences are presented in File S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eImmunohistochemistry (IHC) was performed on paraffin sections using the EnVision G2 Double stain System (DAKO, K5361, DAB+/Permanent Red). Briefly, slides were dewaxed, rehydrated, and subjected to antigen retrieval. After blocking endogenous enzymes, primary antibodies (DUSP5, 1:50, Proteintech #30256-1-AP) or negative control IgG were applied. Subsequent steps included incubation with polymer/HRP, DAB solution, and secondary antibody(DD13,Xiamen Talent Biomedical Technology Co.,Ltd.) Slides were counterstained with hematoxylin, dehydrated, and mounted. IHC images were captured by using confocal microscopy (Leica SP5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot analyses\u003c/h2\u003e \u003cp\u003eFor Western Blot analysis, cell samples or placental tissue samples (5\u0026ndash;10 ug) were homogenized in 2x SDS-sample buffer. Samples were heated, further homogenized, and loaded onto 4%\u0026ndash;15% gradient or 10% SDS gels. The intensity of protein bands was quantified using Quantity One software (Bio-Rad, Hercules, CA). Protein bands were quantified using ImageJ or Quantity One software (Tanon, China). GAPDH served as an internal control in the assays. The antibodies used in this experiment and the conditions of Western Blot are shown in Table S2. The uncropped Western Blot images are shown in File S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCCK8 Assay\u003c/h2\u003e \u003cp\u003eSeed HTR-8/SVneo cells into 96-well plates at an appropriate density with 100\u0026micro;L culture medium per well, and set 3\u0026ndash;5 replicate wells for each group. Include blank control wells (only culture medium without cells) to eliminate background interference.\u003c/p\u003e \u003cp\u003eIncubate the plate at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e until cells adhere (usually 24h), then perform specific treatments (e.g., transfection with MELTF-AS1 overexpression/knockdown vectors).After the treatment period, add 10\u0026micro;L CCK-8 reagent to each well (avoid generating bubbles), and incubate the plate for another 1\u0026ndash;4 h at 37\u0026deg;C.Measure the absorbance (OD value) of each well at 450 nm using a microplate reader. The OD value is positively correlated with the number of viable cells, reflecting cell proliferation activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eColony Formation Assay\u003c/h2\u003e \u003cp\u003eCells were dispersed into a single-cell suspension at 48h after transfected. The colony-forming assay was carried out by incubating 1\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells at 37\u0026deg;C for two weeks in a culture dish containing 10% FBS medium. The cells were then stabilized, and dyed using 0.1% crystal violet, and the colonies counted manually. Experiments were repeated three times for all groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assays\u003c/h2\u003e \u003cp\u003eTranswell chambers (Millipore, Darmstadt, Germany) used for cell invasion assays were first pretreated with 50 \u0026micro;L of a 1:9 Matrigel/DMEM solution (BD, New Jersey, USA). Subsequently, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were dispersed in DMEM without FBS (1 mL) and 200 \u0026micro;L of cellular solution was placed in the upper chamber. Following this, DMEM containing 10% FBS (600 \u0026micro;L) was placed into the lower chamber to act as a chemotactic agent. 48h later, residual cells in the upper chamber were scraped off, while invading cells were immobilized in 4% paraformaldehyde and then dyed in 2% crystal violet. The invading cells were counted by light microscopy (D-35578, Leica, Weztlar, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eAfter total RNA was extracted from the sample, mRNA was enriched with magnetic beads with Oligo (dT). A fragmentation Buffer was added to the obtained mRNA to make the fragments into short fragments. The first cDNA strand was synthesized using random hexamers based on the fragment mRNA as a template. The second strand of cDNA was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I, which was purified and eluted with EB buffer solution through terminal repair, base A and sequencing joint, then recovered by agarose gel electrophoresis and amplified by PCR to complete the entire library preparation. The constructed library was sequenced by Illumina HiSeq2000. The experiment was commissioned by Sangon Biotech Co., Ltd.(China).The sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive under the accession number : [SRR36422258], [SRR36422257], [SRR36422255], [SRR36422256], [SRR36422254], [SRR36422253].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eNuclear-cytoplasmic separation assay\u003c/h2\u003e \u003cp\u003eCollect target cells (e.g., HTR-8/SVneo) in logarithmic growth phase, wash 2\u0026ndash;3 times with pre-cooled PBS to remove culture medium residues, and centrifuge at 800\u0026times;g for 5min at 4\u0026deg;C to obtain cell pellets.Resuspend cell pellets in ice-cold cytoplasmic lysis buffer (containing NP-40, RNase inhibitor, and protease inhibitor) to disrupt the cell membrane without damaging the nuclear envelope. Incubate on ice for 5\u0026ndash;10 min, then centrifuge at 3,000\u0026times;g for 10min at 4\u0026deg;C. Transfer the supernatant (cytoplasmic fraction) to a new pre-cooled tube and store on ice.Resuspend the remaining pellet (crude nuclei) in ice-cold nuclear wash buffer (with protease inhibitor) to remove residual cytoplasm. Centrifuge at 3,000\u0026times;g for 5min at 4\u0026deg;C, discard the supernatant, and repeat washing 2times. Resuspend the purified nuclear pellet in nuclear lysis buffer (containing SDS or RIPA, RNase/protease inhibitors), incubate on ice for 15\u0026ndash;20 min to lyse the nuclear membrane, and centrifuge at 12,000\u0026times;g for 15min at 4\u0026deg;C. Collect the supernatant (nuclear fraction).Detect the expression of cytoplasmic marker and nuclear marker in both fractions by Western blot or qPCR to confirm effective separation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eQuantitative data are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). All statistical analyses were performed using a two-tailed Student\u0026rsquo;s t-test or ANOVA. Correlation analyses between variables were performed using the Pearson rank correlation test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant, and the level of significance was assigned as * if p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and as ** if p\u0026thinsp;\u0026le;\u0026thinsp;0.01, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.J., Y.S. and X.D. designed and conducted experiments, collected and analyzed the data, and wrote the original manuscript. L.J., Y.S. and M.L. carried out experiments and collected and analyzed the data. L.J., Y.S.,M.L., H.K. and X.K. agnelli, performed the experiments and collected the data. D.C. and L.C, provided knowledge insights and critical discussion on this project. All authors have made critical revisions to the important knowledge content in the manuscript and have read and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive under the accession number: [SRR36422258], [SRR36422257], [SRR36422255], [SRR36422256], [SRR36422254], [SRR36422253].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been reported in accordance with the ARRIVE 2.0 guidelines. All experimental protocols have been approved by [The Ethics Committee of Nantong First People\u0026apos;s Hospital] (approval number: [2023KT158])\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAdditional information\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare this study was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSay, L., et al., \u003cem\u003eGlobal causes of maternal death: a WHO systematic analysis.\u003c/em\u003e Lancet Glob Health, 2014. \u003cstrong\u003e2\u003c/strong\u003e(6): p. e323-33.\u003c/li\u003e\n\u003cli\u003eFan, C., et al., \u003cem\u003eTET1 modulates trophoblast function by regulation of ODC1 in preeclampsia.\u003c/em\u003e Sci Rep, 2025. \u003cstrong\u003e15\u003c/strong\u003e(1): p. 38208.\u003c/li\u003e\n\u003cli\u003eGuo, J., et al., \u003cem\u003eTIGAR deficiency exacerbated preeclampsia by impairing autophagy and accelerating trophoblasts apoptosis upon excessive oxidative stress via the NRF2-ARE pathway.\u003c/em\u003e Life Sci, 2025: p. 124062.\u003c/li\u003e\n\u003cli\u003eTan, B., et al., \u003cem\u003eEndothelial progenitor cells control remodeling of uterine spiral arteries for the establishment of utero-placental circulation.\u003c/em\u003e Dev Cell, 2024. \u003cstrong\u003e59\u003c/strong\u003e(14): p. 1842-1859.e12.\u003c/li\u003e\n\u003cli\u003eVarberg, K.M. and M.J. Soares, \u003cem\u003eParadigms for investigating invasive trophoblast cell development and contributions to uterine spiral artery remodeling.\u003c/em\u003e Placenta, 2021. \u003cstrong\u003e113\u003c/strong\u003e: p. 48-56.\u003c/li\u003e\n\u003cli\u003eBakrania, B.A., et al., \u003cem\u003ePreeclampsia: Linking Placental Ischemia with Maternal Endothelial and Vascular Dysfunction.\u003c/em\u003e Compr Physiol, 2020. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 1315-1349.\u003c/li\u003e\n\u003cli\u003eHu, H., et al., \u003cem\u003eNeutrophil extracellular traps induce trophoblasts pyroptosis via enhancing NLRP3 lactylation in SLE pregnancies.\u003c/em\u003e J Autoimmun, 2025. \u003cstrong\u003e153\u003c/strong\u003e: p. 103411.\u003c/li\u003e\n\u003cli\u003eCerillo, A., et al., \u003cem\u003eThe Role of lncRNAs in Complicated Pregnancy: A Systematic Review.\u003c/em\u003e Genes (Basel), 2025. \u003cstrong\u003e16\u003c/strong\u003e(8).\u003c/li\u003e\n\u003cli\u003eZhang, J., et al., \u003cem\u003eLncRNA-ATB Contributes to Severe Preeclampsia by Modulating the p53/MDM2 Pathway via PABPC1.\u003c/em\u003e Faseb j, 2025. \u003cstrong\u003e39\u003c/strong\u003e(12): p. e70736.\u003c/li\u003e\n\u003cli\u003eChen, F., et al., \u003cem\u003eMesenchymal Stem Cell-Derived Exosomal Long Noncoding RNA MALAT1-201 Regulated the Proliferation, Apoptosis and Migration of Trophoblast Cells via Targeting miR-141.\u003c/em\u003e Ann Clin Lab Sci, 2022. \u003cstrong\u003e52\u003c/strong\u003e(5): p. 741-752.\u003c/li\u003e\n\u003cli\u003eLi, Q., et al., \u003cem\u003eMALAT1 modulates trophoblast phenotype via miR-101-3p/VEGFA axis.\u003c/em\u003e Arch Biochem Biophys, 2023. \u003cstrong\u003e744\u003c/strong\u003e: p. 109692.\u003c/li\u003e\n\u003cli\u003eLi, Q., et al., \u003cem\u003eLncRNA MALAT1 affects the migration and invasion of trophoblast cells by regulating FOS expression in early-onset preeclampsia.\u003c/em\u003e Pregnancy Hypertens, 2020. \u003cstrong\u003e21\u003c/strong\u003e: p. 50-57.\u003c/li\u003e\n\u003cli\u003eWu, H.Y., et al., \u003cem\u003eLncRNA MALAT1 regulates trophoblast cells migration and invasion via miR-206/IGF-1 axis.\u003c/em\u003e Cell Cycle, 2020. \u003cstrong\u003e19\u003c/strong\u003e(1): p. 39-52.\u003c/li\u003e\n\u003cli\u003eGao, W.L., et al., \u003cem\u003eThe imprinted H19 gene regulates human placental trophoblast cell proliferation via encoding miR-675 that targets Nodal Modulator 1 (NOMO1).\u003c/em\u003e RNA Biol, 2012. \u003cstrong\u003e9\u003c/strong\u003e(7): p. 1002-10.\u003c/li\u003e\n\u003cli\u003eYuan, S., et al., \u003cem\u003eA PHF19-YTHDC1 condensate switches EZH2-mediated gene suppression to activation for prostate cancer progression.\u003c/em\u003e Proc Natl Acad Sci U S A, 2025. \u003cstrong\u003e122\u003c/strong\u003e(43): p. e2510386122.\u003c/li\u003e\n\u003cli\u003eZhou, X., et al., \u003cem\u003eMiR-26a-5p/EZH2 Mediates Wnt2 Promoter Methylation to Regulate Trophoblast Dysfunction.\u003c/em\u003e Comb Chem High Throughput Screen, 2025.\u003c/li\u003e\n\u003cli\u003eGan, X., et al., \u003cem\u003eAngiopoietin-2 regulates the phenotypic switch of vascular smooth muscle cells.\u003c/em\u003e Faseb j, 2025. \u003cstrong\u003e39\u003c/strong\u003e(5): p. e70434.\u003c/li\u003e\n\u003cli\u003eReddi, K.K., et al., \u003cem\u003eASAH1 facilitates TNBC by DUSP5 suppression-driven activation of MAP kinase pathway and represents a therapeutic vulnerability.\u003c/em\u003e Cell Death Dis, 2024. \u003cstrong\u003e15\u003c/strong\u003e(6): p. 452.\u003c/li\u003e\n\u003cli\u003eXie, Z., et al., \u003cem\u003eBAP1-mediated MAFF deubiquitylation regulates tumor growth and is associated with adverse outcomes in colorectal cancer.\u003c/em\u003e Eur J Cancer, 2024. \u003cstrong\u003e210\u003c/strong\u003e: p. 114278.\u003c/li\u003e\n\u003cli\u003eRushworth, L.K., et al., \u003cem\u003eDual-specificity phosphatase 5 regulates nuclear ERK activity and suppresses skin 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\u003cstrong\u003e14\u003c/strong\u003e: p. 1060253.\u003c/li\u003e\n\u003cli\u003eMeng, S., et al., \u003cem\u003eIGFBP2 Modulates Trophoblast Function and Epithelial-Mesenchymal Transition in Preeclampsia via the PI3K/AKT Signaling Pathway.\u003c/em\u003e Curr Issues Mol Biol, 2025. \u003cstrong\u003e47\u003c/strong\u003e(7).\u003c/li\u003e\n\u003cli\u003eWang, H., et al., \u003cem\u003eG protein-coupled estrogen receptor promotes human extravillous trophoblast invasion via YAP-Snail-mediated CYR61 expression.\u003c/em\u003e Cell Signal, 2025. \u003cstrong\u003e135\u003c/strong\u003e: p. 112033.\u003c/li\u003e\n\u003cli\u003eJiang, S., et al., \u003cem\u003ePreeclampsia-Associated lncRNA INHBA-AS1 Regulates the Proliferation, Invasion, and Migration of Placental Trophoblast Cells.\u003c/em\u003e Mol Ther Nucleic Acids, 2020. \u003cstrong\u003e22\u003c/strong\u003e: p. 684-695.\u003c/li\u003e\n\u003cli\u003eSong, X., et al., \u003cem\u003eDysregulation of LncRNAs in Placenta and Pathogenesis of Preeclampsia.\u003c/em\u003e Curr Drug Targets, 2017. \u003cstrong\u003e18\u003c/strong\u003e(10): p. 1165-1170.\u003c/li\u003e\n\u003cli\u003eOgoyama, M., et al., \u003cem\u003eLncRNA H19-Derived miR-675-5p Accelerates the Invasion of Extravillous Trophoblast Cells by Inhibiting GATA2 and Subsequently Activating Matrix Metalloproteinases.\u003c/em\u003e Int J Mol Sci, 2021. \u003cstrong\u003e22\u003c/strong\u003e(3).\u003c/li\u003e\n\u003cli\u003eTang, C., et al., \u003cem\u003eImpact of knockout of dual-specificity protein phosphatase 5 on structural and mechanical properties of rat middle cerebral arteries: implications for vascular aging.\u003c/em\u003e Geroscience, 2024. \u003cstrong\u003e46\u003c/strong\u003e(3): p. 3135-3147.\u003c/li\u003e\n\u003cli\u003evan Kammen, C.M., et al., \u003cem\u003eReduced uterine perfusion pressure as a model for preeclampsia and fetal growth restriction in murine: a systematic review and meta-analysis.\u003c/em\u003e Am J Physiol Heart Circ Physiol, 2024. \u003cstrong\u003e327\u003c/strong\u003e(1): p. H89-h107.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Preeclampsia, MELTF-AS1, EZH2, DUSP5, trophoblast cell function","lastPublishedDoi":"10.21203/rs.3.rs-8076487/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8076487/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eTo explore the mechanism by which lncRNA MELTF-AS1 binds EZH2 to downregulate target gene DUSP5, thereby promoting trophoblast function and inhibiting the occurrence of preeclampsia (PE).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eBioinformatic re-analysis of dataset GSE183466 identified the lncRNA MELTF-AS1 as a key dysregulated transcript in preeclampsia. Transcriptome sequencing after MELTF-AS1 knockdown identified DUSP5. CCK-8, colony formation, and Transwell assays evaluated effects on HTR-8/SVneo cell proliferation/invasion. Nuclear-cytoplasmic separation localizedthe action site of MELTF-AS1. RIP-qPCR/CHIP-qPCR clarified binding mechanisms of MELTF-AS1/DUSP5, with reduced uterine perfusion pressure mice verifying conclusions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIn this study, Bioinformatic analysis identified that MELTF-AS1 was significantly downregulated in the PE group. Transcriptome sequencing after MELTF-AS1 knockdown showed that DUSP5 was significantly upregulated. \u0026nbsp;MELTF-AS1 enhanced cell proliferation/invasion, while DUSP5 inhibited them. \u0026nbsp;Nuclear-cytoplasmic separation assay revealed that MELTF-AS1 was expressed in both the nucleus and cytoplasm. Further RIP-qPCR and CHIP-qPCR showed that MELTF-AS1 could bind to EZH2, promoting the enrichment of EZH2 at the DUSP5 promoter region and increasing the level of H3K27me3 modification, thereby reducing DUSP5 transcriptional level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThis study reveals the regulatory mechanism mediated by MELTF-AS1, and the MELTF-AS1/DUSP5 regulatory pathway may provide new predictive and therapeutic intervention strategies for PE.\u003c/p\u003e","manuscriptTitle":"MELTF-AS1 Regulates DUSP5 and Trophoblast Function via EZH2 to Inhibit Preeclampsia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 09:15:26","doi":"10.21203/rs.3.rs-8076487/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-02T13:57:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-10T09:48:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-23T16:09:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292881545914767641917773533806013480811","date":"2026-01-22T08:11:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218833762451306482108327060678068549604","date":"2026-01-19T04:39:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-19T04:07:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T03:59:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-18T09:13:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-13T10:19:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-13T10:13:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f736981-465a-4ebc-9fd4-266230c55f4f","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61357240,"name":"Biological sciences/Cell biology"},{"id":61357241,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-05-15T17:38:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 09:15:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8076487","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8076487","identity":"rs-8076487","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}