SIRT7 drives energy metabolic shifts in endometriosis via interaction with TUFM and Rhoa/Rock/Akt pathway activation

Purpose Endometriosis is characterized by the ectopic growth of endometrial-like tissue outside the uterus and altered energy metabolism, but the specific mechanisms involved remain unclear. This study aimed to investigate the impact of Sirtuins7 (SIRT7) on metabolic homeostasis to better understand the metabolic alterations underlying endometriosis.

Integrated metabolomic, transcriptomic, and proteomic analyses revealed metabolic dysregulation in endometriosis. Seahorse XF technology was used to assess metabolic rates. The effects of SIRT7 on endometriosis were validated both in vitro and in vivo. Immunoprecipitation and mass spectrometry were used to identify the interaction between SIRT7 and Tu translation elongation factor, mitochondrial (TUFM). Cellular phenotypes, including proliferation, migration, and apoptosis, were evaluated by CCK-8 assays, Transwell and scratch assays, and flow cytometry.

Multiomics analyses and Seahorse XF tests revealed a metabolic shift from mitochondrial respiration to glycolysis in ectopic tissues. SIRT7 was found to be upregulated in ectopic lesions and significantly influenced the progression of endometriosis. In addition, an interaction between SIRT7 and TUFM has been observed. The metabolic reprogramming, proliferation and migration ability of endometrial stromal cells were greatly reduced by SIRT7 knockdown and TUFM overexpression. The effects of TUFM deficiency were reversed by SIRT7 knockdown. The results also demonstrated that SIRT7 promoted the glycolytic enzymes GBE1 and PYGL, suppressed the mitochondrial enzymes IDH1 and SDHB, and activated the RhoA/ROCK/AKT pathway and the EMT process, thereby facilitating the progression of endometriosis.

These findings underscore the critical involvement of SIRT7 in the energy metabolism reprogramming of ectopic cells. Our research provides valuable insights for the development of nonsurgical treatment strategies for endometriosis. Similar content being viewed by others

Endometriosis (EMS) is a complex gynaecological disorder characterized by the presence of endometrial-like tissue outside the uterine cavity, leading to chronic inflammation, pain, and infertility [1]. The pathogenic mechanisms underlying EMS are complex and multifactorial and involve factors such as retrograde menstruation, genetics, immunological responses, and metabolic dysregulation [2]. Despite extensive research on the molecular mechanisms of this disease, the role of metabolism in its pathogenesis remains poorly understood. Previous studies reported that the metabolic switch in EMS from oxidative to glycolytic metabolism provides the energy and biosynthetic precursors necessary for the survival and proliferation of ectopic cells [3, 4]. Moreover, increased mitochondrial stress and the production of reactive oxygen species (ROS) in ectopic cells lead to increased expression of proinflammatory factors [5]. The metabolic plasticity of ectopic endometrial stromal cells (ESCs) enables them to exhibit enhanced malignant characteristics such as increased proliferation and invasion. Metabolic regulation plays a key role in the development and progression of EMS; however, studies on the specific mechanisms involved remain limited. Sirtuins, a family of NAD+-dependent deacetylases, are pivotal in response to various stressors [6, 7]. One family member, SIRT7, has been identified as an important regulator of cellular homeostasis. SIRT7 is involved in various cellular processes, including gene expression regulation [8], metabolic control [9], and mitochondrial stress responses [10]. SIRT7 maintains tumour phenotypes, with increased expression in many cancer types [11]. With respect to glucose metabolism regulation, SIRT7 directly promotes gluconeogenesis by binding to the G6PC promoter and deacetylating histone H3 lysine 18 (H3K18) in the G6PC promoter [12]. In addition, SIRT7 functionally inhibits NRF1, thereby preventing mitochondrial biogenesis [13]. Nevertheless, the energy regulatory mechanism of its role in EMS progression is not well understood and needs further study. The nuclear-encoded mitochondrial protein Tu translation elongation factor, mitochondrial (TUFM), is well known for its role in autophagy modulation [14]. Recent studies indicate a broader involvement of the TUFM in regulating various biological functions [15]. In lung cancer, TUFM knockdown diminishes mitochondrial respiratory chain activity, increases glycolysis and ROS production, and promotes epithelial‒mesenchymal transition (EMT), migration, invasion, and metastasis [16]. However, the specific mechanism by which TUFM participates in the progression of EMS is still unclear, and its underlying molecular mechanism warrants investigation. In this study, we propose the scientific hypothesis that the interplay between SIRT7 and TUFM may influence the energy metabolism, proliferation, and migration of endometrial stromal cells in EMS. This study provides deeper insights into disease pathogenesis and new clues for exploring nonsurgical treatments for EMS.

Patient samples This study was reviewed and approved by the Medical Ethics Committee of the First Affiliated Hospital of Xiamen University (Approval Number KY2021-031), and written informed consent was obtained from all patients. All procedures were conducted in accordance with the Declaration of Helsinki. All participants in this study were women who were of reproductive age, had normal menstrual cycles and had not received hormone therapy during the three months preceding surgery. Individuals with ovarian endometriosis were diagnosed through pathology postsurgery, whereas control individuals devoid of a history of endometriosis were validated by ultrasonography. Patients with a history of other gynaecological diseases, such as endometrial polyps, endometritis and any endocrine disorders, were excluded from the study. A total of 14 control patients and 74 patients with endometriosis were enrolled in this study. Clinical information was retrospectively collected from medical records and is summarized in Table 1. As shown, there were no significant differences between the two groups in terms of baseline clinical characteristics or laboratory parameters, including age, BMI, blood pressure, and serum levels of CA125, AMH, and eE2. Normal endometrial samples were obtained via hysteroscopy from nonendometriosis patients. Ectopic lesions and paired eutopic endometrial samples were collected from ovarian endometriosis patients who underwent hysteroscopic–laparoscopic surgery at the Department of Obstetrics and Gynecology, First Affiliated Hospital of Xiamen University, between March 2021 and November 2023. All the fresh samples were processed as follows: a portion of the tissue was immediately transported to the laboratory for primary cell culture, another portion was soaked in 4% paraformaldehyde for fixation and paraffin sectioning, and a third portion was frozen in liquid nitrogen and stored at −80 °C until the extraction of RNA or protein. Multiomics analyses Transcriptomic, proteomic and metabolomic data were obtained from the samples of identical cohorts, and the analysis procedures were described in our previous study [17]. In brief, transcriptome analysis was performed by RNA sequencing, and significance analysis was conducted with DEseq2 with Benjamini–Hochberg false discovery rate (FDR) correction; genes with an adjusted p value 2 were deemed significantly differentially expressed genes (DEGs). The proteome was analysed by nano LC–MS/MS operating in DIA mode, combined with parallel accumulation-serial fragmentation; significance analysis was performed with an unpaired t test, and proteins with a p value 1.5 were regarded as significantly differentially expressed proteins (DEPs). The metabolome analysis used advanced nontargeted metabolomics technology, and the metabolites with variable importance value (VIP) values > 1, p values 1.5 or FC < 0.67 were classified as biologically significant differentially expressed metabolites (DEMs). Cell culture Human endometrial stromal cells (HESCs) were obtained from the American Type Culture Collection (ATCC, CRL-4003). Primary ESCs were isolated from tissues. Endometriotic tissues were cut into small pieces and dissociated with collagenase IV (#A004186-0001; Sangon Biotech, China) at 37 °C for one hour, followed by deoxyribonuclease I (DNase I; #B002004-0005; Sangon Biotech) for half an hour. Then, the cell suspension was filtered through cell strainers to remove undissociated tissue fragments and to separate epithelial cells from stromal cells. The filtrate was centrifuged at 1000×g for 5 min. The remaining cells were resuspended and cultured. All the cells were cultured in DMEM/F12 (#L310KJ, BasalMedia, China) supplemented with 10% foetal bovine serum (FBS, Thermo Fisher, USA) and 1% penicillin–streptomycin (Thermo Fisher) in dishes in a 10% CO2 incubator at 37 °C. Transmission electron microscopy To visualize the ultrastructural morphology of organelles in primary endometrial stromal cells, cells at passage five were selected for transmission electron microscopy (TEM). The cells were washed with phosphate buffer (PB) solution to remove impurities and dead cells and then fixed with 2.5% glutaraldehyde at 4 °C for at least 4 h. Then, 1 ml of PB containing 5% bovine serum albumin (BSA) was added, and the adherent cells were scraped off using cell scrapers and collected into 1.5 ml centrifuge tubes. The cells were then subjected to osmication, stained with uranyl acetate, dehydrated in ethanol, and embedded in epoxy resin. Sections of the samples were cut to a thickness of 70 nm with an ultramicrotome and imaged using TEM (Hitachi H-7650, Japan). Imaging was performed at a magnification of 12,000 × (0.5 μm) to examine the mitochondrial distribution in cells and at 20,000 × (0.2 μm) to assess the mitochondrial structure and quantity. Each section was randomly observed in five different fields. The number of mitochondria was determined by counting, and the length of the mitochondria in the three groups was measured by ImageJ software. Seahorse XF assays Three types of primary ESCs and HESCs were seeded in XFe96 cell culture microplates at a density of 1 × 10^4 cells per well. After allowing the cells to adhere at room temperature for 1 h on a clean bench, they were cultured overnight in a 10% CO2 incubator at 37 °C. Concurrently, probe plates containing sterile water were placed in a 37 °C incubator without CO2 overnight. The next day, the sterile water was discarded from the probe plates, and 200 µL of calibration solution was added. The plates were then incubated at 37 °C in a non-CO2 incubator for 60 min. Assay medium (1 mM pyruvate, 2 mM glutamine, and 10 mM glucose) were prepared by adjusting Seahorse XF DMEM to pH 7.4 and warming it to 37 °C in a water bath. The cell culture medium was removed, and the cells were washed once with the warmed assay medium and incubated with assay medium at 37 °C in a non-CO2 incubator for 60 min prior to the assay. According to the instructions, the compounds used in the mitochondrial stress test were oligomycin (1.5 µM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) (1.5 µM), and rotenone/antimycin A (0.5 µM), which were added to the respective ports of the utility plate. For the glycolytic rate test, the concentrations of the compounds used were rotenone/antimycin A (0.5 µM) and 2-DG (50 mM). First, the probe plate was run on an Agilent Seahorse XF96 Analyser for calibration. After calibration, the hydration plate was replaced with the cell culture microplate, and the real-time analyser was run with Wave software to analyse the data. RNA isolation and qRT‑PCR Total RNA was extracted from tissue or cell samples with TRIzol (#AG21102, Accurate Biology, China) and stored at − 80 °C. cDNA was synthesized from total RNA with Evo M-MLV Reverse Transcription Reagent Premix (#AG11706, Accurate Biology). PCR was performed with the SYBR Green Pro Taq HS Premix Kit (#AG11702, Accurate Biology) in a Lightcycler 480 (Roche; Basel, Switzerland). The results were normalized to GAPDH expression, and the 2−∆∆CT method was used to calculate the relative mRNA level. The primer sequences are listed in Table S1. Western blotting RIPA buffer and 1% PMSF (Sangon Biotech) were used to lyse the cells and tissues. The samples were quantified with a BCA protein assay kit (#P0011, Beyotime Biotechnology, China). The samples were subjected to SDS‒polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes (#IPVH00010, Millipore, Germany). The membranes were incubated with primary antibodies overnight after being blocked with 5% BSA (Sangon Biotech) for 1 h, followed by incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h, and detection with chemiluminescence (ECL) reagents was performed by scanning with the ChemiDoc MP Imaging System (Bio-Rad, USA). The antibodies utilized in the experiment included anti-SIRT7 (Santa Cruz Biotechnology, Cat# sc-365344, RRID: AB_10850175), anti-TUFM (ABclonal, Cat# A6423, RRID: AB_2767025), anti-H3K18Ac (ABclonal, Cat# A7257, RRID: AB_2767801), anti-ROCK2 (Abcam, Cat# ab125025, RRID: AB_10972853), anti-GAPDH (Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), anti-beta actin (Proteintech, Cat# HRP-66009, RRID: AB_2883836), anti-GBE1 (Proteintech, Cat# 20313-1-AP, RRID: AB_10697658), anti-PYGL (Proteintech, Cat# 15851-1-AP, RRID: AB_2175014), anti-IDH1 (Proteintech, Cat# 12332-1-AP, RRID: AB_2123159), anti-SDHA (Proteintech, Cat# 14865-1-AP, RRID: AB_11182164), anti-SDHB (Proteintech, Cat# 10620-1-AP, RRID: AB_2285522), anti-AKT (Proteintech, Cat# 10176-2-AP, RRID: AB_2224574), anti-phospho-AKT (Proteintech, Cat# 66444-1-Ig, RRID: AB_2782958), anti-Flag (Proteintech, Cat# 66008-4-Ig, RRID: AB_2918475), anti-His (Proteintech, Cat# 66005-1-Ig, RRID: AB_11232599), anti-E-cadherin (Cell Signaling Technology, Cat# 14472, RRID: AB_2728770), anti-N-cadherin (Cell Signaling Technology, Cat# 13116, RRID: AB_2687616), and anti-RhoA (Cell Signaling Technology, Cat# 2117, RRID: AB_10693922) antibodies. For immunoprecipitation experiments, approximately 10% of the cellular extract was used as input for subsequent Western blot analysis. Establishment of transfected cell lines The cells were seeded into 6-well plates at 1 × 105 cells per well and grown to 50–60% confluence. Transient knockdown cell lines were generated by transfecting short interfering RNAs (siRNAs), including si-SIRT7 and si-TUFM (RiboBio, China), into cells with Lipofectamine RNAiMAX Reagent (Invitrogen, USA) at a final concentration of 5 nM. After 8 h, the supernatants were replaced, and the cells were collected 48 h posttransfection. Stably transfected cell lines were generated using lentiviruses, including oe-SIRT7, sh-SIRT7, and oe-TUFM constructs, obtained from the Shanghai Genechem company. Cells were infected at a multiplicity of infection (MOI) of 5 with Transfection Reagent A according to the manufacturer’s instructions. Eighteen hours after infection, the viral mixture was replaced with fresh culture medium. Puromycin (3.5 µg/mL) was then applied to select successfully transduced cells for 2–3 days. Western blotting and qRT‒PCR were used to confirm the transfection efficiency. The desired sequences are listed in Table S1. Adeno-associated virus (AAV) injection and endometriosis mouse model Fifty-two female BALB/c mice, aged 6–8 weeks and weighing 15–20 g, were ordered and fed at the Animal Research Laboratory of Xiamen University under suitable conditions. AAV-Con, AAV-SIRT7, AAV-RNAi-Con, and AAV-RNAi-SIRT7 were constructed and packaged by Genechem Company. The sequences are listed in Table S1. AAVs were injected into the uteri of 20 donor mice, which were divided into four groups, to infect the endometrial cells (4 × 1011 v.g. per mouse). Four weeks postinfection, the uterus of one donor mouse per group was extracted for frozen sectioning and Western blot analysis to confirm infection fluorescence and protein expression efficiency. The uterine fragments from the remaining four donor mice in each group were pooled and intraperitoneally injected into eight recipient mice to establish an endometriosis mouse model. Four weeks after surgery, the ectopic lesions were excised, and their size and weight were measured and recorded. The lesions were subsequently divided into two segments: one segment was kept in 10% formalin for haematoxylin and eosin (H&E) staining, while the other was frozen in Liquid nitrogen for a minimum of 30 min and subsequently stored at −80 °C. This study was approved by the Ethics Committee of Xiamen University. Immunofluorescence (IF) staining The cells were fixed with 4% paraformaldehyde at room temperature for 10 min, followed by blocking with 5% BSA in phosphate-buffered saline (PBS) containing 0.3% Triton X-100 at RT for 30 min. The fixed cells were incubated with primary antibodies at 4 °C overnight, followed by incubation with Cy3- or FITC-conjugated secondary antibodies for 1 h. Finally, the nuclei were stained with DAPI for 10 min, and the cells were washed with PBS three times for 5 min each time between each step. Images were acquired under an optical inverted biomicroscope (Olympus, Japan). For mitochondria-specific staining, the MitoTracker Deep Red FM fluorescence probe (C1032, Beyotime Biotechnology) was used to stain the mitochondria of live cells. After staining, the cells were fixed and permeabilized, followed by staining with other antibodies and DAPI. Images were obtained with a laser scanning confocal microscope (Leica TCS SP8, Germany). The antibodies used for IF included anti-vimentin (Cell Signaling Technology, Cat# 5741, RRID: AB_10695459), anti-E-cadherin (Cell Signaling Technology, Cat# 14472, RRID: AB_2728770), anti-SIRT7 (Santa Cruz Biotechnology, Cat# sc-365344, RRID: AB_10850175) and anti-TUFM (ABclonal, Cat# A6423, RRID: AB_2767025) antibodies. Immunohistochemistry (IHC) staining assay The paraffin-embedded tissue sections were deparaffinized and subjected to antigen retrieval, followed by three washes. The sections were then immersed in a 3% hydrogen peroxide solution and incubated in the dark at room temperature for 25 min to block endogenous peroxidase activity. After blocking with 3% BSA, the primary antibody was applied at an appropriate dilution and incubated overnight at 4 °C. The sections were then incubated with the corresponding HRP-labelled secondary antibody at room temperature for 1 h after being washed three times with buffer. Next, the sections were treated with DAB chromogenic solution. The reaction was stopped by rinsing the sections with running water. Counterstaining was performed with haematoxylin to stain the cell nuclei. Finally, the sections were dehydrated, sealed, and observed under a microscope. The antibodies utilized in the experiment included an anti-SIRT7 antibody (Santa Cruz Biotechnology, Cat#sc-365344, RRID: AB_10850175). Immunoprecipitation (IP) and Co-IP HESCs were lysed with NP-40 RIPA buffer (#N8032, Solarbio Science Technology Company, China) supplemented with the protease inhibitor PMSF at 4 °C for 1 h with continuous rotation, followed by centrifugation at 13 000 × g for 15 min. The cellular extract was precleared with Protein A/G Dynabeads (Thermo Fisher) at 4 °C for 1 h. The precleared lysate (500 µl at 1 mg/ml) was then incubated with 1 µg of antibodies or the IgG control at 4 °C overnight on a tube rotator. The mixture was then incubated with Protein A/G beads for 4 h. The beads bound to the protein complex were washed three times with NP-40 buffer and eluted with 1 × SDS‒PAGE loading buffer (#P0285, Beyotime Biotechnology) at 100 °C for 10 min. For cells stably transfected with SIRT7 (Flag-tag) and TUFM (His-tag), the cellular extract was directly incubated with anti-Flag magnetic beads (#HY-K0207, MCE, USA) or anti-His magnetic beads (#HY-K0209, MCE) at 4 °C overnight. After washing with TBST, the beads were eluted with 1 × SDS‒PAGE loading buffer. The eluates were subjected to Western blotting. The following antibodies were used: an anti-SIRT7 antibody (Santa Cruz Biotechnology, Cat#sc-365344, RRID: AB_10850175), an anti-TUFM antibody (ABclonal, Cat#A6423, RRID: AB_2767025), an anti-DYKDDDDK tag polyclonal antibody (Proteintech, Cat# 20543-1-AP, RRID: AB_11232216), and an anti-His-Tag monoclonal antibody (Proteintech, Cat# 66005-1-Ig, RRID: AB_11232599). Liquid chromatography with tandem mass spectrometry (LC‒MS/MS) analysis The precipitated immune complexes were separated by SDS‒PAGE. The gel was dyed with Coomassie Brilliant Blue, and the appropriate bands were cut out for subsequent analysis by Shanghai Applied Protein Technology Co., Ltd. Briefly, the gel was digested with trypsin to obtain peptides. The peptides were analysed using LC‒MS/MS (nanoLC‒QE). Finally, software such as MASCOT was used to analyse the LC‒MS/MS data, providing qualitative identification information for the target protein peptide molecules. RNA sequencing RNA sequencing was performed following shRNA-mediated knockdown of SIRT7. This loss-of-function approach was selected to elucidate genes and pathways directly regulated by SIRT7 that may be masked or compensated for in overexpression models. Total RNA was extracted 48 h posttransfection with TRIzol reagent. The quantity and purity of the RNA were assessed with a NanoDrop 2000 spectrophotometer, whereas RNA integrity was evaluated with an Agilent 2100 Bioanalyzer. An Illumina NovaSeq 6000 instrument from Gene Denovo Biotechnology Co. (Guangzhou, China) was used to sequence the resulting cDNA library. DEGs were identified on the basis of an absolute fold change of ≥ 2 and a false discovery rate (FDR) of less than 0.05. For Gene Ontology (GO) analysis, all DEGs were mapped to GO terms with the Gene Ontology database (http://www.geneontology.org/). CCK-8 proliferation assay The CCK-8 solution was acquired from LLBIO (#HY-K0301, China). Transfected cells were seeded into a 96-well plate at a density of 5 × 10³ cells per well. The cells were categorized into different groups on the basis of the transfection conditions, with 5 parallel wells for each group. After the cells attached, cell viability was assessed at continuous and intermittent time intervals. Then, 100 µl of serum-free culture medium containing 10 µl of the CCK-8 solution was added to each well, and the cells were incubated at 37 °C for 2 h. Then, the optical density (OD) was measured at 450 nm with an ELISA reader spectrophotometer (Dynatec Laboratories, Chantilly, VA). Cell migration assay Transwell and wound-healing assays were used to assess migratory ability. The cell pellet was resuspended in serum-free DMEM, and the cell density was adjusted to 1 × 10⁶ cells/mL. Next, the top chamber of an 8 μm pore size filter membrane was filled with 200 µL of cell solution, and the bottom chamber was filled with 700 µL of DMEM containing 10% FBS in a 24-well plate with Transwell inserts (Corning, USA). The incubation time was dependent on the cell type. Afterwards, the cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet at room temperature. The chamber was dipped into PBS as many times as needed to remove the excess paraformaldehyde or crystal violet. Cotton swabs were used to carefully remove any remaining cells that had not migrated from the top of the membrane, ensuring that the membrane was not damaged. The Transwell membrane was allowed to dry. Finally, the number of cells in different fields was observed and counted under an inverted microscope. The cells were seeded at an appropriate density in a 6-well plate and allowed to reach 100% confluence within 24 h. In a Biosafety cabinet, a 1 mL pipette tip was used to press firmly against the top of the culture plate, which rapidly moved vertically through the cell monolayer to create a wound. The medium and cell debris were carefully aspirated. The culture medium containing 0.1% BSA was slowly added to the well wall. Following the generation of the wound, an initial picture was taken. The culture plate was then placed in a 37 °C incubator with 10% CO2. Pictures were taken every 12 h. The images were imported into ImageJ software to calculate the wound area, migration rate (%) = (scratch area at 0 h − scratch area at 12 h)/scratch area at 0 h * 100%, and a bar chart was generated to represent the changes in wound closure over time. Annexin V-FITC/PI apoptosis assay YF®488-Annexin V and propidium iodide (PI) apoptosis kits (#Y6002, UE, China) were used to detect apoptotic cells. In accordance with the instructions, the cells were placed into a flow tube, washed once with PBS, and centrifuged at 300 × g for 5 min, after which the supernatant was discarded. One hundred microlitres of diluted 1× Annexin V Binding Buffer was added to resuspend the cells. Then, 2.5 µL of Annexin V-FITC Reagent and 2.5 µL of PI Reagent were added to the cell suspension, and the mixture was incubated at room temperature in the dark for 20 min. Subsequently, 400 µL of diluted 1 × Annexin V Binding Buffer was added before flow cytometric analysis. Statistical analysis Statistical analyses were performed with GraphPad Prism 8 software (San Diego, USA) or R software. Comparisons between two groups were performed with the unpaired two-tailed Student’s t test. For comparisons involving more than two groups, one-way or two-way analysis of variance (ANOVA) was used. The results are expressed as the average ± SD of at least three independent experiments. Statistical significance was defined as p < 0.05 and shown as *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001, with ns indicating no significance. All experiments were independently repeated at least three times unless otherwise indicated.

Primary ectopic stromal cells exhibit mitochondrial stress and metabolic reprogramming In our previous study, by integrating metabolomics, transcriptomics and proteomics data from the same samples of endometriosis, metabolic dysregulation of carbohydrate metabolism was observed in EMS. The carbohydrate pathways mainly included the glycolytic process occurring in the cytoplasm and the TCA cycle occurring in the mitochondria. A metabolic map (Figure S1) integrating the identified differentially expressed metabolites, genes, and proteins from three omics datasets revealed significant changes in the expression of key enzymes involved in carbohydrate metabolism in ectopic tissue. The values for the expression of genes, proteins, and metabolites are shown in Table S2. This observed imbalance in energy metabolism across the omics analyses inspired further investigation. Primary stromal cells were extracted from the endometria of nonendometriosis patients (Norm) and endometriosis patients (Eut), as well as from the ectopic lesions (Ect) of endometriosis patients. Immunofluorescence staining experiments were performed to verify that primary cells express the stromal cell marker vimentin and lack the epithelial cell marker E-cadherin, confirming their identity as stromal cells (Figure S2). The observation of mitochondrial morphology using TEM of the three primary endometrial stromal cell types revealed that ectopic stromal cells presented a greater mitochondrial count and shorter mitochondrial lengths (Fig. 1A). Flow cytometric analysis revealed significantly elevated levels of ROS in ectopic stromal cells (Fig. 1B). Furthermore, Seahorse mitochondrial stress tests performed on live cells revealed higher oxygen consumption rate (OCR) values and respiration rates in primary ectopic cells than in the other two primary cell types and in HESC model cells (Fig. 1C). The glycolytic rate test revealed higher proton efflux rate (PER) values and glycolytic rates in ectopic cells (Fig. 1D). The metabolic index, calculated from the OCR/PER ratio, was reduced in primary ectopic cells (Fig. 1E). These mitochondrial functions and metabolic phenotypes collectively indicates a state of mitochondrial stress and a metabolic transition from mitochondrial respiration to glycolysis in primary ectopic stromal cells. SIRT7 is upregulated in ectopic tissues and drives metabolic reprogramming The sirtuin family is a group of important molecules involved in homeostatic regulation and adaptation to metabolic stress. To assess their expression patterns in endometriosis, the mRNA expression levels of all seven sirtuin family members were measured in human endometrial tissue samples using qRT‒PCR. Among them, SIRT7 was found to be the most significantly elevated family member in ectopic lesions compared with normal and eutopic tissues (Fig. 2A). The elevated expression of SIRT7 in ectopic tissues was further validated in an expanded cohort by PCR and confirmed at the protein level by Western blotting (Fig. 2B-C). Immunohistochemical analysis revealed that SIRT7 was primarily localized in stromal cells (Fig. 2D). To identify effective shRNAs for stable knockdown of SIRT7, we screened multiple lentiviral clones. As shown in Figure S3A, PCR analysis demonstrated that shRNA clones #1 and #3 significantly reduced SIRT7 mRNA expression. However, Western blot analysis (Figure S3B) revealed that only clone #1 efficiently suppressed SIRT7 protein levels. Therefore, shRNA #1 was selected for subsequent experiments. On the basis of the baseline expression levels of SIRT7, SIRT7 was overexpressed in primary Norm and Eut cells, where its endogenous expression was low, and knocked down in primary Ect cells, where its expression was comparatively high. The transfection efficiencies of the stably transfected cells were confirmed by PCR and Western blot analysis (Fig. 2E-F). To evaluate disease-specific effects, paired Eut and Ect primary stromal cells from the same endometriosis patients were used following lentiviral transduction, thereby minimizing donor-related variability. SIRT7 overexpression led to elevated ROS levels, suppressed mitochondrial DNA (mt-DNA) expression, and reduced metabolic indices, indicating increased mitochondrial stress and a metabolic transition from mitochondrial respiration to glycolysis. However, the knockdown of SIRT7 rescued these phenotypes and led to a shift back to reliance on mitochondrial respiration (Fig. 2G-I). These findings indicate that SIRT7 plays a key role in the metabolic remodelling of ESCs by regulating mitochondrial homeostasis and energy metabolism. SIRT7 promotes the proliferation and migration of ESCs To determine the involvement of SIRT7 in the progression of EMS, we examined key cellular behaviours, including apoptosis, proliferation, and migration. Apoptosis was assessed by flow cytometry (Fig. 3A), proliferation was evaluated with CCK-8 assays (Fig. 3B), and cell migration was assessed with Transwell assays (Fig. 3C) and wound healing assays (Fig. 3D). The results revealed that stable overexpression of SIRT7 notably suppressed apoptosis while increasing proliferation and migration. Conversely, stable knockdown of SIRT7 led to increased apoptosis and inhibited cell proliferation and migration. Additionally, we conducted transient knockdown of SIRT7 in primary ESCs and confirmed its effectiveness (Figure S4), supporting the reproducibility of the observed phenotypes. Overall, these findings indicate an essential function for SIRT7 in modulating the proliferation and migration of ESCs, suggesting its potential contribution to the invasive behaviour associated with endometriosis. SIRT7 promotes lesion development in EMS and regulates key cellular functions, as revealed by transcriptomic profiling An experimental flowchart illustrating the AAV injection procedure and the establishment of the endometriosis mouse model is shown in Fig. 4A. Four weeks after injection, frozen sections of the transfected uterine tissues were examined, confirming fluorescence and successful transfection (Figure S5A). Upon sacrifice, lesions in the abdominal cavity were observed (Fig. 4B). H&E staining confirmed these lesions as endometriotic tissues (Figure S5B), and Western blot analysis validated the transfection efficiency of SIRT7 overexpression and knockdown (Figure S5C-D). Subsequently, in growth assessments of the lesions, SIRT7 overexpression was shown to significantly increase the number of ectopic foci compared to that of the control (Fig. 4C), while SIRT7 knockdown markedly reduced the volume, weight, and count of lesions (Fig. 4D). These findings indicate a direct role for SIRT7 in driving the progression of EMS. To further elucidate the biological functions underlying the pro-endometriotic effects of SIRT7, we performed transcriptomic analysis following its knockdown. Although SIRT7 overexpression is positively correlated with endometriosis progression, we reasoned that loss-of-function analysis would more directly reveal genes and pathways critically dependent on SIRT7 activity. This approach enabled us to identify key regulatory networks affected by SIRT7 depletion, thereby providing mechanistic insight into its role in the metabolic and phenotypic transformation of stromal cells. To evaluate sample variation and reproducibility, we performed principal component analysis (PCA), which revealed clear clustering of the SIRT7 knockdown and control groups (Figure S5E), supporting the robustness of our RNA-seq data. DEGs were then filtered using an adjusted p value 1. On the basis of these criteria, a total of 1388 upregulated genes and 748 downregulated genes were identified in the SIRT7-knockdown cells compared with the control cells (Fig. 4E). GO enrichment analysis of all DEGs revealed functional tendencies in terms of biological processes (BPs), molecular functions (MFs), and cellular components (CCs) (Fig. 4F). Specifically, SIRT7 knockdown was associated mainly with changes in intracellular structures, organelles, and nuclear organization, suggesting potential effects on cell structure and organelle function. Metabolic processes (GO:0008152) were the most enriched biological pathways, indicating that SIRT7 may regulate cellular homeostasis through metabolic modulation. In terms of molecular function, enriched terms were related to protein binding (GO:0005515), implying that SIRT7 may influence intracellular protein function and interaction networks and that protein‒protein interactions may serve as a key regulatory mechanism under pathological conditions. These findings offer biological insights into the regulatory role of SIRT7 and offer a clear direction for further investigations in this area. To investigate the role of SIRT7 in cellular metabolism, we generated a curated list of SIRT7-regulated metabolic genes by intersecting DEGs with metabolism-related gene sets defined by the GO term “metabolic process”. These genes were subjected to GO enrichment analysis to identify significantly affected pathways (Fig. 4G). All the Listed pathways had q values below 1e-05, indicating a high level of statistical significance. Notably, pathways related to the cellular response, migration, and motility regulation were significantly enriched, suggesting that SIRT7-mediated metabolic regulation may provide the essential energy and material basis for higher-order biological processes such as cell migration. These findings further support our previous observations of the critical role of SIRT7 in regulating cell motility and migration. TUFM interacts with and colocalizes with SIRT7 GO enrichment analysis of SIRT7-associated transcriptomic alterations revealed “protein binding” as one of the top enriched molecular function terms, suggesting that SIRT7 may influence endometrial stromal cell behaviour through interactions with other proteins. On the basis of this finding, IP followed by MS analysis was performed to identify candidate SIRT7-interacting proteins in HeSCs. This approach led to the identification of 135 candidate proteins potentially associated with SIRT7. To refine the candidate pool, these 135 proteins were cross-analysed with 675 metabolism-related and 853 mitochondria-related differentially expressed genes derived from the transcriptomic dataset of EMS tissues, which yielded six candidate genes (Fig. 5A). Among these genes, only LDHA and TUFM were differentially expressed in the EMS tissue proteomic data (Fig. 5B), indicating their potential relevance to disease pathology. Subsequent co-IP assays using endogenous proteins were performed in HESCs to evaluate the interactions between SIRT7 and these two candidates. The results confirmed a specific interaction between SIRT7 and TUFM, whereas LDHA was excluded (Fig. 5C). To further validate this interaction, tagged protein co-IP assays were conducted in HESCs. Flag-tagged SIRT7 and His-tagged TUFM each successfully pulled down their respective endogenous binding partners (Fig. 5D). Moreover, in a coexpression cell model, reciprocal co-IP using Flag and His tags further confirmed the protein‒protein interaction between SIRT7 and TUFM (Fig. 5E). IF staining was performed to examine the subcellular localization of SIRT7 and TUFM in HESCs (Fig. 5F). The results revealed overlapping fluorescence signals in the nucleus, cytoplasm, and mitochondria, suggesting that SIRT7 and TUFM may colocalize in these compartments. Although this observation suggests the possibility of compartment-specific functional interplay between the two proteins, further studies are needed to confirm direct interactions in these locations. Additionally, TUFM expression was significantly reduced in ectopic EMS tissues at both the mRNA and protein levels (Fig. 5G-H). Loss of TUFM induces SIRT7 upregulation and promotes metabolic reprogramming and cell behaviour through SIRT7 To gain deeper insight into the relationship between TUFM and SIRT7, endometrial stromal cell models with TUFM knockdown and overexpression were constructed. Successful transfection was confirmed by PCR (Fig. 6A) and Western blotting (Fig. 6B), which validated TUFM expression at both the RNA and protein levels. PCR analysis was then performed to examine the transcriptional regulatory relationship between SIRT7 and TUFM. The results revealed that only sh-SIRT7 promoted TUFM RNA expression, whereas oe-SIRT7 had no effect. Conversely, changes in TUFM expression had no significant effect on SIRT7 RNA levels (Fig. 6C). Further investigation using WB to assess protein-level regulation revealed an interesting pattern: TUFM expression remained unchanged in both oe-SIRT7 and sh-SIRT7 cells. However, TUFM knockdown led to increased SIRT7 protein levels, whereas TUFM overexpression resulted in decreased SIRT7 protein expression (Fig. 6D). These findings suggest that TUFM may not regulate SIRT7 at the transcriptional level but rather may influence its posttranslational modification or degradation, thereby affecting SIRT7 protein stability. In summary, TUFM acts as an upstream factor that negatively regulates SIRT7 protein expression in a unidirectional manner. The functional role of TUFM in ESCs was further investigated through analysis of metabolic phenotypes following TUFM modulation. Knockdown of TUFM significantly increased ROS levels and decreased the metabolic index, whereas TUFM overexpression reduced ROS accumulation and increased the metabolic index (Fig. 6E-F). These findings suggest that TUFM deficiency induces mitochondrial stress and promotes a metabolic shift towards glycolysis in ESCs. Subsequent experiments were performed to assess the effects of the TUFM on cellular behaviour. TUFM knockdown significantly promoted cell proliferation and migration, whereas TUFM overexpression suppressed these phenotypes (Fig. 6G-I), suggesting that TUFM plays a negative regulatory role in the aggressive behaviour of ESCs. To further explore the potential synergistic role of TUFM and SIRT7 in regulating cellular functions, we conducted combinatorial transfection experiments. Specifically, SIRT7 was overexpressed (oe-SIRT7) or silenced (sh-SIRT7) in TUFM-overexpressing or TUFM-silenced cells, respectively, to assess the compensatory effects of SIRT7 modulation. The results demonstrated that overexpression of SIRT7 (oeTUFM–oeSIRT7) effectively reversed the inhibitory effects of TUFM overexpression on ESC proliferation and migration. Conversely, knockdown of SIRT7 (siTUFM–shSIRT7) abrogated the increased proliferative and migratory abilities induced by TUFM silencing (Fig. 7A-C). These findings indicate that the regulatory effects of TUFM on ESC proliferation and migration are mediated, at least in part, through SIRT7. The TUFM-SIRT7 axis regulates metabolic enzymes and the Rhoa/Rock/Akt pathway To identify the specific downstream targets regulated by the TUFM–SIRT7 axis, we screened DEGs associated with metabolic reprogramming and phenotypic changes on the basis of RNA-seq data and further validated them through experimental assays. Western blot analysis (Fig. 8A) revealed that SIRT7 overexpression increased the levels of glycogen metabolism enzymes, including glycogen synthase (GBE1) and glycogen phosphorylase (PYGL), while it decreased the expression of mitochondrial metabolic enzymes such as succinate dehydrogenase subunits A and B (SDHA, SDHB) and isocitrate dehydrogenase 1 (IDH1). These results directly linked SIRT7 to the metabolic shift from mitochondrial respiration to glycolysis. Additionally, SIRT7 activation increased the RhoA/ROCK/AKT signalling pathway and facilitated EMT, as indicated by decreased E-cadherin and increased N-cadherin expression. Collectively, these findings suggest a dual regulatory role of SIRT7 in mediating both metabolic shifts and phenotypic changes in ESCs, thereby influencing both energy metabolism and cellular behaviour. Given that TUFM has been implicated in regulating SIRT7 expression, we further explored whether TUFM regulates these metabolic and signalling proteins through SIRT7. The results (Fig. 8B) revealed that TUFM overexpression significantly reduced the levels of GBE1 and PYGL and increased the levels of mitochondrial metabolic enzymes, including SDHA, SDHB, and IDH1. Conversely, TUFM knockdown led to the upregulation of glycolytic enzymes and the suppression of mitochondrial enzymes, which is consistent with the metabolic shift observed in SIRT7-overexpressing cells. In parallel, TUFM modulation also affected the RhoA/ROCK/AKT signalling pathway and EMT markers, with TUFM knockdown increasing AKT and AKT phosphorylation and inducing the E-cadherin to N-cadherin switch, while TUFM overexpression reversed these effects. These results further support that TUFM regulates functional pathways consistent with those modulated by SIRT7. Subsequent rescue experiments were performed to assess whether the molecular changes induced by TUFM could be reversed. The results (Fig. 8C-D) revealed that SIRT7 modulation effectively “rescued” the TUFM-induced alterations in protein expression. These findings indicate that SIRT7 functions as a critical downstream effector of TUFM in regulating metabolic reprogramming and the cellular phenotype.

In summary, our findings demonstrate that TUFM deficiency promotes SIRT7 overexpression, leading to mitochondrial stress and metabolic reprogramming in ectopic ESCs, thereby contributing to the pathogenesis and progression of EMS (Fig. 8E). Elevated SIRT7 promotes a metabolic shift from mitochondrial respiration to glycolysis by upregulating glycogen metabolism enzymes (GBE1 and PYGL) and downregulating mitochondrial metabolic enzymes (SDHA, SDHB, and IDH1). Concurrently, SIRT7 activates the RhoA/ROCK/AKT signalling pathway and induces EMT, as evidenced by decreased E-cadherin and increased N-cadherin expression. These combined effects lead to increased proliferation and migration of ESCs, ultimately contributing to ectopic lesion growth in EMS. This study highlights the critical role of the TUFM–SIRT7 axis in linking metabolic reprogramming with cellular behaviour and disease progression. To sustain cell survival, endometriotic cells must adapt their metabolism to meet cellular demands and environmental conditions. In hypoxic ectopic lesions, endometriotic cells survive by switching from oxidative phosphorylation to glycolysis, as shown in a nonhuman primate model [18]. Peritoneal mesothelial cells isolated from the pelvic peritoneum of women with EMS presented significantly lower mitochondrial respiration and higher glycolysis levels [19]. In contrast to the traditional view that mitochondrial respiratory dysfunction occurs in ectopic tissue, our study revealed that primary ectopic ESCs are in a state of mitochondrial stress activation, accompanied by elevated ROS levels and increased mitochondrial respiration rates. Most studies have reported increased ROS levels in patients with EMS compared with controls [20, 21]. A recent single-cell study revealed that genes related to both the glycolytic and oxidative pathways of energy production were activated in ectopic stromal cells [22]. Moreover, studies have revealed that the use of oxidative phosphorylation (OXPHOS) inhibitors [23] and glycolysis inhibitors [19] can attenuate the progression of EMS, further supporting the dual involvement of both metabolic pathways in disease development. Consistently, we observed that ectopic cells exhibited a significant metabolic shift from mitochondrial respiration to glycolysis, a transition consistent with the Warburg effect. This phenomenon, commonly observed in cancerous and proliferating cells, involves reliance on glycolysis even in the presence of oxygen and functional mitochondria [24]. Given the critical role of energy metabolism in disease progression, our study focused on elucidating the molecular mechanism underlying this metabolic shift, and we hope that these findings can contribute to the development of a new metabolic-directed strategy for nonhormonal treatment of EMS. SIRT7 is recognized as a crucial energy sensor, and many studies have underscored its role in glucose sensing and metabolic regulation [25]. For example, during glucose deprivation, SIRT7 Shifts from the nucleolus to the nucleoplasm, facilitating the hyperacetylation of polymerase-associated factor 53 (PAF53), which inhibits rDNA transcription to conserve energy [26]. Additionally, SIRT7 increases gluconeogenesis by modulating H3K18 deacetylation of the glucose-6-phosphatase (G6PC) promoter [12]. In addition to glucose, lipids also serve as crucial substrates for energy metabolism, and accumulating evidence suggests that SIRT7 plays a significant regulatory role in this metabolic pathway, particularly in liver disease and cancer progression. The overexpressed SIRT7 has been shown to interact with the transcription factor Myc, suppressing the transcription of ribosomal proteins, thereby mitigating endoplasmic reticulum (ER) stress and ultimately improving hepatic steatosis in vivo [27]. Conversely, SIRT7 knockout mice exhibit severe mitochondrial dysfunction, as hyperacetylation of GABPβ1 impairs the transcription of mitochondrial genes, disrupting mitochondrial homeostasis and leading to hepatic lipid accumulation and steatosis [28]. Furthermore, SIRT7 directly interacts with PPAR-γ2, a master regulator of adipocyte differentiation, and deacetylates it at lysine 382. This modification weakens the transcriptional activity of PPAR-γ2, leading to dysregulation of adipogenic gene expression and alterations in lipid accumulation within adipose tissue [29, 30]. Collectively, these findings underscore the pivotal role of SIRT7 in regulating glucose and lipid metabolism through diverse transcriptional and posttranslational mechanisms. However, further studies are needed to elucidate the underlying mechanisms involved. At present, the role of SIRT7 in regulating metabolic pathways in EMS remains unclear. In this study, we explored the regulatory function of SIRT7 in energy metabolism and demonstrated that SIRT7 promotes a metabolic shift from mitochondrial respiration to glycolysis in ESCs by modulating the expression of key rate-limiting enzymes involved in these pathways. Specifically, SIRT7 limits mitochondrial respiration by suppressing the expression of the mitochondrial enzymes IDH1 and SDH, which are key enzymes of the TCA cycle. IDH1 catalyses the conversion of isocitrate to α-ketoglutarate (α-KG), which plays an important role in maintaining mitochondrial respiration [31]. Mutations in IDH1 reduce energy flux in the TCA cycle and induce glycolysis [32, 33]. Deacetylase inhibitors have been shown to promote IDH1 expression, thereby suppressing ROS levels and reducing mitochondrial stress [34]. SDHB catalyses the oxidation of succinate to fumarate, and loss of SDHB causes abnormal accumulation of succinate and impaired TCA cycle function, causing a metabolic shift towards aerobic glycolysis to meet the high energetic and biosynthetic demands of tumour cells [35,36,37]. On the other hand, SIRT7 promoted the expression of glycolytic enzymes such as GBE1 and PYGL, thereby driving the transition towards glycolytic metabolism. GBE1 and PYGL are involved in glycolysis and glycogenolysis, and their expression is elevated in various cancer tissues under hypoxic conditions [38,39,40]. GBE1 targets the NF-κB pathway, shifting the glucose metabolism pattern of cancer cells towards glycolysis and enhancing the Warburg effect to drive progression [41, 42]. Elevated PYGL mobilizes accumulated glycogen to fuel glycolysis, inducing EMT and facilitating liver metastasis [43]. In summary, this coordinated regulation mediated by SIRT7 facilitates a switch to a glycolytic phenotype, which may support the survival and proliferation of ectopic ESCs under the hypoxic or inflammatory conditions typical of endometriotic lesions. These findings provide novel insights into the mechanisms by which SIRT7 orchestrates energy metabolic reprogramming in EMS and suggest that targeting SIRT7 and its downstream metabolic targets could represent a potential therapeutic strategy for this disease. TUFM, encoded by a nuclear gene, is synthesized in the cytoplasm and imported into the mitochondria, where it regulates the expression of the mitochondrial genome by controlling the translation of mtDNA-encoded proteins [44]. TUFM was reported to serve as a critical signal transducer in the communication between the mitochondrial genome and nuclear genes. TUFM knockdown was found to decrease mtDNA expression, activate the AMPK–GSK3β/β-catenin pathway, and promote glycolysis [45], highlighting its role in coordinating mitochondrial stress responses to maintain cellular homeostasis. Recent studies have also emphasized that TUFM is essential for mitochondrial respiratory function and metabolic regulation [46]. In lung cancer, downregulation of TUFM reduces mitochondrial respiratory chain activity and increases ROS production, leading to EMT and promoting the migration and invasion of cancer cells [16]. Consistent with these findings, our results demonstrated that TUFM knockdown in ESCs triggered mitochondrial stress and promoted glycolysis. Notably, TUFM silencing led to elevated SIRT7 protein levels and induced a metabolic reprogramming pattern similar to that observed upon SIRT7 overexpression. Furthermore, SIRT7 knockdown reversed the metabolic alterations caused by TUFM depletion, suggesting a functional link between TUFM and SIRT7 in regulating cellular metabolism. Given that TUFM influences SIRT7 protein levels without altering its mRNA expression, we speculate that TUFM may modulate SIRT7 protein stability, potentially through mechanisms involving posttranslational modifications or proteasomal degradation. Further investigations, including protein stability assays and ubiquitination analyses, are warranted to clarify this precise regulatory relationship. In addition, our immunofluorescence data revealed partial colocalization between TUFM and SIRT7, suggesting that TUFM may also regulate the subcellular localization of SIRT7. Future experiments examining changes in SIRT7 distribution upon TUFM depletion will be critical for determining whether TUFM modulates SIRT7 compartmentalization and function. This may represent a novel regulatory mechanism by which TUFM influences mitochondrial function and metabolic reprogramming through spatial control of SIRT7 activity. The RhoA/ROCK/AKT signalling pathway has been recognized as a key regulator of metabolic reprogramming and EMT [47]. A classic hallmark of EMT progression is the downregulation of E-cadherin, which reduces intercellular adhesion and leads to the loss of epithelial characteristics, whereas the upregulation of N-cadherin enhances the migratory and invasive capacities of cells [48]. Our previous work demonstrated that RhoA-dependent signalling contributes to the pathological progression of EMS by upregulating the expression of EMT-associated cytoskeletal proteins [49]. Moreover, activated RhoA and its downstream effector ROCK have been shown to mediate the expression and translocation of GLUT1 to the plasma membrane, thereby affecting glucose uptake and stimulating the Warburg effect [50, 51]. Moreover, AKT serves as a central hub in the PI3K–AKT–mTOR pathway, driving metabolic reprogramming through multiple mechanisms. It upregulates GLUT1 expression and activates key glycolytic enzymes, including Hexokinase 2 (HK2) and pyruvate kinase M2 (PKM2), increasing glycolytic flux [52]. Additionally, AKT regulates oxidative phosphorylation by inducing pyruvate dehydrogenase kinase 1 (PDK1), which inhibits the pyruvate dehydrogenase complex, thereby diverting pyruvate away from the TCA cycle towards lactate production under hypoxic conditions [53]. Collectively, these studies highlight a critical link between RhoA/ROCK/AKT signalling and metabolic reprogramming, suggesting that similar mechanisms may be involved in the metabolic alterations observed in EMS. Our findings indicate that the TUFM–SIRT7 axis coordinately regulates the RhoA/ROCK/AKT signalling pathway and the EMT process, thereby influencing cell proliferation and migration. This finding further strengthens the evidence for a functional connection between TUFM and SIRT7. Although changes in pathway activity were observed following the modulation of SIRT7 expression, the direct mechanistic relationship between SIRT7 and components of this pathway remains to be elucidated. Moreover, intracellular signalling is highly interconnected, and other pathways may work in concert with or counteract the RhoA/ROCK/AKT axis to regulate metabolism and cell behaviour in EMS. Pathways such as the AMPK/mTOR [54] and HIF-1α [55] pathways have been widely implicated in metabolic regulation and may interact with SIRT7-mediated signalling cascades. Further investigations are warranted to clarify the precise molecular mechanisms underlying these regulatory relationships. Increasing evidence suggests that metabolic reprogramming not only affects short-term cellular behaviours but also contributes to the establishment of a profibrotic, chronically inflamed microenvironment that facilitates lesion persistence and progression. In various disease models, dysregulated glycolysis and mitochondrial dysfunction have been shown to sustain inflammatory cytokine production and activate profibrotic signalling pathways, ultimately driving disease chronicity [57]. Interestingly, SIRT7 has been reported to play crucial roles in the regulation of both fibrosis and inflammation in other pathological conditions. For example, SIRT7 has been confirmed to promote inflammation in hyperglycaemic endothelial cells via the modulation of gene transcription [58] and to increase cardiac fibrosis by inhibiting mitochondrial biogenesis in cardiomyocytes [59]. Furthermore, the RhoA/ROCK/AKT signalling pathway, which was confirmed to be activated by SIRT7 in our study, has been widely implicated in tissue fibrosis and chronic inflammation [60]. Fibrosis and chronic inflammation are well-established pathological features of EMS [56]. Given these observations, future studies are needed to determine whether SIRT7-driven metabolic changes directly influence fibrotic remodelling and chronic inflammation in endometriotic lesions. Although our study focused primarily on the molecular mechanisms by which SIRT7 and TUFM regulate the progression of EMS, the clinical translation of these findings is an important direction for future research. Given the aberrant expression of SIRT7 and TUFM observed in ectopic endometrial tissues, assessing the expression levels of SIRT7 and TUFM in clinical samples could help identify patient subgroups who may benefit from targeted therapeutic strategies. Moreover, the development of small-molecule inhibitors or modulators specifically targeting SIRT7 or TUFM may offer a novel avenue for nonsurgical treatment of EMS. Future studies should explore noninvasive detection strategies and validate the clinical applicability of these potential biomarkers.

In conclusion, our study identified SIRT7 as a critical driver of metabolic reprogramming and ectopic lesion growth in EMS. SIRT7 increased cellular proliferation and migration by modulating key metabolic enzymes and activating the RhoA/ROCK/AKT signalling pathway. Furthermore, TUFM contributed to the regulation of metabolic homeostasis and cellular functions by affecting SIRT7 protein expression. Together, these findings reveal a novel TUFM–SIRT7 axis that mediates oxidative stress and metabolic reprogramming in EMS, underscoring the pivotal role of metabolic regulation in disease pathogenesis and offering new insights into the underlying mechanisms of EMS. Data availability The data are available from the corresponding author upon reasonable request. Abbreviations - EMS: - Endometriosis - ROS: - Reactive oxygen species - ESCs: - Endometrial stromal cells - HESCs: - Human endometrial stromal cells - EMT: - Epithelial‒mesenchymal transition - Norm/NC: - Eutopic endometria of nonendometriosis patients - Eut/EU: - Eutopic endometria of endometriosis patients - Ect/EC: - Ectopic endometria of endometriosis lesions - TUFM: - Tu translation elongation factor, mitochondrial - AAV: - Adeno-associated virus

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We sincerely thank Dr. Qiansheng Huang, Dr. Weidong Zhou, and Dr. Rongfeng Wu for their valuable feedback and insightful comments on this research. Funding This work was supported by the National Natural Science Foundation of China (no. 82271678, no. 82171638 and no. 82301852). Author information Authors and Affiliations Contributions Huaying Zhang and Jiahao Chen designed the study, interpreted the results, and drafted the manuscript. Mengjie Yang and Xinyu Ding conducted the data analysis. Guolin Chai and Ruofan Huang performed the animal experiments. Dianchao Lin collected the clinical samples and patient information. Zhixiong Huang and Qionghua Chen revised the manuscript and provided financial support. All the authors read and approved the final manuscript. Huaying Zhang and Jiahao Chen contributed equally to this work. Corresponding authors Ethics declarations Ethical approval This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Xiamen University (Approval Number KY2021-031). Informed consent was obtained from all individual participants included in the study. All procedures were conducted in accordance with the Declaration of Helsinki. Competing interests The authors have no relevant financial or nonfinancial interests to disclose. Additional information Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Huaying Zhang and Jiahao Chen are co-first authors. Supplementary Information Below is the link to the electronic supplementary material. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. About this article Cite this article Zhang, H., Chen, J., Yang, M. et al. SIRT7 drives energy metabolic shifts in endometriosis via interaction with TUFM and Rhoa/Rock/Akt pathway activation. Cell. Mol. Life Sci. 82, 381 (2025). https://doi.org/10.1007/s00018-025-05886-4 Received: Revised: Accepted: Published: Version of record: DOI: https://doi.org/10.1007/s00018-025-05886-4