{"paper_id":"7741c09e-26c7-462f-bf08-6072c9a26c23","body_text":"Front. Biosci. (Landmark Ed) 2024; 29(12): 417\nhttps://doi.org/10.31083/j.fbl2912417\nCopyright: © 2024 The Author(s). Published by IMR Press.\nThis is an open access article under the CC BY 4.0 license .\nPublisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nOriginal Research\nThe PKM2/HIF-1α Axis is Involved in the Pathogenesis of\nEndometriosis via TGF-β1 under Endometrial Polyps\nJianjuan Li1,*,†, Li Liu 2,†, Ruiqi Fan 1\n1Department of Reproductive Medicine, Dongying People’s Hospital, 257091 Dongying, Shandong, China\n2Department of Obstetrics, Dongying People’s Hospital, 257091 Dongying, Shandong, China\n*Correspondence: 18554602275@163.com (Jianjuan Li)\n†These authors contributed equally.\nAcademic Editor: Jordi Sastre-Serra\nSubmitted: 26 July 2024 Revised: 5 November 2024 Accepted: 15 November 2024 Published: 17 December 2024\nAbstract\nBackground: Endometriosis patients exhibit a cancer-like glycolytic phenotype. The pyruvate kinase M2 (PKM2)/hypoxia-inducible\nfactor-1 alpha (HIF-1 α) axis plays important roles in glycolysis-related diseases, but its role in patients with endometrial polyps (EPs)\ncombined with endometriosis has not been validated. Methods: EP samples were collected from patients with and without endometrio-\nsis. PKM2, HIF-1 α, and transforming growth factor-beta 1 (TGF- β1) levels were detected by immunohistochemistry (IHC), quan-\ntitative polymerase chain reaction, western blotting, and/or immunofluorescence. Primary endometrial stromal cells (ESCs) and non-\nendometriotic patient-derived ESCs (NESCs) were isolated from patients with EP with or without endometriosis. PKM2 loss-of-function\nassays in ESCs and gain-of-function assays in NESCs were performed to assess the function of PKM2. The effects of PKM2 and TGF-\nβ1 on the promoter activity of HIF-1 α were determined by dual-luciferase reporter assay. Results: PKM2 was overexpressed in ESCs\ncompared to NESCs. Furthermore, PKM2 knockdown repressed viability, decreased migration and invasion, and restrained glycolysis\nof ESCs, accompanied by reduced HIF-1 α levels and weakened promoter activity of HIF-1 α. In addition, PKM2 overexpression had\nthe opposite effect on these indicators in NESCs. Of note, an anti-TGF- β1 Ab reversed the PKM2-overexpression-mediated effects on\ncell viability, migration, and invasion, but not glycolysis or HIF-1 α promoter activity, in NESCs. Additionally, PKM2, HIF-1 α, and\nTGF-β1 levels were higher in EP samples with endometriosis than in EP samples without endometriosis, and there were positive cor-\nrelations between PKM2, HIF-1 α, and TGF- β1 IHC scores in all EP samples. Conclusions: PKM2/HIF-1 α-axis-dependent glycolysis\nparticipates in the pathogenesis of EP combined with endometriosis by mediating TGF- β1 signaling.\nKeywords: EP; endometriosis; glycolysis; PKM2; HIF-1 α\n1. Introduction\nEndometriosis is a benign gynecological disorder\ncharacterized by the presence of endometrial glands and\nmesenchyme outside the uterine cavity and myometrium\n[1]. It is an estrogen-dependent disease that affects approx-\nimately 5–10% of women of reproductive age and is ac-\ncompanied by symptoms such as infertility, dyspareunia,\ndysmenorrhea, and chronic pelvic pain [ 2]. In addition, al-\nthough endometriosis has benign pathological features, it\nalso has cancer-like features, such as diffusion, invasion,\nand hyperplasia.\nEndometrial polyps (EPs) are localized hyperplastic\ngrowths of the endometrial glands and stroma that occur\nin up to 25% of women [ 3]. EPs can cause symptoms,\nsuch as abnormal uterine bleeding and infertility, which\ncan occur in women of any age. Clinical infertility studies\nhave found that the incidence of EPs is higher in patients\nwith endometriosis (46.7–68.4%) than in those without en-\ndometriosis, implying that the presence of EPs may be a\nkey factor in causing infertility in people with endometrio-\nsis [4,5]. Furthermore, patients with EPs and endometrio-\nsis have a higher probability of recurrence after polypec-\ntomy than those without endometriosis, implying that en-\ndometriosis may be associated with the pathogenesis of EPs\n[6]. In comparison to EPs, patients with endometriosis have\na longer latency period of approximately 1–5 years, such\nthat there may be cases where EPs are detected without en-\ndometriotic lesions. Therefore, recognition of the associa-\ntion between endometriosis and EPs is lacking. Exploring\nthe potential mechanism of the response to EP combined\nwith endometriosis may help in the early screening of pa-\ntients with EPs with or without endometriosis.\nRecently, patients with endometriosis have been re-\nported to exhibit a cancer-like glycolytic phenotype [ 7].\nEndometriotic growth is promoted by increased glucose\nmetabolism, and aberrant levels of glycolytic enzymes are\ndetected in endometriosis-derived endometrial stromal cells\n(ESCs) [8,9]. The key role of pyruvate kinase M2 (PKM2)\nin the regulation of glycolysis has been emphasized in a\nprevious study [ 10]. It has been shown that oxaloacetate\nboosts aerobic glycolytic effects by facilitating PKM2 ac-\ntivity [ 11]. In response to the decrease in PKM2 activity,\nmonomeric and dimeric forms of PKM2 translocate into the\nnucleus, where they interact with hypoxia-inducible factor-\n1 alpha (HIF-1 α) and mediate the expression of multiple\n\npro-glycolytic enzymes [ 12]. Available evidence suggests\na pro-promotional role of PKM2 in endometriosis [ 9,13].\nIncreased evidence demonstrates that HIF-1 α expression\nlevels are significantly increased in clinical endometrio-\nsis samples [ 14,15]. Furthermore, the inhibition of HIF-\n1α helps to arrest the progression of endometriosis, indi-\ncating that HIF-1 α plays a key role in endometriosis [ 16].\nAt present, the PKM2/HIF-1 α axis plays a vital role in\nglycolysis-related diseases, but its role in patients with EPs\nwith endometriosis has not been established.\nTherefore, we focused on investigating the function\nand mechanism of action of the PKM2/HIF-1 α axis in EPs\ncombined with endometriosis, which will help to better un-\nderstand the correlation between the two diseases and im-\nprove their clinical diagnosis and treatment.\n2. Methods and Materials\n2.1 Patients’ Samples\nForty-one patients with EPs who underwent hystero-\nscopic surgery at Dongying People’s Hospital during their\nmenstrual augmentation period were enrolled in the study.\nThe excised EP samples were divided into endometrio-\nsis (n = 23) and non-endometriosis (n = 18) groups, de-\npending on the presence or absence of endometriosis. Pa-\ntients with EPs, systemic inflammatory diseases, a history\nof hormonal therapy within 3 months prior to the opera-\ntion, uterine malformations, uterine adhesions, endometrial\ndysplasia, malignant neoplasia, or uterine fibroids were ex-\ncluded. One portion of the EP samples was fixed with\n4% paraformaldehyde (#G1101-500ML; Y uBioLab, Bei-\njing, China) and subjected to paraffin embedding for im-\nmunohistochemical (IHC) analysis, while the other portion\nwas used to isolate primary ESCs. The study was carried\nout in accordance with the guidelines of the Declaration of\nHelsinki and approved by the Ethics Committee of Dongy-\ning People’s Hospital (Approval Number 2024 [019]), and\nwritten informed consent was obtained from all patients or\ntheir families/legal guardians prior to tissue sample collec-\ntion.\n2.2 Tissue IHC Analysis\nProtein expression levels and cellular localization of\nPKM2, HIF-1 α, and transforming growth factor-beta 1\n(TGF-β1) in EP samples derived from endometriosis and\nnon-endometriosis groups were measured using IHC anal-\nysis. Briefly, 4-µm-thick sections were prepared from\nparaffin-fixed samples and mounted on silane-coated glass\nslides. Rehydration was performed in serial dilutions of\nethanol using a dewaxing reagent (#ST975; Beyotime,\nShanghai, China). Endogenous peroxidase activity was\nquenched using 1% hydrogen peroxide (#7722-84-1; Sigma\nAldrich, St Louis, MO, USA) for 15 min. After wash-\ning, the sections were incubated with primary antibod-\nies against PKM2 (#bs-0101R-1; Bioss, Beijing, China),\nHIF-1α (#ab51608; Abcam, Cambridge, MA, USA), and\nTGF-β1 (#ab215715; Abcam, Cambridge, MA, USA) at\n4 °C overnight. Horseradish-peroxidase-conjugated anti-\nrabbit IgG (#ab97051; Abcam, Cambridge, UK) was added\nand the samples were incubated for 25 min. After wash-\ning, the sections were incubated in 3,3-diaminobenzidine\n(#A690009; Sangon, Shanghai, China) in phosphate-\nbuffered saline (PBS) for 10 min and counterstained with\nhematoxylin to allow visualization of the immune com-\nplexes. Images were obtained using a microscope (Olym-\npus, Tokyo, Japan).\nAll slides were scored by an independent pathologist\nwho was not informed of the sample characteristics, and the\nfields of view were randomly selected at a magnification of\n×400. IHC results of PKM2, HIF-1 α, and TGF- β1 were\nquantified using the Allred score [ 17], which is the sum of\nthe scores of the proportion of positive cells (score range,\n0–5) and the response intensity (score range 0–3). The per-\ncentage of positive cells was scored as follows: absence of\npositive cells, 0; 1% positive cells, 1; 2–10% positive cells,\n2; 11–30% positive cells, 3; 31–66% positive cells, 4; and\n67–100% positive cells, 5. The staining intensity was de-\nfined as follows: 0, negative (no staining); 1, weakly pos-\nitive (yellow); 2, moderately positive (brown-yellow); and\n3, strongly positive (brown). Five fields of view were ran-\ndomly selected for each sample and the results were ana-\nlyzed using the semi-quantitative method. To avoid false\npositives or negatives, the primary antibody was replaced\nby PBS as a negative control, and tissues with known posi-\ntive expression of PKM2, HIF-1 α, and TGF- β1 were used\nas positive controls. The EP tissues in each group were di-\nvided into a negative group (scores of 0 or 2) and a positive\ngroup (scores ≥3).\n2.3 Isolation of primary ESCs and Nonendometriotic\nPatient-Derived ESCs (NESCs)\nFresh EP samples collected from patients with EP\nwith or without endometriosis under sterile conditions were\nminced finely and digested enzymatically with 5 mg of\ncollagenase I (500 µg/mL; #17100017; Gibco™, Thermo\nFisher Scientific, Waltham, MA, USA) and 1 mg de-\noxyribonuclease type I (100 µg/mL; #10325ES80; Y easen,\nShanghai, China) for 1 h at 37 °C. After centrifugation,\nthe cells were suspended in DMEM/F12 culture medium\ncontaining 1 nM estradiol (#IE0210; Solarbio, Beijing,\nChina), 0.2% insulin (#P3376; Beyotime), 1% L-glutamine\n(#ST1441-25g; Beyotime), 1% antibiotic solution (#SNA-\n001; Sunncell, Wuhan, China), and 10% dextran-coated\ncharcoal-treated fetal bovine serum (#SH30068.03; Hy-\nClone, Logan, UT, USA). The purity of the isolated ESCs\nwas confirmed >95% (P2-P3) by Immunocytochemistry\n(ICC) staining using antibodies against vimentin (stro-\nmal cell marker) (#FNab09409; Finetest, Wuhan, China)\nand cytokeratin (epithelia cell marker) (#bs-1712R; Bioss).\nNon-endometriotic patient-derived ESCs were designated\nas control cells and named non-endometriotic patient-\n2\n\n\nderived ESCs (NESCs). ESCs and NESCs from P3-P4 were\nused for subsequent experiments.\n2.4 Cell Transfection\nSmall interfering RNAs (siRNAs) targeting PKM2\n(si-PKM2#1, sense: GGAAAGAACAUCAAGAUAA TT,\nantisense: UUAUCUUGAUGUUCUUUCCTT; si-\nPKM2#2, sense: GGAAUGAACGUGGCUCGUUTT,\nantisense: AACGAGCCACGUUCAUUCCTT; or si-\nPKM2#3, sense: GGGUGAACUUGGCCAUGAA TT,\nantisense: UUCAUGGCCAAGUUCACCCTT) were\nutilized to interfere with PKM2 expression in ESCs,\nwith si-NC (sense: UUCUCCGAACGUGUCACGUTT,\nantisense: ACGUGACACGUUCGGAGAA TT) used as\na control. A PKM2-overexpressing (PKM2-OE) plasmid\nwas constructed by inserting the cDNA sequence of PKM2\n(NM_002654.6) into pcDNA3.1, with an empty vector as\na control. siRNAs (10 mM) were transfected into ESCs\nusing Lipofectamine RNAiMAX (Invitrogen, Carlsbad,\nCA, USA). The PKM2-OE plasmid was transfected\nusing Lipofectamine 3000 (Invitrogen) according to the\nmanufacturer’s instructions.\n2.5 Reverse Transcription (RT)-Quantitative Polymerase\nChain Reaction (qPCR)\nTRIzol reagent (#15596026; Thermo Fisher Sci-\nentific) was used to extract total RNA from ESCs or\nNESCs. The purity and concentration of the RNA were\nverified by measuring the absorbance ratio at 260/280 nm.\nSubsequently, cDNA was generated using M-MLV reverse\ntranscriptase (#AE101-03; TransGen Biotech, Beijing,\nChina). qPCR was performed using iTaq Universal SYBR\nGreen Supermix (#1725121; Bio-Rad, Hercules, CA,\nUSA). PCR primers were synthesized by TsingKe (Beijing,\nChina). PKM2 expression was analyzed using the forward\nprimer, 5 ′-A TGTCGAAGCCCCA TAGTGAA-3′, and\nreverse primer, 5 ′-TGGGTGGTGAA TCAA TGTCCA-3′\nand TGF-β1 expression was analyzed using the forward\nprimer, 5 ′-TACCTGAACCCGTGTTGCTCTC-3′ and the\nreverse primer, 5 ′-GTTGCTGAGGTA TCGCCAGGAA-\n3′. The transcript levels of PKM2 were normalized\nto those of the housekeeping gene β-actin (actin),\nwhich was measured using the forward primer, 5 ′-\nCACCA TTGGCAA TGAGCGGTTC-3′ and reverse\nprimer, 5 ′-AGGTCTTTGCGGA TGTCCACGT-3′. The\n2−∆∆Cq method was applied to calculate the relative\nexpression levels.\n2.6 Western Blotting\nCell samples were lysed in ice-cold RIPA buffer\n(#R0010; Solarbio) supplemented with a protease inhibitor\ncocktail (#C600386; Sangon). Protein levels were quan-\ntified using a Pierce BCA kit (#23225; Thermo Fisher\nScientific). Equal amounts of protein (approximately\n50 µg) were separated by 12% sodium dodecyl sulfate-\npolyacrylamide gel electrophoresis and then transferred\nto polyvinylidene fluoride membranes (0.45 µm; #88585;\nThermo Fisher Scientific). Following blocking in 5% fat-\nfree milk, the membranes were incubated overnight at 4\n°C with primary antibodies against PKM2 (#bs-0101R-1;\nBioss), TGF-β1 (#ab215715; Abcam), or actin (#bs-0061R;\nBioss). After incubation, the membranes were incubated\nwith a horseradish-peroxidase-labeled goat anti-rabbit sec-\nondary antibody (#ab97051; Abcam). Protein bands were\ndetected using Dura Extended Duration Substrate (#34075;\nThermo Fisher Scientific). Band intensity was evaluated\nusing ImageJ software (NIH, Bethesda, MD, USA; version\n1.5) and normalized to the band intensity of actin.\n2.7 Cell Counting Kit-8 (CCK-8) Assay\nESCs/NESCs were plated on 96-well plates and incu-\nbated with or without an anti-TGF- β1 antibody (1 µg/mL)\nfor 48 h. To each well, 250 µL of CCK-8 solution (#C0039;\nBeyotime) was added. Two hours later, the absorbance\nat 450 nm was recorded using a plate reader (Biotek,\nWinooski, VT, USA; Elx808).\n2.8 Wound-Healing Assays\nESCs/NESCs (1 × 105) were seeded in 12-well plates\nand incubated until a subconfluent monolayer was formed.\nA sterile pipette tip (200 µL) was used to make a scratch-\nwound in the confluent monolayers. The cells were\nfurther cultured in Dulbecco’s modified Eagle medium\n(DMEM)/F12, with or without an anti-TGF-β1 antibody for\n24 h. Images were obtained using an inverted microscope\nand analyzed using ImageJ software.\n2.9 Transwell Invasion Assay\nESCs/NESCs (1 × 105) suspended in serum-free\nDMEM/F12 were seeded into the upper chambers of 24-\nwell Transwell plates pre-coated with Matrigel (#356234;\nCorning, New Y ork, NY , USA) and diluted at a ratio\nof 1:3. To the lower chamber, 10% fetal-bovine-serum-\nsupplemented DMEM/F12 (600 µL), with or without an\nanti-TGF-β1 antibody, was added. Non-invasive cells were\nremoved from the upper chambers after 24 h of incubation,\nand the remaining cells were fixed with 4% paraformalde-\nhyde and stained with 0.1% crystal violet (#E607309; San-\ngon) for 30 min. Observation and photography were per-\nformed under an inverted microscope, and counting was\nperformed using ImageJ software.\n2.10 Measurement of Glucose Uptake and Lactate\nProduction\nTransfected and non-transfected ESCs/NESCs (1 ×\n105) were incubated for 24 h under different treatments,\nfollowed by collection of the culture media. Quantifica-\ntion of glucose and lactate levels in the cell culture medium\nwas performed using a glucose assay kit (#GAGO20-1KT;\nSigma Aldrich, St Louis, MO, USA) or a lactate assay kit\n3\n\n(#ab65330; Abcam), respectively, according to the manu-\nfacturer’s instructions.\n2.11 Measurement of TGF-β1\nTGF-β1 levels in the supernatants of ESCs/NESCs\nwere determined using an enzyme-linked immunosorbent\nassay (ELISA) kit (#PT880; Beyotime) according to the\nmanufacturer’s instructions.\n2.12 Immunofluorescence (IF)\nCells coated on glass covers were allowed to grow\novernight to prepare the slides, which were then fixed\nwith 4% paraformaldehyde. After permeabilization with\n0.2% Triton X-100 (#9002-93-1; Solarbio), the cells were\nblocked with 1% bovine serum albumin (#9048-46-8; So-\nlarbio) and then incubated with a primary antibody against\nHIF-1α (#ab51608; Abcam) for 12 h at 4 °C. They were\nthen washed with PBS and incubated with a fluorescently\nlabeled secondary antibody (#ab150079; Abcam) for 1 h\nin the dark. Cell nuclei were stained with 4 ′,6-diamidino-\n2-phenylindole (#E607303; Sangon) at a concentration of\n1.43 µM (blue). The cells were observed and imaged using\na fluorescence microscope (Olympus).\n2.13 Determination of HIF-1 α Gene Promoter Activity\nTransfected and non-transfected ESCs/NESCs were\ntransiently transfected with the pGL3-HIF-1 α-promoter\nvector (0.5 µg) together with the Renilla luciferase plas-\nmid phRL-TK (#E2231; Promega, Madison, WI, USA) us-\ning Fugene HD transfection reagent (#E2311; Promega)\nand incubated with or without an anti-TGF- β1 anti-\nbody. A double-luciferase reporter assay system (#E1910;\nPromega) was used to detect luciferase activity.\n2.14 Statistical Analysis\nData presented in this paper represent at least three in-\ndependent experiments and are expressed as the mean ±\nstandard deviation. Statistical analyses were performed us-\ning GraphPad Prism software (version 8.0; GraphPad, San\nDiego, CA, USA). The normality of the data was deter-\nmined using the Shapiro-Wilk test. Comparisons between\ntwo groups were conducted using an unpaired Student’s t-\ntest. Data from more than two groups were analyzed using\none-way analysis of variance, followed by Tukey’s post-hoc\ntest. The correlation of IHC scores among PKM2, HIF-1 α,\nand TGF- β1 in all EP samples was analyzed using Pear-\nson’s correlation coefficient. p < 0.05 was considered sta-\ntistically significant.\n3. Results\n3.1 The Expression Level of PKM2 is Positively\nCorrelated with HIF-1α and TGF-β1 in EP Samples from\nPatients with EPs and Endometriosis\nTo explain the relationship between PKM2, HIF-1 α,\nand TGF- β1 in patients with EPs and endometriosis, we\nperformed IHC staining of EP samples from patients with\nEPs, with or without endometriosis (Fig. 1A). IHC staining\nidentified significantly higher protein levels of PKM2 ( p <\n0.0001), HIF-1α (p < 0.0001), and TGF-β1 (p < 0.0001) in\nEP samples combined with endometriosis (n = 23) than in\nEP samples without endometriosis (n = 18), with PKM2 and\nHIF-1α primarily localized in nucleus and cytoplasm, but\nTGF-β1 preferentially localized in the cytoplasm (Fig. 1A–\nD). Additionally, a positive correlation on IHC scores in all\nEP samples was observed between HIF-1 α and PKM2 (r =\n0.8951, p < 0.0001), HIF-1 α and TGF- β1 (r = 0.7563, p\n< 0.0001), as well as PKM2 and TGF- β1 (r = 0.7859, p\n= 0.002) (Fig. 1E–G). These results suggested that PKM2,\nHIF-1α, and TGF- β1 may be involved in the pathogenesis\nof EPs combined with endometriosis.\n3.2 PKM2 is Highly Expressed in Primary ESCs\nTo analyze PKM2 function, we isolated ESCs and\nNESCs from EP samples obtained from patients with and\nwithout endometriosis. The purity of the ESCs and NESCs\nwas determined by ICC staining using anti-vimentin and\nanti-cytokeratin antibodies. As illustrated in Fig. 2A, the\npurity of ESCs and NESCs exceeded 95% after passaging\nfor 2–3 generations. Subsequently, PKM2 expression at\nthe transcriptional and translational levels were assessed.\nThe data also show higher mRNA ( p = 0.0021) and pro-\ntein (p = 0.0388) levels of PKM2 in ESCs than in NESCs\n(Fig. 2B,C). Taken together, these results suggested that\nhigh PKM2 levels may be related to EPs combined with\nendometriosis.\n3.3 PKM2-Dependent Glycolysis Affects the Proliferative,\nMigratory, and Invasive Capacities of ESCs\nConsidering the up-regulation of PKM2 in ESCs, we\ninvestigated the function of PKM2 by interfering with\nPKM2 expression in ESCs using si-PKM2#1, si-PKM2#2,\nor si-PKM2#3. All three siRNAs repressed PKM2 at both\ntranscriptional and protein levels, and si-PKM2#2 ( p =\n0.0002 and p < 0.0001; si-PKM2#1, p = 0.0078 and p =\n0.0427; si-PKM2#3, p = 0.0158 and p = 0.0017), which\nhad the best knockdown efficiency, was selected for sub-\nsequent analyses (Fig. 3A,B). We observed higher viability\nin ESCs in comparison to NESCs ( p < 0.0001), but the vi-\nability of ESCs was impaired after PKM2 knockdown ( p\n= 0.0002), as evidenced by CCK-8 assays (Fig. 3C). Fur-\nthermore, ESCs possessed stronger migratory ( p < 0.0001)\nand invasive ( p = 0.0022) abilities than NESCs; however,\nPKM2 down-regulation reduced the migratory (p < 0.0001)\nand invasive ( p = 0.0027) abilities of ESCs (Fig. 3D,E).\nAs an important regulator of glycolytic enzymes, PKM2\npromotes lactate production and metabolic reprogramming.\nTherefore, we investigated the effect of PKM2 on glycol-\nysis in ESCs. The results showed a striking increase in\nglucose uptake ( p < 0.0001) and lactate production ( p <\n0.0001) in ESCs versus NESCs; however, these features\n4\n\n\nFig. 1. Pyruvate kinase M2 (PKM2) levels are positively correlated with hypoxia-inducible factor-1 alpha (HIF-1 α) and trans-\nforming growth factor-beta 1 (TGF- β1) levels in patients with endometrial polyp (EP). (A) Representative images ( ×200 and\n×400) of immunohistochemical (IHC) staining for PKM2, HIF-1 α, and TGF- β1 in EP samples from patients with EPs with or without\nendometriosis. Scale bars: 100 µm. (B–D) Scatterplots showing IHC scores for PKM2, HIF-1 α, and TGF- β1 in patients with EP with\n(n = 23) and without (n = 18) endometriosis ( ∗∗∗p < 0.001; unpaired Student’s t-test). (E–G) Correlation analysis of PKM2, HIF-1 α,\nand TGF-β1 IHC scores in all EP samples (n = 23). Bars represent the mean ± standard deviation (SD).\n5\n\nFig. 2. High levels of PKM2 are observed in primary ESCs. (A) Representative images of ICC staining for vimentin and cytokeratin\nin primary endometrial stromal cells (ESCs) and non-endometrial patient-derived ESCs (NESCs). Scale bars: 100 µm. (B,C) Relative\nmRNA and protein levels of PKM2 in primary ESCs and NESCs were detected by reverse transcription (RT)-quantitative polymerase\nchain reaction (qPCR) and western blotting, respectively (n = 3; ∗p < 0.05 and ∗∗p < 0.01; unpaired Student’s t-test). All bars represent\nthe mean ± SD.\nof ESCs were undermined upon PKM2 knockdown (glu-\ncose uptake, p < 0.0001; lactate production, p < 0.0001)\n(Fig. 3F,G). Previous reports have demonstrated the regu-\nlatory role of PKM2 in TGF- β1 signaling [ 18,19], we de-\ntermined the effect of PKM2 on TGF- β1 in ESCs. As\nexpected, a greater amount of TGF- β1 was released from\nESCs than NESCs (p < 0.0001), but the silencing of PKM2\nreduced the release of TGF- β1 from ESCs ( p < 0.0001)\n(Fig. 3H). Consistently, TGF- β1 mRNA and protein lev-\nels were strongly elevated in ESCs compared with those in\nNESCs ( p = 0.0001 and p = 0.0006), yet PKM2 silencing\nrepressed TGF-β1 mRNA and protein levels in ESCs ( p =\n0.0002 and p = 0.0004) (Fig. 3I,J). Collectively, these re-\nsults showed that PKM2-dependent glycolysis affects the\nproliferation, migration, invasion, and TGF-β1 secretion of\nESCs.\n3.4 PKM2 Silencing Restrains the Transcriptional Activity\nof HIF-1α in ESCs\nPKM2 has been reported to interact with HIF-1 α\nand stimulate the HIF-1 α transcriptional activation do-\nmain function. Therefore, we further determined whether\nPKM2 can mediate the transcriptional activity of HIF-1 α\nin ESCs. IF staining showed higher levels of HIF-1 α in\nESCs relative to NESCs, whereas PKM2 down-regulation\nrepressed HIF-1α expression in ESCs (Fig. 4A). To further\nvalidate this relationship, we constructed the luciferase re-\nporter gene vector pGL3-HIF-1 α-promoter encompassing\nthe promoter of the HIF-1α gene. Dual-luciferase reporter\nassays showed that the luciferase activity of the pGL3-HIF-\n1α-promoter vector was stronger in ESCs than in NESCs (p\n= 0.0004) (Fig. 4B). However, the luciferase activity of the\npGL3-HIF-1α-promoter vector was repressed in ESCs co-\ntransfected with si-PKM2 (p = 0.0027) (Fig. 4B). All results\nshowed that PKM2 promoted the transcriptional activity of\nHIF-1α in ESCs.\n3.5 Glycolysis Mediated by PKM2 Enhances the\nProliferation, Migration, and Invasion of NESCs via\nTGF-β1\nSince up-regulation of PKM2 promotes proliferation,\nmigration and invasion of ESCs, we introduced PKM2 into\nNESCs to verify that PKM2 is a key factor in EPs com-\nbined with endometriosis. Transfection of the PKM2-OE\nvector strongly upregulated PKM2 mRNA and protein lev-\nels in NESCs (both p < 0.0001) (Fig. 5A,B). PKM2 over-\nexpression significantly increased TGF-β1 mRNA and pro-\ntein levels ( p < 0.0001 and p = 0.0026) (Fig. 5C,D). Func-\ntional analyses demonstrated that PKM2 overexpression in-\ncreased the viability, migration, and invasion of NESCs (all\np < 0.0001), but these effects were reversed by the introduc-\ntion of an anti-TGF-β1 antibody (all p < 0.0001) (Fig. 5E–\nG), Furthermore, the up-regulation of PKM2 markedly in-\ncreased glucose uptake and lactate production by NESCs\n(both p < 0.0001), but the addition of an anti-TGF- β1 anti-\nbody had no effect on PKM2-mediated increases in glucose\nuptake and lactate production ( p = 0.9991 and p = 0.6620)\n(Fig. 5H,I). Importantly, PKM2 up-regulation enhanced the\nluciferase activity of the pGL3-HIF-1α-promoter in NESCs\n(p < 0.0001), but the addition of an anti-TGF- β1 antibody\n6\n\n\nFig. 3. PKM2-dependent glycolysis affects the proliferation, migration, and invasion of ESCs. (A,B) The PKM2-interference\nefficiency of si-PKM2#1, si-PKM2#, and si-PKM2#3 in ESCs was detected by reverse transcription (RT)-quantitative polymerase chain\nreaction (qPCR) and western blotting (n = 3; ns,p > 0.05 vs. control, #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. the negative control small\ninterfering RNAs (siRNAs) [si-NC]; one-way analysis of variance [ANOV A]). (C–E) The viability, migration, and invasion of NESCs,\nESCs, and ESCs transfected with si-NC or si-PKM2 were determined by Cell Counting Kit-8 (CCK-8), wound-healing, and Transwell\ninvasion assays (n = 3; ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. NESCs, ns, p > 0.05 vs. ESCs, and ##p < 0.01 and ###p < 0.001 vs. ESCs\n+ si-NC; one-way ANOV A). Scale bars: 100 µm. (F,G) Glucose uptake and lactate production were measured in different subgroups\nof cells using respective kits (n = 3; ∗∗∗p < 0.001 vs. NESCs, ns, p > 0.05 vs. ESCs, and ###p < 0.001 vs. ESCs + si-NC; one-way\nANOV A). (H) The amount of TGF-β1 released from different subgroups of cells was measured by enzyme-linked immunosorbent assay\n(ELISA; n = 3; ∗∗∗p < 0.001 vs. NESCs, ns, p > 0.05 vs. ESCs, and ###p < 0.001 vs. ESCs + si-NC; one-way ANOV A). (I,J) Relative\nmRNA and protein levels of TGF- β1 were determined by RT-qPCR and western blotting (n = 3; ∗∗∗p < 0.001 vs. NESCs, ns, p > 0.05\nvs. ESCs, and ###p < 0.001 vs. ESCs + si-NC; one-way ANOV A). Bars represent the mean ± SD.\n7\n\nFig. 4. PKM2 mediates the transcriptional activity of HIF-1 α. (A) Representative images of immunofluorescence staining for HIF-\n1α in NESCs, ESCs, and ESCs transfected with si-NC or si-PKM2. Scale bars: 100 µm. (B) Dual-luciferase reporter assays were used\nto determine the luciferase activity of the pGL3-HIF-1α-promoter vector in NESCs, ESCs, and ESCs transfected with si-NC or si-PKM2\n(n = 3; ∗∗∗p < 0.001 vs. NESCs, ns, p > 0.05 vs. ESCs, and ##p < 0.001 vs. ESCs + si-NC; one-way ANOV A). Bars represent the mean\n± SD.\ndid not affect the luciferase activity of this promoter vec-\ntor in NESCs overexpressing PKM2 ( p = 0.1994), suggest-\ning that TGF- β1 is a downstream effector molecule of the\nPKM2/HIF-1α axis in NESCs (Fig. 5J). Together, these\ndata showed that PKM2/HIF-1α axis-dependent glycolysis\ncontributes to NESC proliferation, migration, and invasion\nvia TGF-β1.\n4. Discussion\nA previous study has established a strong relation-\nship between endometriosis and EPs [ 20]; however, the\npathogenic mechanism underlying the frequent emergence\nof EPs in patients with endometriosis has not yet been\nfully clarified. One of the most widely accepted theories is\nthat estrogen affects the development of both endometrio-\nsis and EPs [ 21], and a close correlation exists between\nthe imbalance between the proliferation and apoptosis of\nESCs and these two diseases [ 22]. In addition, these two\ndisorders are associated with cytokine secretion, immune-\ninflammatory responses, oxidative stress, microecological\nimbalances, and metabolic disorders. Notably, there is a\nsignificant overlap in the pathogenesis of these diseases.\nThe pathogenesis of endometriosis combined with EPs may\nbe a consequence of their interactions in the same patholog-\nical environment. Another possibility is that the emergence\nof one disease leads to a change in the microenvironment,\nwhich triggers the subsequent occurrence of another disease\nwhen the environmental change reaches a certain thresh-\nold. Therefore, investigating the underlying mechanisms\nof EPs combined with endometriosis is essential to enhance\nthe recognition of the two diseases, prevent clinical under-\ndiagnosis, and reduce the risk of the mutual induction of the\ntwo diseases.\nIncreased glycolysis is closely associated with en-\ndometriosis progression [ 23]. Horne et al . [ 24] reported\nthat mitochondrial respiration was significantly reduced,\nand glycolysis levels were higher in peritoneal mesothelial\ncells derived from the pelvic peritoneum of patients with en-\ndometriosis. In parallel, ESCs derived from patients with\nendometriosis possess metabolic reprogramming changes\n[25,26]. Moreover, oral administration of the pyruvate de-\nhydrogenase (PDH) activator dichloroacetate reduces the\nlactate concentration in mouse peritoneal fluid and shrinks\nendometriotic lesions in mouse models of endometriosis\n[24]. PKM2 is a rate-limiting enzyme in glycolysis and\nit promotes the progression of endometriosis. Wang et al .\n[27] reported that PKM2 is overexpressed in ovarian en-\ndometriosis, and PKM2 up-regulation mediated by PIM2\nfacilitates the fibrosis and glycolysis of ESCs. Further-\nmore, PKM2 down-regulation repressed metastasis, pro-\nliferation, and glycolysis in ESCs derived from patients\nwith endometriosis, via the m6A-dependent regulation of\nfat mass and obesity-associated gene-mediated autophagy-\nrelated protein 5 (A TG5) expression [ 13]. In addition, nu-\nclear factor kappa B-induced transcription of PKM2 is re-\npressed by cinnamic acid in ESCs derived from patients\nwith endometriosis, thus repressing glycolysis, invasion,\n8\n\n\nFig. 5. The PKM2/HIF-1 α axis enhances the viability, migration, and invasion of NESCs via TGF- β1. (A,B) The transfection\nefficiency of the PKM2- overexpression (OE) vector in NESCs was evaluated by RT-qPCR and western blotting (n = 3; ns, p > 0.05\n> 0.05 vs. control and ∗∗∗p < 0.001 vs. vector; one-way ANOV A). (C,D) Relative mRNA and protein levels of TGF- β1 in NESCs\nand NESCs transfected with empty vector or the PKM2-OE were determined by RT-qPCR and western blotting (n = 3; ns, p > 0.05\n0.05 vs. control and ∗∗p < 0.01, ∗∗∗p < 0.001 vs. vector; one-way ANOV A). (E–G) The viability, migration, and invasion of cells\nin different subgroups (ESCs, NESCs, NESCs + vector, NESCs + PKM2-OE, and NESCs + PKM2-OE + anti-TGF- β1 antibody [Ab])\nwere determined by CCK-8, wound healing, and Transwell invasion assays (scale bar = 100 µm) (n = 3; ns, p > 0.05 and ∗∗∗p < 0.001;\none-way ANOV A). (H,I) Measurement of glucose uptake and lactate production in cells from different subgroups was performed using\nappropriate kits (n = 3; ns, p > 0.05 and ∗∗∗p < 0.001; one-way ANOV A). (J) Luciferase activity of the pGL3-HIF-1α promoter vector\nwas determined using a dual-luciferase reporter assay in cells with different treatments (n = 3; ns, p > 0.05 and ∗∗∗p < 0.001; one-way\nANOV A). Bars represent the mean ± SD.\n9\n\nand viability [9]. These data highlight the promotional role\nof PKM2 in endometriosis; however, its involvement in the\nregulation of EPs in combination with endometriosis re-\nmains unclear. In the present study, we isolated primary\nESCs and NESCs from EP patients, with or without en-\ndometriosis, to explore the role of PKM2. Functional ex-\nperiments showed that PKM2 silencing repressed the via-\nbility, migration, invasion, and glycolysis of primary ESCs.\nHowever, PKM2 overexpression contributed to the viabil-\nity, migration, invasion, and glycolysis of NESCs, suggest-\ning that PKM2 may promote behavioral changes in NESCs\ntowards ESCs.\nIt has been found that the local hypoxic microenviron-\nment may also be an important factor in the development of\nendometriosis. Severe hypoxic stress is encountered when\nendometrial tissues are shed from the uterus retrogradely to\nthe peritoneal cavity and implanted into the ovary or peri-\ntoneum. Increasing evidence suggests that HIF-1 α is up-\nregulated in endometriosis and may be involved in the in-\nvasive process of ESCs. Previous studies reported that HIF-\n1α is highly expressed in the ectopic endometrium [ 15,28]\nand it facilitates ESC invasion, migration, and adhesion\n[15,29]. Feng et al. [ 30] demonstrated that nuclear translo-\ncation of PKM2 is responsible for mediating the function\nof HIF-1 α during the aerobic glycolytic transition. A re-\ncent study showed that the interaction of PKM2 with HIF-\n1α results in activation of HIF-1 α transcriptional activity,\nin a process dependent on the AC020978-induced nuclear\ntranslocation of PKM2 [ 31]. In this study, high levels of\nHIF-1α and PKM2 were detected in nuclei in EP samples\nfrom patients with EPs and endometriosis, and they were\npositively correlated with IHC scores in all EP samples, in-\ncluding those with and without endometriosis. In addition,\nHIF-1α was down-regulated in primary ESCs with PKM2\nknocked down, and this was coupled with lower levels of\nHIF-1α promoter activity. These findings indicated that\nPKM2 mediates the transcriptional activity of HIF-1 α in\npatients with EP and endometriosis.\nAs a multifunctional growth factor, TGF- β1 modu-\nlates diverse biological processes, including cell prolifer-\nation, differentiation, and angiogenesis [ 32]. It has been\nreported that the levels of TGF- β1 in serum, menstrual\nblood, and peritoneal fluid of patients with endometrio-\nsis were substantially higher than those of healthy women\n[33,34]. Moreover, high local levels of TGF- β1 can cre-\nate a suitable microenvironment, which may help ectopic\nendometrial cells in the pelvis to escape immune surveil-\nlance and survive through the regulatory effects of TGF- β1\non natural killer cells and macrophages [ 35]. In addition,\nhigh levels of TGF- β1 may exert a role in the formation\nof EPs [ 36]. The present study verified that TGF- β1 was\nhighly localized in the cytoplasm in EP samples from the\nendometriosis group, and there was a positive correlation\namong IHC scores of TGF- β1, PKM2, and HIF-1 α in all\nEP samples. Furthermore, the elevated secretion of TGF-\nβ1 by ESCs was reversed following PKM2 knockdown. In\naddition, the promoting effects of PKM2 on NESC viabil-\nity, migration, and invasion were counteracted by the addi-\ntion of an anti-TGF- β1 antibody. However, the anti-TGF-\nβ1 antibody did not affect the PKM2-mediated increase in\nglucose uptake, lactate production, or the promoter activity\nof HIF-1 α in NESCs, suggesting that TGF- β1 is a down-\nstream effector molecule of PKM2 in NESCs. These results\nmanifested that TGF- β1 may be involved in the pathogen-\nesis of EP combined endometriosis. Notably, PKM2 par-\nticipates in several diseases by regulating TGF- β1 signal-\ning [18,19]. HIF-1 α binds to the MH2 structural domain of\nphosphorylated mothers against decapentaplegic homolog\n3 (SMAD3) to convert the TGF- β function to glycolysis\n[37]. Moreover, miR-122-5p-mediated down-regulation of\nHIF-1α represses TGF- β1-induced cardiac fibroblast dif-\nferentiation [38]. Shi et al. [ 39] reported that the restoration\nof epidermal cell autophagy by bone marrow mesenchymal\nstem cells facilitates wound healing via the HIF-1 α/TGF-\nβ1/SMAD pathway in diabetes mellitus. All of this ev-\nidence indicates that TGF- β1 may be a signaling factor\nacting downstream of PKM2 and HIF-1 α. Thus, we in-\nferred that the PKM2/HIF-1α axis is related to EPs with en-\ndometriosis via TGF- β1. However, one of the limitations\nof the present study is the absence of confirmation of the\nrelationship between HIF-1 α and TGF- β1 by performing\nrelevant experiments, which were explored in the future by\ndual-luciferase reporter assays and rescue experiments. In\naddition, the small size of the clinical sample is a limitation\nof this study, as it may have made the results more sub-\nject to chance. Moreover, the selectivity of a single-center\nstudy has bias. Therefore, an expanded sample size from\nmultiple-centers is needed to further validate the results in\nthe future. Challengingly, clinical samples and related data\ncould not be collected from patients with mild endometrio-\nsis combined with EPs because they were only diagnosed\nbut not treated.\nImmune cells play a major role in the development\nof endometriosis and EPs, as evidenced by the secretion\nof cytokines associated with processes such as endothe-\nlial cell angiogenesis and proliferation, as well as a de-\ncreased ability to clear ectopic endothelial cells [ 40,41]. T\nlymphocytes are the primary component of cellular immu-\nnity, in which T-helper 17 cells 17 (Th17) are a subpop-\nulation derived from the differentiation of CD4 + T cells.\nTh17 cells can initiate an inflammatory response rapidly\nthrough neutrophil recruitment, activation, and migration\n[42]. Over-immunization with Th17 may induce uncon-\ntrolled neutrophil infiltration at the maternal-fetal interface\n[43]. Emerging evidence supports the involvement of Th17\ncells in the development of endometriosis lesions [ 44]. En-\ndometriosis may be associated with increased numbers and\nupregulated activity of Th17 cells in the peritoneum and\nectopic endometrial implants [ 45,46]. Moreover, the in-\ncreased number of Th17 cells in the peritoneal fluid dur-\n10\n\n\ning advanced stages of endometriosis may promote the de-\nvelopment of lesions [ 47]. The increased Th17 response is\nobserved in recurrent EPs [ 48]. Interestingly, PKM2 is re-\nquired for the regulation of Th17 cell differentiation and\nfunction [ 49,50]. Whether PKM2 regulates the aberrant\nproperties of ESCs by mediating Th17 cell activity in EPs\ncombined with endometriosis remains unclear and is a di-\nrection for future exploration.\n5. Conclusions\nIn summary, PKM2-dependent glycolysis facilitates\nbehavioral changes in NESCs towards ESCs by increasing\nthe transcriptional activity of HIF-1α and promoting the se-\ncretion of TGF- β1. This study helps to better understand\nthe pathogenesis of EPs combined with endometriosis and\nimplies the possibility of the combined targeting of PKM2,\nHIF-1α, and TGF- β1 for the early diagnosis and manage-\nment of EPs combined with endometriosis, with the aim of\nimproving the pregnancy rate of patients.\nAvailability of Data and Materials\nThe datasets used and/or analyzed during the current\nstudy are available from the corresponding author on rea-\nsonable request.\nAuthor Contributions\nJJL: Conceptualization, Formal analysis, Methodol-\nogy, Writing - original draft. LL: Conceptualization, For-\nmal analysis, Methodology, Writing - original draft. RQF:\nData curation, Investigation, Project administration, V ali-\ndation, Writing - review & editing. All authors read and ap-\nproved the final manuscript. All authors have participated\nsufficiently in the work and agreed to be accountable for all\naspects of the work.\nEthics Approval and Consent to Participate\nThe study was carried out in accordance with the\nguidelines of the Declaration of Helsinki and approved by\nthe Ethics Committee of Dongying People’s Hospital (Ap-\nproval Number 2024 [019]), and informed written con-\nsent was received from all patients or their families/legal\nguardians prior to tissue sample collection.\nAcknowledgment\nNot applicable.\nFunding\nThis research received no external funding.\nConflict of Interest\nThe authors declare no conflict of interest.\nReferences\n[1] Nezhat C, Khoyloo F, Tsuei A, Armani E, Page B, Rduch T,et al.\nThe Prevalence of Endometriosis in Patients with Unexplained\nInfertility. Journal of Clinical Medicine. 2024; 13: 444. https:\n//doi.org/10.3390/jcm13020444.\n[2] Capozzi V A, Scarpelli E, dell’Omo S, Rolla M, Pezzani A, Mor-\nganelli G, et al. 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