{"paper_id":"bc19e0bc-d967-42e9-9805-bf4b90d74c51","body_text":"R E S E A R C H Open Access\nHSF1 promotes endometriosis development\nand glycolysis by up-regulating PFKFB3\nexpression\nYixin Wang, Jing Xiu, Tingting Yang, Chune Ren * and Zhenhai Yu *\nAbstract\nBackground: Endometriosis is a chronic hormonal inflammatory disease characterized by the presence of\nendometrial tissue outside the uterus. Endometriosis often causes infertility, which brings physical and mental pain\nto patients and their families.\nMethods: We examined the functions of heat shock factor 1 (HSF1) in endometriosis development through cell\ncount assay, cell-scratch assay and clone formation experiments. We used quantitative real-time PCR (qRT-PCR) and\nWestern blot (WB) to detect HSF1 expression. Glucose and lactate levels were determined using a glucose (GO)\nassay kit and a lactate assay kit. Furthermore, we used a HSF1 inhibitor-KRIBB11 to establish a mouse model of\nendometriosis.\nResults: Our data demonstrated that HSF1 promoted endometriosis development. Interestingly, HSF1 enhanced\nglycolysis via up-regulating PFKFB3 expression in endometriosis cells, which was a key glycolysis enzyme. Consistently,\nthe HSF1 inhibitor KRIBB11 could abrogate endometriosis progression in vivo and in vitro.\nConclusions: Findings indicate that HSF1 plays an important role in endometriosis development, which might become a\nnew target for the treatment of endometriosis.\nElectronic supplementary material:Supplementary data are available.\nKeywords: HSF1, PFKFB3, Inhibitor, Glycolysis, Endometriosis\nBackground\nEndometriosis is a disease with features of chronic inflam-\nmation, which is defined as the functional endometrial\nstroma and glands outside the uterine cavity [ 1]. The main\nclinical manifestations of endometriosis are lower abdom-\ninal pain, dysmenorrhea, infertility, sexual discomfort, ab-\nnormal menstruation, local periodic pain, and bleeding.\nThere are approximately 6– 10% of women of childbearing\nage suffering from endometriosis in the world, and the in-\nfertility rate among them is as high as 50%, seriously af-\nfecting the women health [ 2]. Endometriosis is mainly\naffected by estrogen and progesterone, which promote\nendometrial tissue proliferation, survival, and inflamma-\ntion [ 3]. In addition, the development of endometriosis is\nalso related to progesterone resistance [ 4]. The most com-\nmon theory of endometriosis is the implantation theory,\nwhich may be related to genetic and immune inflamma-\ntory factors [ 5, 6]. However, there is still no effective drug\nto treat endometriosis.\nIn eukaryotes, there are many stressors that can cause\nprotein damage and induce an evolutionally conserved\ncytoprotective mechanism, the heat shock response\n(HSR), to maintain protein stability [ 7]. The heat shock\nfactor 1 (HSF1) plays a central role in refolding or de-\ngrading intracellular proteins [ 8]. HSF1 is a transcription\n© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,\nwhich permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give\nappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if\nchanges were made. The images or other third party material in this article are included in the article's Creative Commons\nlicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons\nlicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain\npermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.\nThe Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the\ndata made available in this article, unless otherwise stated in a credit line to the data.\n* Correspondence: chuneren@163.com; tomsyu@163.com\nDepartment of Reproductive Medicine, Affiliated Hospital of Weifang Medical\nUniversity, Weifang, Shandong Province, People ’s Republic of China\nWang et al. Reproductive Biology and Endocrinology           (2021) 19:86 \nhttps://doi.org/10.1186/s12958-021-00770-9\n\nfactor that can respond to endogenous and exogenous\ncellular stresses by inducing HSP expression, which\ncould facilitate the refolding of misfolded proteins [ 9].\nHSF1 also plays an important role in tumor develop-\nment, which seriously affects its prognosis [ 10]. For ex-\nample, HSF1 is highly expressed in prostate cancer, and\nplays its functions by increasing expression of its down-\nstream effector HSP27 [ 11]. Other tumors such as colo-\nrectal cancer, breast cancer, oral cancer, and liver cancer\nalso have a high HSF1 expression [ 7]. Furthermore,\nHSF1 can improve the tumor microenvironment to pro-\nmote its survival [ 12]. Therefore, HSF1 can be used as a\nnew therapeutic target. However, the roles of HSF1 in\nendometriosis are still largely unknown.\nThe increases of glucose metabolism are beneficial to\nthe endometriosis development, and abnormal expres-\nsions of glycolysis enzymes were detected in the endo-\nmetriosis cells [ 13]. Most normal cells mainly rely on the\noxidative metabolism to produce energy, but tumor cells\nstill choose to glycolysis pathway even in the sufficient\noxygen [ 14]. The glycolysis pathway could produce en-\nergy quickly, which could satisfy the cell rapid prolifera-\ntion [ 15]. Lactate produced from glycolysis promotes\nangiogenesis, cell invasion and immunosuppression,\nwhich promotes tumorigenesis [ 16]. Interestingly, endo-\nmetriosis cell made a shift from oxidative phosphoryl-\nation to aerobic glycolysis, which inhibits the production\nof reactive oxide species and then activates survival sig-\nnals [ 17]. Therefore, glycolysis could be considered as a\ntarget for the treatment of endometriosis. In the process\nof glycolysis, there is a key enzyme named 6-\nPhosphofructo-2-kinase/Fructose-2, 6-Biphosphatase 3\n(PFKFB3), which belongs to a family of bio-functional\nproteins [ 18]. There are four members of PFKFB family,\namong which PFKFB3 has the highest catalytic activ-\nity in glycolysis [ 19]. PFKFB3, as a key enzyme of gly-\ncolysis, regulates the process of glycolysis and plays\nan important role in the occurrence and development\nof many diseases [ 20, 21]. Therefore, PFKFB3 has be-\ncome a potential target for drug development [ 22].\nHowever, the role of PFKFB3 in endometriosis re-\nmains unclear.\nEndometriosis is a benign disease, but it has some\nclinical characteristics similar to the tumor, such as im-\nplantable, invasive, and distant metastasis. Moreover,\nHSF1 was previously reported to be overexpressed in\nendometriosis [ 23]. Therefore, we hypothesized that\nHSF1 also regulated the endometriosis development. To\ntest this hypothesis, we manipulated HSF1 expression in\nendometriosis cells, and used a constructed mouse\nmodel. Our data demonstrated that HSF1 promoted\nendometriosis development via enhancing PFKFB3 ex-\npression. Our study provides a new idea for the clinical\ntreatment of endometriosis by targeting HSF1.\nMaterials and methods\nCell culture and antibodies\nThe endometriotic epithelial cell line (11Z) was established\nby Professor Anna Strazinski-Powitz [ 24]. The human\nendometrial stromal cell line (ESC) was established by Dr.\nKrikun [ 25]. All cell lines were cultured in Dulbecco ’ s\nModified Eagle Medium/Ham’ s F-12 50/50 Mix (DMEM/\nF-12) supplemented with 10% FBS (Gibco, Carlsbad, CA,\nUSA) with 100 μg/mL penicilin and 100 μg/mL strepto-\nmycin at 37 °C and 5% CO2.\nMouse anti- β-actin (A1978) was from Sigma-Aldrich,\nand dilution: 1:5000. Mouse anti-HSF1 (sc-17,757) was\nfrom Santa Cruz, and dilution: 1:1000. Rabbit anti-\nPFKFB3 (ab181861) was purchase from Abcam, and di-\nlution: 1:2000. KRIBB11 were obtained from Med Chem\nExpress (MCE), 50 mg/kg.\nSiRNA and transfection\nThe sequence of small interfering (si) RNAs against\nHSF1 was 5 ′- GCAGGUUGUUCAUAGUCAGAA-3 ′.\nThe sequence of Control (Negative Control) was 5 ′-\nUUCUCCGAACGGUCACGU-3′ [26]. The transfection\nwas performed as described previously [ 27].\nWestern blot\nThe indicated cells were collected and lysed on ice using\nlysis buffer (Beyotime, Shanghai, China, P0013), and\nwere centrifuged at 12000 rpm at 4 °C for 15 min. Then,\n5 × loading buffer was added to the sample, and boiled\nfor 10 min. The protein was separated by SDS-PAGE and\ntransferred to PVDF membrane. After blocking, immuno-\nblot assay was performed using indicated antibodies,\nwhich was performed as described previously [ 28].\nQuantitative real-time PCR\nThe isolation of total RNA from cells and the synthesis\nof cDNA were described above [ 29]. Quantitative real-\ntime PCR used SYBR Green PCR Master Mix (Takara)\nwith CFX96 Real-Time PCR detection system (Bio-Rad,\nshanghai, China).\nCell proliferation assay\nThe cells were transfected with the indicated plasmids.\nAfter 24 h, the transfected cells were reseeded in 24-well\nplates. The cell numbers were counted every 24 h for 4\ndays [ 28].\nColony-formation assay\nThe cells were transfected with indicated plasmids. After\n24 h, 500 transfected cells were reseeded in new six-well\nplates. After cultured for 10 – 14 days, the cells were fixed\nwith 4% paraformaldehyde for 15 min. Then, the cells\nwere stained with crystal violet for 20 min, and photo-\ngraphed [ 30].\nWang et al. Reproductive Biology and Endocrinology           (2021) 19:86 Page 2 of 10\n\nCell-scratch assay\nThe cells were transfected with indicated plasmids. After\n24 h, the transfected cells were reseeded in new 6-well\nplates. The pipette tip was used to draw a line and washed\nwith PBS. After 24 h, cells were photographed [ 31].\nGlucose consumption and lactate production\nThe cells were transfected with indicated plasmids. After\n24 h, the transfected cells were reseeded in new 6-well\nplates. After 1 day, and the culture mediums were col-\nlected to determine the concentration of glucose and\nlactic acid using glucose (GO) assay kit (Sigma,\n#GAGO20-1KT) and lactate assay kit (Biovision,\n#K627– 100). The methods were performed as described\npreviously [ 28, 30].\nAnimal experiments\nAnimal experiments have been approved by ethics Com-\nmittee of Weifang Medical University. We used 5-week-\nold BALB/c female mice, and the donor mice ( n =5 )\nwere injected with estradiol benzoate to promote endo-\nmetrial development. Estradiol benzoate was diluted\nwith oil and injected intramuscularly into the thigh of\ndonor mice, 3 μg/mouse, 2 times for 1 week. After 1\nweek, the uterus from donor mice was cut into pieces\nand intraperitoneally injected into the recipient mice.\nAfter 1 week, the mice in the experimental group ( n =7 )\nwere intraperitoneally injected HSF1 inhibitor KRIBB11,\nand the mice in the control group (n = 7) were injected\nwith normal saline, 2 times a week for 1 month. Then,\nthe mice were sacrificed to observe the endometrial\nlesion.\nTissue collection and immunohistochemistry\nAll tissues were obtained from endometriosis mouse\nmodel. The sections were embedded in paraffin, dried\nand dewaxed with xylene, then hydrated in ethanol.\nAntigen extract was heated in a microwave oven for 30\nmin, and incubated with 3% H 2O2 for 20 min to block\nendogenous peroxidase. Primary antibody was incubated\novernight at 4 °C, and secondary antibody was incubated\nin the next day. After staining with DAB, the nucleus\nwas stained with hematoxylin. Then, it is dehydrated in\nethanol and xylene. The immunohistochemistry was per-\nformed as described previously [ 27]. The immunostain-\ning intensity was quantified using the Image J [ 32].\nStatistical analysis\nAll statistical analyses were done using Graphpad Prism\n5.0 software. The statistical analyses were presented as\nmean ± SEM, and performed by two-tailed unpaired Stu-\ndent’ s t-test. P values < 0.05 were considered to be statis-\ntically significant. n.s. was not significant.\nResults\nHSF1 promotes cell proliferation, cell migration and clone\nformation in endometriosis cells\nTo determine whether HSF1 plays an important role in\nendometriosis, we manipulated HSF1 expression in\nendometriosis cells. We found that HSF1 overexpression\nsignificantly promoted cell proliferation in endometriosis\ncells (Fig. 1A). Moreover, cell-scratch tests and clone\nformation experiments revealed that HSF1 overexpres-\nsion promoted cell migration and growth in endometri-\nosis cells (Fig. 1B and C). Conversely, HSF1 knockdown\ninhibited the growth of endometriosis cells (Fig. 1D and\nF), and inhibited cell migration (Fig. 1E). These findings\nsuggest that HSF1 positively regulates cell proliferation\nand migration in endometriosis cells.\nHSF1 enhances glycolysis in endometriosis cells\nEndometriosis cells need high glycolysis in the process\nof rapid metastasis and growth [ 13]. To determine the\nfunctions of HSF1 in glycolysis, we overexpressed or\nknocked down HSF1 in endometriosis cells. Interest-\ningly, we found that HSF1 could increase both glucose\nconsumption and lactate production (Fig. 2A and B).\nSubsequently, to determine whether HSF1 inhibitor\nKRIBB11 could suppress glucose metabolism, we cul-\ntured endometriosis cells with KRIBB11. Consistently,\nKRIBB11 reduced the glucose consumption and lactic\nacid generation in endometriosis cells (Fig. 2A and B).\nThese data show that HSF1 enhances glycolysis in endo-\nmetriosis cells.\nHSF1 promotes PFKFB3 expression in endometriosis cells\nIn the previous experiment of the current study, we\nshowed that HSF1 promotes glycolysis in endometriosis\ncell. Therefore, we hypothesized that regulation of gly-\ncolysis by HSF1 might depend on key glycolytic en-\nzymes. So we selected three key enzymes in glycolysis to\nverify our hypothesis, PFKFB3, PKM2 and HK2. Expos-\ning cells to heat-shock in a time-dependent manner, the\nPFKFB3 expression were increased (Fig. 3A and B). But\nheat-shock activation had little effect on the PKM2 and\nHK2 expressions (Supplementary Fig. 1A and B). In\naddition, overexpression HSF1 increased PFKFB3 ex-\npression (Fig. 3C and D). Conversely, HSF1 knockdown\nresulted in a decrease in PFKFB3 expression (Fig. 3E and\nF). Taken together, our results indicate that HSF1 pro-\nmotes PFKFB3 expression in endometriosis cells.\nKRIBB11 inhibits endometriosis cell growth by targeting\nHSF1\nKRIBB11, a specific inhibitor of HSF1, effectively inhibits\nHSF1 activity, leading to cell cycle arrest in the G2/M\nphase, cell apoptosis, and inhibition of tumor cell prolif-\neration [ 33]. Cells were seeded in 24-well plates and\nWang et al. Reproductive Biology and Endocrinology           (2021) 19:86 Page 3 of 10\n\nFig. 1 (See legend on next page.)\nWang et al. Reproductive Biology and Endocrinology           (2021) 19:86 Page 4 of 10\n\n(See figure on previous page.)\nFig. 1 HSF1 promotes the cell proliferation, cell migration and clone formation in endometriosis cells. ( A) 11Z and ESC cells were transfected with\nHA-tagged HSF1 or empoty vector. After one day, cells were re-plated in 24-well plates, and cell counts were performed every 24 h to analyses\ncell growth. ( B) 11Z and ESC cells were transfected with HA-tagged HSF1 or empoty vector. After one day, cells were re-plated in 6-well plates to\nperform scratch test assay. ( C) 11Z and ESC cells were transfected with HA-tagged HSF1 or empoty vector. After one day, cells were re-plated in\n6-well plates, and were cultured for 10 –14 days to observe the cell clone formation. ( D-F) 11Z and ESC cells were transfected with siRNA-HSF1 or\nNC. Cell counting, scratching, and cloning were performed. All date are mean ± SD of three independent experiments (* P < 0.05)\nFig. 2 HSF1 enhances glycolysis in endometriosis cells. ( A, B) 11Z and ESC cells were transfected with HA-tagged HSF1 or empoty vector, siRNA\nor NC. Cells were re-plated in 6-well plates. After 24 h, glucose and lactic acid concentrations in culture medium were determine using glucose\nand lactic acid kits. 11Z and ESC cells were cultured with KRIBB11 for 24 h, and the concentration of glucose and lactic acid in the super-medium\nwas determined. All date are mean ± SD of three independent experiments (* P < 0.05)\nWang et al. Reproductive Biology and Endocrinology           (2021) 19:86 Page 5 of 10\n\ngiven an increasing concentration of KRIBB11. The IC 50\nvalues of two cell lines were measured (Fig. 4A). As we\nexpected, KRIBB11 inhibited the growth of endometri-\nosis cells (Fig. 4B and C). Cell-scratch tests indicated\nthat KRIBB11 inhibited the migration of endometrial\ncells (Fig. 4D). Western blot showed that PFKFB3 pro-\ntein level was reduced after HSF1 inhibition by KRIBB11\n(Fig. 4E). Thus, these data reveal that HSF1-specific in-\nhibitor KRIBB11 reduces the key glycolytic enzyme\nPFKFB3 expression by inhibiting HSF1, and ultimately\ninhibits endometriosis cell growth.\nKRIBB11 plays a therapeutic role in a mouse model of\nendometriosis\nTo determine whether KRIBB11 regulates endometriosis\ncell growth in vivo, the endometria of donor mice were\ncut up and intraperitoneally injected into recipient mice.\nAfter one week, a mouse model of endometriosis was\nestablished, the experimental group was treated with\nKRIBB11 and the control group was injected with nor-\nmal saline (Fig. 5A). At the end, the mice were sacrificed\nto observe ectopic lesion. Interestingly, all control mice\nwere observed the endometriosis tissues, but only two in\nFig. 3 HSF1 promotes PFKFB3 expression in endometriosis cells. ( A, B) 11Z and ESC cells were heat shocked in a time-dependent manner. The\nexpression of PFKFB3 was determined by Western blot and qRT-PCR. ( C, D) 11Z and ESC cells were transfected with HA-tagged HSF1 or empoty\nvector, and the expressions of PFKFB3 were detected. ( E, F) 11Z and ESC cells were transfected with siRNA-HSF1 or NC. The expressions of\nPFKFB3 were detected after 2 days. All date are mean ± SD of three independent experiments (*P < 0.05)\nWang et al. Reproductive Biology and Endocrinology           (2021) 19:86 Page 6 of 10\n\nthe mice treated with KRIBB11 were observed (Fig. 5B).\nEctopic lesions from mice treated with KRIBB11 grew\nsignificantly slower than those in control group. Consist-\nently, the weight of ectopic lesions from mice treated\nwith KRIBB11 was lower than in the control group (Fig.\n5C). Using immunohistochemical staining, we found that\nHSF1 expression was significantly lower in the mice\ntreated with KRIBB11 (Fig. 5D). Taken together, HSF1-\nspecific inhibitor KRIBB11 plays a therapeutic role in\nthe mouse model of endometriosis.\nDiscussion\nEndometriosis is an age-related disease of the reproductive\nsystem, and its prevalence is up to 10% in premenopausal\nwomen worldwide [ 6]. The diagnosis of endometriosis is\ndifficult, because experienced obstetricians and gynecolo-\ngists are required to correctly assess the clinical symptoms\nof this disease [ 34]. In recent years, more studies have\nbeen published on how to treat endometriosis. However,\nthe treatment of endometriosis is still a clinical challenge,\nwhich causes increased burdens to women of childbearing\nage. Because endometriosis cells have similar characteris-\ntics of invasion and metastasis with tumor cells, and HSF1\nis a carcinogen promoting tumor progression, so we\nspeculate that HSF1 plays a similar role in the occurrence\nand development of endometriosis. Our hypothesis is sup-\nported by a series of experiments. Our data show that\nHSF1 promotes endometriosis development, and en-\nhances glycolysis via up-regulating PFKFB3 in endometri-\nosis cells. In mice, we treat with HSF1 inhibitor KRIBB11,\nFig. 4 KRIBB11 inhibits endometriosis cell growth by targeting HSF1. ( A) 11Z and ESC cells were plated in 24-well plates. KRIBB11 was given in a\nconcentration-dependent manner, and the IC 50 value of the drug was measured by cell counting. ( B) 11Z and ESC cells were plated in 24-well\nplates, and the experimental group treated with HSF1 inhibitor KRIBB11. The cell counts were performed every 24 h for 4 days. ( C) 11Z and ESC\ncells were plated in 6-well plates, and the experimental group treated with HSF1 inhibitor KRIBB11. After 10 –14 days, the cell clones were\nanalyzed. (D) 11Z and ESC cells were plated in 6-well plates, and the scratches were made by pipette tip. Experimental group was treated with\nHSF1 inhibitor KRIBB11. After 24 h, the wound healing was analyzed. ( E) 11Z and ESC cells were plated in 6-well plates. The experimental group\nwas treated with HSF1 inhibitor KRIBB11. After 24 h, the cells are lysed for westen blot. All date are mean ± SD of three independent\nexperiments (*P < 0.05)\nWang et al. Reproductive Biology and Endocrinology           (2021) 19:86 Page 7 of 10\n\nwhich could effectively inhibit endometriosis develop-\nment. Taken together, HSF1 is a promising target for\nendometriosis.\nHSF1 is discovered in 1984 as the main regulator of\nHSR. HSF1 is activated after cell stress, which leads to the\nHSP expression to protect cells. After heat shock, HSF1 is\nphosphorylated, trimerized, and transferred to the nucleus\nto induce chaperone gene expression by binding to DNA\nsequence motifs known as heat shock elements (HSEs)\n[35]. HSF1 transcriptional activity mainly depends on the\nformation of trimer in the nucleus, and post-translational\nmodifications also could regulate its transcriptional activ-\nity, such as acetylation, phosphorylation, and methylation\n[36]. Specially, HSF1 is found to play an important role in\nmultiple cancers, which promotes cell invasion, migration,\nand proliferation of tumor cells [ 37]. Cancer cells rely on\nHSR to support their rapid growth and counteract the\nharmful mutations [ 38]. Previous studies have demon-\nstrated that HSF1 is highly expressed in endometrial car-\ncinoma and is closely related to endometrial invasion,\nwhich leads to a poor prognosis in estrogen receptor-\npositive tumors [ 39]. Endometriosis is also a highly\nestrogen-dependent disease [40]. Our study demonstrates\nthat HSF1 plays a crucial role in endometriosis develop-\nment, which is consistent with previous studies.\nMetabonomics can be used as a diagnostic tool to study\nthe metabolic changes under the physiological or patho-\nlogical state of disease [ 41]. The lipid metabolism [ 42],\namino acid metabolism [ 43], and glucose metabolism [ 13]\nin patients with endometriosis are increased. Women with\nendometriosis have high levels of cholesterol compared to\na control group without endometriosis [ 42]. Quantitative\nFig. 5 KRIBB11 plays a therapeutic role in a mouse model of endometriosis. ( A) Endometriosis model was established using 5-week-old BALB/c\nfemale mice. ( B) Mice were sacrificed, the endometriosis tissues and the weight of mice were analyzed (*P < 0.05). ( C) The size of the ectopic\ntissues was observed and heterotopic tissues were weighed (*P < 0.05). ( D) Immunohistochemical staining was performed to determine the HSF1\nexpression in the control group and the experimental group (Scale bars, 50 μm). Quantitative analyses of HSF1 expression was performed. ( E)\nHSF1 promoted glycolysis by up-regulating the expression of PFKFB3, which induced the development of endometriosis\nWang et al. Reproductive Biology and Endocrinology           (2021) 19:86 Page 8 of 10\n\nanalysis of lipid metabolites shows that the concentrations\nof phosphatidylcholine and phosphatidylserine in patients\nwith early endometriosis (I-II) are decreased, while the\nconcentrations of phosphatidylic acid are increased [ 44].\nEndometriosis is largely determined by estrogen synthesis\nand metabolism genetic factors, which increase the risk of\ndeveloping endometriosis [ 45]. Similar to tumor cells,\nWarburg Effect also occurs in stromal cells of endometrial\ntissues, which increases glucose consumption and lactate\nproduction [ 13]. In addition, increased glucose metabol-\nism may be the cause of excessive reactive oxygen species\nin endometriosis [ 46]. Moreover, the expression of both\naerobic and anaerobic glycolytic markers was increased in\nendometriosis patients, which ultimately contribute to\nendometriosis development [ 47]. As a rate-limiting en-\nzyme in glycolysis, PFKFB3 has the highest kinase activity\nto guide glucose into glycolysis. In our study, we find\nPFKFB3 is highly expressed and promotes glycolysis in\nendometriosis cells, which is consistent with previous\nstudy that glycolysis promotes endometriosis develop-\nment. Taken together, our findings provide some new in-\nsights into the functions of HSF1/PFKFB3 axis in\nendometriosis development, which is as a new target to\ntreat endometriosis (Fig. 5E).\nConclusions\nOur data show that HSF1 plays an important role in the\ndevelopment of endometriosis. HSF1 can regulate gly-\ncolysis process by up-regulating the expression of\nPFKFB3 and ultimately promote the growth of endomet-\nriosis, while HSF1 specific inhibitors can inhibit the\nabove effects. This will provide a new way of thinking\nfor the treatment of endometriosis in the future.\nAbbreviations\nHSF1: Heat shock factor 1; HSR: Heat shock response; HSP: Heat shock\nprotein; HSEs: heat shock elements; PCR: Polymerase chain reaction; qRT-\nPCR: Quantitative real-time PCR; WB: Western blot; PFKFB3: 6-Phosphofructo-\n2-kinase/Fructose-2, 6-Biphosphatase 3; DMEM/F-12: Dulbecco ’s Modified\nEagle Medium/Ham ’s F-12 50/50 Mix; FBS: Fetal bovine serum; CO 2: Carbon\ndioxide; RNA: Ribonucleic acid; siRNA: Small interfering RNA;\nDNA: Deoxyribonucleic acid; cDNA: Complementary DNA; PBS: Phosphate\nbuffer saline; PKM2: pyruvate kinase 2; HK2: Hexokinase 2; IC 50: 50% inhibiting\nconcentration; p-TEFb: Positive transcription elongation factor b;\nSEM: Standard error of the means; SD: Standard deviation; n.s.: Not significant\nSupplementary Information\nThe online version contains supplementary material available at https://doi.\norg/10.1186/s12958-021-00770-9.\nAdditional file 1: Supplementary Figure 1. Heat-shock activation had\nlittle effect on the PKM2 and HK2 expressions. (A) 11Z cells were heat-\nshocked for 0 or 10 min, and qRT-PCR was performed to analyze the HK2\nand PKM2 mRNA levels. (B) ESC cells were heat-shocked for 0 or 10 min,\nand qRT-PCR was performed to analyze the HK2 and PKM2 mRNA levels.\nAcknowledgments\nNot applicable.\nAuthors’ contributions\nZ.Y. and C.R. designed research; Z.Y. and Y.W. wrote and revised the paper.\nAll authors read and approved the final manuscript.\nFunding\nThe study was supported by research grants from National Natural Science\nFoundation of China (Grant no. 81972489) and National Natural Science\nFoundation of Shandong Province (Grant no. ZR2020YQ58).\nAvailability of data and materials\nThe data used in this study are available from the corresponding author on\nreasonable request.\nDeclarations\nEthics approval and consent to participate\nAll procedures performed in this study involving were in accordance with\nthe ethical standards of the institutional research committee of Weifang\nmedical university.\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors declare that they have no competing interests.\nReceived: 18 December 2020 Accepted: 28 May 2021\nReferences\n1. Mehedintu C, Plotogea MN, Ionescu S, Antonovici M. Endometriosis still a\nchallenge. J Med Life. 2014;7(3):349 –57.\n2. Giudice LC. Clinical practice. Endometriosis. N Engl J Med. 2010;362(25):\n2389–98. https://doi.org/10.1056/NEJMcp1000274.\n3. Olsarova K, Mishra GD. Early life factors for endometriosis: a systematic\nreview. Hum Reprod Update. 2020;26(3):412 –22. https://doi.org/10.1093/\nhumupd/dmaa002.\n4. 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