HSF1 promotes endometriosis development and glycolysis by up-regulating PFKFB3 expression

HSF1 promotes endometriosis development and glycolysis by up-regulating PFKFB3 expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research HSF1 promotes endometriosis development and glycolysis by up-regulating PFKFB3 expression Yixin Wang, Jing Xiu, Tingting Yang, Chune Ren, Zhenhai Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-134073/v2 This work is licensed under a CC BY 4.0 License Status: Under Review Version 2 posted 8 You are reading this latest preprint version Show more versions Abstract Background Endometriosis is a chronic hormonal inflammatory disease characterized by the presence of endometrial tissue outside the uterus. Endometriosis often causes infertility, which affects the body and mind of patients and their families. Methods We examined the functions of heat shock factor 1 (HSF1) in endometriosis development through cell count, scratch and clone formation experiments. We used quantitative real-time PCR (qRT-PCR) and Western blot (WB) to detect the functions of HSF1 in endometriosis cells. Glucose and lactate levels were determined using a glucose (GO) assay kit and a lactate assay kit. Furthermore, we established a mouse model of endometriosis by using a HSF1 inhibitor-KRIBB11. Results Our study demonstrated that HSF1 was highly expressed in endometriosis, and promoted endometriosis development. Interestingly, we found that HSF1 promoted glycolysis in endometriosis cells. Further, HSF1 enhanced glycolysis by up-regulating PFKFB3 in endometriosis cells, which was a key enzyme in glucose metabolism. Moreover, the HSF1 inhibitor KRIBB11 could abrogate endometriosis progression in vivo and in vitro . Conclusions Findings indicate that HSF1 plays an important role in the development of endometriosis, which might become a new target for the treatment of endometriosis and provide a new idea for the clinical treatment of endometriosis. Electronic supplementary material Supplementary data are available. Endocrinology & Metabolism Sexual & Reproductive Medicine HSF1 KRIBB11 PFKFB3 Glycolysis Endometriosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Endometriosis is a disease with features of chronic inflammation, and it is defined as the functional endometrial stroma and glands outside the uterine cavity [ 1 ]. The main clinical manifestations of endometriosis are lower abdominal pain, dysmenorrhea, infertility, sexual discomfort, abnormal menstruation and local periodic pain, bleeding and a mass. Approximately 6%-10% of women with endometriosis develop the disease, and the infertility rate is as high as 50%, seriously affecting the physical health of these women [ 2 ]. Endometriosis is mainly affected by estrogen and progesterone, which promotes endometrial tissue proliferation, survival, and inflammation [ 3 ]. Further, the development, progression, infertility, and chronic pelvic pain of endometriosis are associated with progesterone resistance [ 4 ]. The most common theory leading to endometriosis is the implantation theory, which may also be related to genetic factors and immune inflammatory factors [ 5 , 6 ]. However, there is still no clear treatment for endometriosis. In eukaryotes, various in vivo and in vitro stressors cause protein damage which induces an evolutionally conserved cellular protective mechanism, the heat shock response (HSR), to maintain protein stability [ 7 ]. The molecular chaperone heat shock factor 1 (HSF1) plays a central role in this process, helping to refold or degrade intracellular proteins [ 8 ]. HSF1 is an evolutionarily conserved transcription factor that can respond to endogenous and exogenous cellular stresses by inducing HSP expression and ultimately maintaining intracellular protein stability. HSF1 responds to stress by up-regulating HSP27 and HSP40, however, HSP70 and HSP90 facilitate the refolding of misfolded proteins [ 9 ]. HSF1 also plays an important role in various fields of tumor biology, promoting the occurrence and development of tumors and affecting the prognosis [ 10 ]. For example, HSF1 is highly expressed in prostate cancer PC-3 cells, and plays its functions by increasing levels of its downstream effector HSP27 [ 11 ]. Other tumors such as colorectal cancer, breast cancer, oral cancer, and liver cancer have also demonstrated high HSF1 expression [ 7 ]. Furthermore, HSF1 can change the survival microenvironment of tumors, promoting their survival under harsh microenvironments and being related to their prognosis [ 12 ]. Therefore, HSF1 can be used as a tumor marker and a new therapeutic target. However, the roles of HSF1 in endometriosis are still largely unknown. In glycolysis process, there is a key enzyme called 6-Phosphofructo-2-kinase/Fructose-2, 6-Biphosphatase 3 (PFKFB3), which belongs to a family of bio-functional proteins and is involved in fructose-2, 6-bisphosphate synthesis and degradation [ 13 ]. There are four members of the PFKFB family, but PFKFB3 has the highest kinase/phosphatase ratio in glycolysis [ 14 ]. So many studies on glucose metabolism in cancer are based on PFKFB3, which has become a potential target for much drug development [ 15 ]. PFKFB3 is widely expressed in tissues, particularly in solid tumors, proliferative tissues, leukemic cells, and transformed cells [ 13 ]. As a key enzyme in glycolysis, PFKFB3 regulates glycolysis and plays an important role in the development of many diseases [ 16 ]. However, the underlying mechanisms of PFKFB3 functions in endometriosis remain unclear. Morphologically, endometriosis is a benign disease, but it has some clinical characteristics similar to the tumor process, such as implantable, invasive, and distant metastasis. Moreover, HSF1 was previously reported to be overexpressed in endometriosis [ 17 ]. Therefore, we hypothesized that HSF1 also regulated the development of endometriosis. To test this hypothesis, we manipulated HSF1 expression in endometriosis cells, and used a constructed mouse model, which suggested that HSF1 influenced the development of endometriosis. Our study provides a new idea for the clinical treatment of endometriosis by targeting HSF1. Materials And Methods Cell culture and antibodies The endometriotic epithelial cell line (11Z) was established by Professor Anna Strazinski-Powitz [ 18 ]. The human endometrial stromal cell line (ESC) was established by Dr. Krikun [ 19 ]. All cell lines were cultured in Dulbecco’s Modified Eagle Medium/Ham’s F-12 50/50 Mix (DMEM/F-12) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) with 100mg/mL penicilin and 100mg/mL streptomycin at 37℃ and 5% CO 2 . Mouse anti-β-actin (A1978) was from Sigma-Aldrich, and dilution: 1:5000. Mouse anti-HSF1 (sc-17757) was from Santa Cruz, and dilution: 1:1000. Rabbit anti-PFKFB3 (ab181861) was purchase from ABCAM, and dilution: 1:2000. KRIBB11 were obtained from Med Chem Express (MCE), 50mg/kg. SiRNA and transfection The sequence of small interfering (si) RNAs against HSF1 was 5’- GCAGGUUGUUCAUAGUCAGAA-3’. The sequence of siRNA-NC (Negative Control) was 5’-UUCUCCGAACGGUCACGU-3’ [ 20 ]. The method was performed as described previously [ 21 ]. Western blot The indicated cells were collected and lysed on ice using lysis buffer, and were centrifuged at 12000rpm at 4℃ for 15min. Then, 5´loading buffer was added to the sample, and boiled for 10min. The western blot method was performed as described previously [ 22 ]. Quantitative real-time PCR The isolation of total RNA from cells and the synthesis of cDNA were described above [ 23 ]. Performing quantitative real-time PCR using SYBR Green PCR Master Mix (Takara) with CFX96 Real-Time PCR detection system (Bio-Rad, shanghai, China). Cell proliferation assay The indicated cells were transfected with the indicated plasmids, and reseeded in 24-well plates. The cell numbers were counted every 24hr for 4 days [ 22 ]. Colony-formation assay The 500 indicated cells were seeded in six-well plates, and cultured at 37℃ in 5% CO 2 for 10-14 days. After the clones were formed, the culture medium was removed and fixed at room temperature with 4% paraformaldehyde for 15min. After the fixation, the cells were stained with crystal violet, and then photographed [ 24 ]. Wound healing assay The indicated cells were inoculated in 6-well plates and cultured in medium until overgrown. The pipette tip was used to draw a fine line and washed with PBS. After 24hr, cells were photographed again [ 25 ]. Glucose consumption and lactate production The indicated cells were seeded in 6-well plates, and the culture mediums were collected after 24hr to determine the concentration of glucose and lactic acid. The methods were performed as described previously [ 22 , 24 ]. Animal experiments Animal experiments have been approved by ethics Committee of Weifang Medical University. We used 5-week-old BALB/c female mice, and the donor mice (n=5) were injected with estradiol benzoate to promote endometrial development. Estradiol benzoate was diluted with oil and injected intramuscularly into the thigh of donor mice, 3mg/mouse, 2 times for one week. After one week, the uterus of donor mice was cut into pieces and intraperitoneally injected into experimental (n=7) and control mice (n=7). After one week, the mice in the experimental group were intraperitoneally injected HSF1 inhibitor KRIBB11, and the mice in the control group were injected with normal saline in the same amount 2 times a week for one month. Then, the mice were sacrificed to observe the endometrial lesion. Tissue Collection and Immunohistochemistry All tissues were derived from mice model of endometriosis. The sections were embedded in paraffin, dried and dewaxed with xylene. The immunohistochemistry was performed as described previously [ 21 ]. The immunostaining intensity was quantified using the Image J [ 26 ]. Statistical analysis All statistical analyses were used Graphad Prism 5.0 software. The statistical analyses were presented as mean ± SEM, and performed by two-tailed unpaired Student’s t-test. P<0.05 is significant (*p<0.05). n.s.= not significant. Results HSF1 promotes the cell proliferation, cell migration and clone formation in endometriosis cells Endometriosis and tumorigenesis share similar characteristics, and previous studies have shown that HSF1 plays an important role in tumorigenesis [ 27 ]. To determine whether HSF1 plays a similar role in endometriosis, we performed a series of experiments in endometriosis cells. HSF1 overexpression significantly promoted the cell proliferation, indicating that HSF1 played a significant role in endometriosis (Fig. 1A). Moreover, cell-scratch tests and clone formation experiments revealed that HSF1 overexpression promoted the cell migration and growth in endometriosis cells (Fig. 1B and C). Furthermore, HSF1 knockdown inhibited the growth of endometriosis cells (Fig. 1D and F), and also inhibited the cell migration (Fig. 1E). These findings suggest that HSF1 positively regulates the cell proliferation and migration in endometriosis cells. HSF1 enhances glycolysis in endometriosis cells The endometriosis cells require high glycolysis during its rapid metastasis and growth [ 28 ]. To validate the effects of HSF1 on glycolysis, we overexpressed or knocked down HSF1 in endometriosis cells. Interestingly, we found that HSF1 could increase both glucose consumption and lactate production (Fig. 2A and B). Subsequently, to determine whether the HSF1 inhibitor KRIBB11 could suppress glucose metabolism, we cultured endometriosis cells with KRIBB11. As we expected, KRIBB11 reduced the glucose consumption and lactic acid generation in endometriosis cells (Fig. 2A and B). These data show that HSF1 enhances glycolysis in endometriosis cells. HSF1 promotes PFKFB3 expression in endometriosis cells In previous studies, we confirmed that HSF1 promoted glycolysis in endometriosis cells. Therefore, we hypothesized that HSF1 regulation glycolysis may depend on key glycolytic enzymes. By treating cells with heat shock in a time-dependent manner, the expression levels of PFKFB3 were increased (Fig. 3A and B). But HSF1 activation had little effect on the PKM2 and HK2 expressions (Supplementary Fig. 1A and B). In addition, overexpression HSF1 increased the expression of PFKFB3 (Fig. 3C and D). Furthermore, HSF1 knockdown resulted in a decrease in the expression of PFKFB3 (Fig. 3E and F). Taken together, our results indicate that HSF1 promotes PFKFB3 expression in endometriosis cells. KRIBB11 inhibits endometriosis cell growth by targeting HSF1 KRIBB11, a specific inhibitor of HSF1, effectively inhibits HSF1 activity, leading to cell cycle arrest in the G2/M phase, cell apoptosis, and inhibition of tumor cell proliferation [ 29 ]. The Cells were seeded onto 24-well plates were treated with increasing concentrations of KRIBB11, and the IC 50 values of the two cell lines were measured (Fig. 4A). As we expected, KRIBB11 inhibited the growth of endometriosis cells (Fig. 4B and C). Cell-scratch tests indicated that KRIBB11 inhibited the migration of endometrial cells (Fig. 4D). Moreover, western blot showed that the PFKFB3 protein level was reduced after HSF1 inhibition by KRIBB11 (Fig. 4E). Thus, these data reveal that the HSF1-specific inhibitor KRIBB11 reduces the expression of the key glycolytic enzyme PFKFB3 by inhibiting HSF1 expression, and ultimately inhibits the proliferation of endometriosis cells. KRIBB11 plays a therapeutic role in a mouse model of endometriosis To determine whether KRIBB11 regulates endometriosis in vivo , the endometria of donor mice were cut up and intraperitoneally injected into recipient mice, and a mouse model of endometriosis was established after one week (Fig. 5A). Two days after the last injection, the mice were sacrificed, and the abdominal cavity was opened to observe the ectopic lesion. Interestingly, the endometriosis tissues were observed in all control mice, but only two in seven experimental mice (Fig. 5B). Ectopic lesions without KRIBB11 grew significantly faster than those in the experimental group, and the weight of ectopic lesions with KRIBB11 was substantially lower than that of ectopic lesions in the control group (Fig. 5C). We performed immunohistochemical staining of ectopic tissue collected from mice, and HSF1 expression was significantly lower in the mice with KRIBB11 (Fig. 5D). The above results indicate that the HSF1-specific inhibitor KRIBB11 plays a therapeutic role in the mouse model of endometriosis. Discussion Endometriosis is an age-related disease of the reproductive system, and its prevalence is up to 10% in premenopausal women worldwide [ 6 ]. The diagnosis of endometriosis is difficult, because experienced obstetricians and gynecologists are required to assess the clinical symptoms of the disease and assess the existence of ectopic endometrium in the abdominal cavity and pelvis [ 30 ]. In recent years, more studies have been published on how to treat endometriosis. However, the treatment of endometriosis is still a challenge in clinical, which causes increased burdens to women of childbearing age. Moreover, endometriosis has the characteristics of invasion and metastasis, which is similar to tumor behavior. HSF1 is an oncogene to promote tumor progress, so we speculate that HSF1 plays a similar role in the development of endometriosis. Our hypothesis is supported by the finding that HSF1 promotes endometriosis development through a series of experiments, including glycolysis, cell counting, cloning, and cell scratching. Because endometriosis cells must get more energy to support rapid cell proliferation, glycolysis must be enhanced. As we expected, we find that HSF1 up-regulates PFKFB3 expression to promote glycolysis, thus accelerating the development of endometriosis. By increasing the expression of PFKFB3, the efficiency of glycolysis can be rapidly improved. Importantly, HSF1 regulates glucose metabolism through PFKFB3, ultimately influencing the development of endometriosis. Interestingly, these effects are abrogated using the HSF1-specific inhibitor KRIBB11, which bind to HSF1 to prevent HSF1-dependent recruitment of p-TEFb to HSP70 promoters [ 31 ]. The effects of KRIBB11 are demonstrated in vitro and in vivo . Therefore, HSF1 is a potential target for the treatment of endometriosis. However, the regulatory role of HSF1 in endometriosis is still elusive. Other mechanisms may be studied in the future. Our findings provide some new insights into the functions of HSF1 in endometriosis, which identifies a new pathway to treat endometriosis (Fig. 5E). Conclusions We have verified the important roles of HSF1 in endometriosis, which will provide new ideas for the treatment of endometriosis in the future. Abbreviations HSF1: Heat shock factor 1 HSR: Heat shock response HSP: Heat shock protein PCR: Polymerase chain reaction qRT-PCR: Quantitative real-time PCR WB: Western blot PFKFB3: 6-Phosphofructo-2-kinase/Fructose-2, 6-Biphosphatase 3 DMEM/F-12: Dulbecco’s Modified Eagle Medium/Ham’s F-12 50/50 Mix FBS: Fetal bovine serum RNA: Ribonucleic acid siRNA: Small interfering RNA DNA: Deoxyribonucleic acid cDNA: Complementary DNA PBS: Phosphate buffer saline PKM2: pyruvate kinase 2 HK2: Hexokinase 2 IC 50 : 50% inhibiting concentration p-TEFb: Positive transcription elongation factor b SEM: Standard error of the means SD: Standard devistion n.s.: Not significant Declarations Ethics approval and consent to participate All procedures performed in this study involving were in accordance with the ethical standards of the institutional research committee of Weifang medical university. Consent for publication Not applicable. Availability of supporting data The data used in this study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding The study was supported by research grants from National Natural Science Foundation of China (Grant no. 81972489) and National Natural Science Foundation of Shandong Province (Grant no. ZR2020YQ58). Authors’ contributions Z.Y. and C.R. designed research; Z.Y. and Y.W. wrote and revised the paper. All authors read and approved the final manuscript. Acknowledgments Not applicable. Authors’ information Affiliations Department of Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, 261000, Shandong Province, P.R. China. Yixin Wang, Jing Xiu, Tingting Yang, Chune Ren and Zhenhai Yu Corresponding author Correspondence to Chune Ren and Zhenhai Yu. References Mehedintu C, Plotogea MN, Ionescu S, Antonovici M: Endometriosis still a challenge. 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Qi X, Zhang Y, Ji H, Wu X, Wang F, Xie M, Shu L, Jiang S, Mao Y, Cui Y, Liu J: Knockdown of prohibitin expression promotes glucose metabolism in eutopic endometrial stromal cells from women with endometriosis. Reprod Biomed Online 2014, 29: 761-770. Yoon YJ, Kim JA, Shin KD, Shin DS, Han YM, Lee YJ, Lee JS, Kwon BM, Han DC: KRIBB11 inhibits HSP70 synthesis through inhibition of heat shock factor 1 function by impairing the recruitment of positive transcription elongation factor b to the hsp70 promoter. J Biol Chem 2011, 286: 1737-1747. Malvezzi H, Marengo EB, Podgaec S, Piccinato CA: Endometriosis: current challenges in modeling a multifactorial disease of unknown etiology. J Transl Med 2020, 18: 311. Kijima T, Prince T, Neckers L, Koga F, Fujii Y: Heat shock factor 1 (HSF1)-targeted anticancer therapeutics: overview of current preclinical progress . Expert Opin Ther Targets 2019, 23: 369-377. Supplementary Files FigureS1.pdf Supplementary Figure 1. (A) 11Z cells were heat-shocked for 0 or 10min, and qRT-PCR was performed to analyze the HK2 and PKM2 mRNA levels. (B) ESC cells were heat-shocked for 0 or 10min, and qRT-PCR was performed to analyze the HK2 and PKM2 mRNA levels. Cite Share Download PDF Status: Under Review Version 2 posted Editorial decision: Major revision 20 Feb, 2021 Review # 1 received at journal 09 Feb, 2021 Reviewer # 2 agreed at journal 07 Feb, 2021 Reviewers invited by journal 01 Feb, 2021 Reviewer # 1 agreed at journal 01 Feb, 2021 Editor assigned by journal 31 Jan, 2021 Submission checks completed at journal 31 Jan, 2021 Editor invited by journal 31 Jan, 2021 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-134073","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":10653931,"identity":"d906203b-6c8a-45ae-8baf-ea2c497858d2","order_by":0,"name":"Yixin Wang","email":"","orcid":"","institution":"Weifang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yixin","middleName":"","lastName":"Wang","suffix":""},{"id":10653932,"identity":"9049a70f-36f4-49c7-8677-d155e95382bb","order_by":1,"name":"Jing Xiu","email":"","orcid":"","institution":"Weifang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Xiu","suffix":""},{"id":10653933,"identity":"0ad07ea9-8919-42f7-a735-f9f6c39d07f5","order_by":2,"name":"Tingting Yang","email":"","orcid":"","institution":"Weifang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Yang","suffix":""},{"id":10653934,"identity":"6a9c084a-1565-4c02-8f1e-86b30ccddaab","order_by":3,"name":"Chune Ren","email":"","orcid":"","institution":"Weifang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chune","middleName":"","lastName":"Ren","suffix":""},{"id":10653935,"identity":"c68007d9-bea2-4393-a6ca-e8078548f552","order_by":4,"name":"Zhenhai Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAp0lEQVRIiWNgGAWjYPACGx5+/gYi1fJAqDQZyRkHSNNy2MagIYFILfbs3cmfedvO8xgwHGD88DGHGFt4zm6T5m27zWPO3MAsOXMbMVokcrcxg7RYNhxgY+YlUstmoMPO8RgcSCBeywagww6QouXM2W2Sc84l80jOONhMnF/Y23s3f3hTZmfPz9988MNHYrSAABMkchgbiFQPUvuDeLWjYBSMglEwEgEAyg8ykTMMJRAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8928-6890","institution":"Weifang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhenhai","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2020-12-22 13:08:13","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-134073/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-134073/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5828899,"identity":"dba65101-2953-4f85-a956-e574ba6742fe","added_by":"auto","created_at":"2021-02-10 16:20:25","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2662922,"visible":true,"origin":"","legend":"HSF1 promotes the cell proliferation, cell migration and clone formation in endometriosis cells. \n(A) 11Z and ESC cells were transfected with HA-tagged HSF1 or empoty vector. After one day, cells were re-plated in 24-well plates, and cell counts were performed every 24hr to analyses cell 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 perform 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 6-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 NC. Cell counting, scratching, and cloning were performed. All date are mean ± SD of three independent experiments (*P\u003c0.05).","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-134073/v2/a38d5a784e0359c4754610d0.jpg"},{"id":5829216,"identity":"a26d9c1d-611e-4deb-9b10-32e89d80d035","added_by":"auto","created_at":"2021-02-10 16:26:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1290136,"visible":true,"origin":"","legend":"HSF1 enhances glycolysis in endometriosis cells. \n(A, B) 11Z and ESC cells were transfected with HA-tagged HSF1 or empoty vector, siRNA or NC. Cells were re-plated in 6-well plates. After 24hr, glucose and lactic acid concentrations in culture medium were determine using glucose and lactic acid kits. 11Z and ESC cells were cultured with KRIBB11 for 24hr, and the concentration of glucose and lactic acid in the super-medium was determined. All date are mean ± SD of three independent experiments (*P\u003c0.05).","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-134073/v2/5855912091114f6b1a51eda7.jpg"},{"id":5828901,"identity":"cf85dd33-cb28-46ee-960f-99cd89fc4c88","added_by":"auto","created_at":"2021-02-10 16:20:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1334069,"visible":true,"origin":"","legend":"HSF1 promotes PFKFB3 expression in endometriosis cells. (A, B) 11Z and ESC cells were heat shocked in a time-dependent manner. The expression of PFKFB3 was determined by Western blot and qRT-PCR. (C, D) 11Z and ESC cells were transfected with HA-tagged HSF1 or empoty vector, and the expressions of PFKFB3 were detected. (E, F) 11Z and ESC cells were transfected with siRNA-HSF1 or NC. The expressions of PFKFB3 were detected after 2 days. All date are mean ± SD of three independent experiments (*P\u003c0.05).","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-134073/v2/ccdf5351bf4264e1aa993c5a.jpg"},{"id":5829067,"identity":"79b71b56-0059-421a-bbad-1889456a224d","added_by":"auto","created_at":"2021-02-10 16:23:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2420763,"visible":true,"origin":"","legend":"KRIBB11 inhibits endometriosis cell growth by targeting HSF1 A) 11Z and ESC cells were plated in 24-well plates. KRIBB11 was given in a concentration-dependent manner, and the IC50 value of the drug was measured by cell counting. (B) 11Z and ESC cells were plated in 24-well plates, and the experimental group treated with HSF1 inhibitor KRIBB11. The cell counts were performed every 24hr for 4 days. (C) 11Z and ESC cells were plated in 6-well plates, and the experimental group treated with HSF1 inhibitor KRIBB11. After 10-14 days, the cell clones were analyzed. (D) 11Z and ESC cells were plated in 6-well plates, and the scratches were made by pipette tip. Experimental group was treated with HSF1 inhibitor KRIBB11. After 24hr, the wound healing was analyzed. (E) 11Z and ESC cells were plated in 6-well plates. The experimental group was treated with HSF1 inhibitor KRIBB11. After 24hr, the cells are lysed for westen blot. All date are mean±SD of three independent experiments (*P\u003c0.05).","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-134073/v2/eb7ea66933395468d66582d1.jpg"},{"id":5828903,"identity":"2a37262a-231e-4331-99e6-c4f4b8a50272","added_by":"auto","created_at":"2021-02-10 16:20:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2078862,"visible":true,"origin":"","legend":"KRIBB11 plays a therapeutic role in a mouse model of endometriosis. (A) Endometriosis model was established using 5-week-old BALB/c female mice. (B) Mice were sacrificed, the endometriosis tissues and the weight of mice were analyzed (*P\u003c0.05). (C) The size of the ectopic tissues was observed and heterotopic tissues were weighed (*P\u003c0.05). (D) Immunohistochemical staining was performed to determine the HSF1 expression in the control group and the experimental group (Scale bars, 50μm). Quantitative analyses of HSF1 expression was performed. (E) HSF1 promoted glycolysis by up-regulating the expression of PFKFB3, which induced the development of endometriosis.","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-134073/v2/a36f60156cd93aad147e4de0.jpg"},{"id":13657971,"identity":"28753df5-740d-4b41-868f-d94eaf34a66f","added_by":"auto","created_at":"2021-09-17 10:12:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1567539,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-134073/v2/7b9561b2-ce18-45b3-b10f-a1a8b4134a20.pdf"},{"id":5829066,"identity":"801cf4cd-2024-4a95-8aef-a4c5fc0fc11d","added_by":"auto","created_at":"2021-02-10 16:23:25","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":51555,"visible":true,"origin":"","legend":"Supplementary Figure 1. (A) 11Z cells were heat-shocked for 0 or 10min, and qRT-PCR was performed to analyze the HK2 and PKM2 mRNA levels. (B) ESC cells were heat-shocked for 0 or 10min, and qRT-PCR was performed to analyze the HK2 and PKM2 mRNA levels.","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-134073/v2/d46fce115b82e2e13d3f7849.pdf"}],"financialInterests":"","formattedTitle":"HSF1 promotes endometriosis development and glycolysis by up-regulating PFKFB3 expression","fulltext":[{"header":"Background","content":"\u003cp\u003eEndometriosis is a disease with features of chronic inflammation, and it is defined as the functional endometrial stroma and glands outside the uterine cavity [\u003ca href=\"#_ENREF_1\"\u003e1\u003c/a\u003e]. The main clinical manifestations of endometriosis are lower abdominal pain, dysmenorrhea, infertility, sexual discomfort, abnormal menstruation and local periodic pain, bleeding and a mass. Approximately 6%-10% of women with endometriosis develop the disease, and the infertility rate is as high as 50%, seriously affecting the physical health of these women [\u003ca href=\"#_ENREF_2\"\u003e2\u003c/a\u003e]. Endometriosis is mainly affected by estrogen and progesterone, which promotes endometrial tissue proliferation, survival, and inflammation [\u003ca href=\"#_ENREF_3\"\u003e3\u003c/a\u003e]. Further, the development, progression, infertility, and chronic pelvic pain of endometriosis are associated with progesterone resistance [\u003ca href=\"#_ENREF_4\"\u003e4\u003c/a\u003e]. The most common theory leading to endometriosis is the implantation theory, which may also be related to genetic factors and immune inflammatory factors [\u003ca href=\"#_ENREF_5\"\u003e5\u003c/a\u003e, \u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e]. However, there is still no clear treatment for endometriosis.\u003c/p\u003e\n\u003cp\u003eIn eukaryotes, various \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e stressors cause protein damage which induces an evolutionally conserved cellular protective mechanism, the heat shock response (HSR), to maintain protein stability [\u003ca href=\"#_ENREF_7\"\u003e7\u003c/a\u003e]. The molecular chaperone heat shock factor 1 (HSF1) plays a central role in this process, helping to refold or degrade intracellular proteins [\u003ca href=\"#_ENREF_8\"\u003e8\u003c/a\u003e]. HSF1 is an evolutionarily conserved transcription factor that can respond to endogenous and exogenous cellular stresses by inducing HSP expression and ultimately maintaining intracellular protein stability. HSF1 responds to stress by up-regulating HSP27 and HSP40, however, HSP70 and HSP90 facilitate the refolding of misfolded proteins [\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e]. HSF1 also plays an important role in various fields of tumor biology, promoting the occurrence and development of tumors and affecting the prognosis [\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e]. For example, HSF1 is highly expressed in prostate cancer PC-3 cells, and plays its functions by increasing levels of its downstream effector HSP27 [\u003ca href=\"#_ENREF_11\"\u003e11\u003c/a\u003e]. Other tumors such as colorectal cancer, breast cancer, oral cancer, and liver cancer have also demonstrated high HSF1 expression [\u003ca href=\"#_ENREF_7\"\u003e7\u003c/a\u003e]. Furthermore, HSF1 can change the survival microenvironment of tumors, promoting their survival under harsh microenvironments and being related to their prognosis [\u003ca href=\"#_ENREF_12\"\u003e12\u003c/a\u003e]. Therefore, HSF1 can be used as a tumor marker and a new therapeutic target. However, the roles of HSF1 in endometriosis are still largely unknown.\u003c/p\u003e\n\u003cp\u003eIn glycolysis process, there is a key enzyme called 6-Phosphofructo-2-kinase/Fructose-2, 6-Biphosphatase 3 (PFKFB3), which belongs to a family of bio-functional proteins and is involved in fructose-2, 6-bisphosphate synthesis and degradation [\u003ca href=\"#_ENREF_13\"\u003e13\u003c/a\u003e]. There are four members of the PFKFB family, but PFKFB3 has the highest kinase/phosphatase ratio in glycolysis [\u003ca href=\"#_ENREF_14\"\u003e14\u003c/a\u003e]. So many studies on glucose metabolism in cancer are based on PFKFB3, which has become a potential target for much drug development [\u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e]. PFKFB3 is widely expressed in tissues, particularly in solid tumors, proliferative tissues, leukemic cells, and transformed cells [\u003ca href=\"#_ENREF_13\"\u003e13\u003c/a\u003e]. As a key enzyme in glycolysis, PFKFB3 regulates glycolysis and plays an important role in the development of many diseases [\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e]. However, the underlying mechanisms of PFKFB3 functions in endometriosis remain unclear.\u003c/p\u003e\n\u003cp\u003eMorphologically, endometriosis is a benign disease, but it has some clinical characteristics similar to the tumor process, such as implantable, invasive, and distant metastasis. Moreover, HSF1 was previously reported to be overexpressed in endometriosis [\u003ca href=\"#_ENREF_17\"\u003e17\u003c/a\u003e]. Therefore, we hypothesized that HSF1 also regulated the development of endometriosis. To test this hypothesis, we manipulated HSF1 expression in endometriosis cells, and used a constructed mouse model, which suggested that HSF1 influenced the development of endometriosis. Our study provides a new idea for the clinical treatment of endometriosis by targeting HSF1.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe endometriotic epithelial cell line (11Z) was established by Professor Anna Strazinski-Powitz [\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e]. The human endometrial stromal cell line (ESC) was established by Dr. Krikun [\u003ca href=\"#_ENREF_19\"\u003e19\u003c/a\u003e]. All cell lines were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium/Ham\u0026rsquo;s F-12 50/50 Mix (DMEM/F-12) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) with 100mg/mL penicilin and 100mg/mL streptomycin at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eMouse anti-\u0026beta;-actin (A1978) was from Sigma-Aldrich, and dilution: 1:5000. Mouse anti-HSF1 (sc-17757) was from Santa Cruz, and dilution: 1:1000. Rabbit anti-PFKFB3 (ab181861) was purchase from ABCAM, and dilution: 1:2000. KRIBB11 were obtained from Med Chem Express (MCE), 50mg/kg.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSiRNA and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence of small interfering (si) RNAs against HSF1 was 5\u0026rsquo;- GCAGGUUGUUCAUAGUCAGAA-3\u0026rsquo;. The sequence of siRNA-NC (Negative Control) was 5\u0026rsquo;-UUCUCCGAACGGUCACGU-3\u0026rsquo; [\u003ca href=\"#_ENREF_20\"\u003e20\u003c/a\u003e]. The method was performed as described previously [\u003ca href=\"#_ENREF_21\"\u003e21\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe indicated cells were collected and lysed on ice using lysis buffer, and were centrifuged at 12000rpm at 4℃ for 15min. Then, 5\u0026acute;loading buffer was added to the sample, and boiled for 10min. The western blot method was performed as described previously [\u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolation of total RNA from cells and the synthesis of cDNA were described above [\u003ca href=\"#_ENREF_23\"\u003e23\u003c/a\u003e]. Performing quantitative real-time PCR using SYBR Green PCR Master Mix (Takara) with CFX96 Real-Time PCR detection system (Bio-Rad, shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe indicated cells were transfected with the indicated plasmids, and reseeded in 24-well plates. The cell numbers were counted every 24hr for 4 days [\u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony-formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 500 indicated cells were seeded in six-well plates, and cultured at 37℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e for 10-14 days. After the clones were formed, the culture medium was removed and fixed at room temperature with 4% paraformaldehyde for 15min. After the fixation, the cells were stained with crystal violet, and then photographed [\u003ca href=\"#_ENREF_24\"\u003e24\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe indicated cells were inoculated in 6-well plates and cultured in medium until overgrown. The pipette tip was used to draw a fine line and washed with PBS. After 24hr, cells were photographed again [\u003ca href=\"#_ENREF_25\"\u003e25\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlucose consumption and lactate production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe indicated cells were seeded in 6-well plates, and the culture mediums were collected after 24hr to determine the concentration of glucose and lactic acid. The methods were performed as described previously [\u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e, \u003ca href=\"#_ENREF_24\"\u003e24\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments have been approved by ethics Committee of Weifang Medical University. We used 5-week-old BALB/c female mice, and the donor mice (n=5) were injected with estradiol benzoate to promote endometrial development. Estradiol benzoate was diluted with oil and injected intramuscularly into the thigh of donor mice, 3mg/mouse, 2 times for one week. After one week, the uterus of donor mice was cut into pieces and intraperitoneally injected into experimental (n=7) and control mice (n=7). After one week, the mice in the experimental group were intraperitoneally injected HSF1 inhibitor KRIBB11, and the mice in the control group were injected with normal saline in the same amount 2 times a week for one month. Then, the mice were sacrificed to observe the endometrial lesion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue Collection and Immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll tissues were derived from mice model of endometriosis. The sections were embedded in paraffin, dried and dewaxed with xylene. The immunohistochemistry was performed as described previously [\u003ca href=\"#_ENREF_21\"\u003e21\u003c/a\u003e]. The immunostaining intensity was quantified using the Image J [\u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were used Graphad Prism 5.0 software. The statistical analyses were presented as mean \u0026plusmn; SEM, and performed by two-tailed unpaired Student\u0026rsquo;s t-test. P\u0026lt;0.05 is significant (*p\u0026lt;0.05). n.s.= not significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHSF1 promotes the cell proliferation, cell migration and clone formation in endometriosis cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEndometriosis and tumorigenesis share similar characteristics, and previous studies have shown that HSF1 plays an important role in tumorigenesis [\u003ca href=\"#_ENREF_27\"\u003e27\u003c/a\u003e]. To determine whether HSF1 plays a similar role in endometriosis, we performed a series of experiments in endometriosis cells. HSF1 overexpression significantly promoted the cell proliferation, indicating that HSF1 played a significant role in endometriosis (Fig. 1A). Moreover, cell-scratch tests and clone formation experiments revealed that HSF1 overexpression promoted the cell migration and growth in endometriosis cells (Fig. 1B and C). Furthermore, HSF1 knockdown inhibited the growth of endometriosis cells (Fig. 1D and F), and also inhibited the cell migration (Fig. 1E). These findings suggest that HSF1 positively regulates the cell proliferation and migration in endometriosis cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSF1 enhances glycolysis in endometriosis cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe endometriosis cells require high glycolysis during its rapid metastasis and growth [\u003ca href=\"#_ENREF_28\"\u003e28\u003c/a\u003e]. To validate the effects of HSF1 on glycolysis, we overexpressed or knocked down HSF1 in endometriosis cells. Interestingly, we found that HSF1 could increase both glucose consumption and lactate production (Fig. 2A and B). Subsequently, to determine whether the HSF1 inhibitor KRIBB11 could suppress glucose metabolism, we cultured endometriosis cells with KRIBB11. As we expected, KRIBB11 reduced the glucose consumption and lactic acid generation in endometriosis cells (Fig. 2A and B). These data show that HSF1 enhances glycolysis in endometriosis cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSF1 promotes PFKFB3 expression in endometriosis cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn previous studies, we confirmed that HSF1 promoted glycolysis in endometriosis cells. Therefore, we hypothesized that HSF1 regulation glycolysis may depend on key glycolytic enzymes. By treating cells with heat shock in a time-dependent manner, the expression levels of PFKFB3 were increased (Fig. 3A and B). But HSF1 activation had little effect on the\u003cem\u003e PKM2\u003c/em\u003e and \u003cem\u003eHK2\u003c/em\u003e expressions (Supplementary Fig. 1A and B). In addition, overexpression HSF1 increased the expression of PFKFB3 (Fig. 3C and D). Furthermore, HSF1 knockdown resulted in a decrease in the expression of PFKFB3 (Fig. 3E and F). Taken together, our results indicate that HSF1 promotes PFKFB3 expression in endometriosis cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKRIBB11 inhibits endometriosis cell growth by targeting HSF1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKRIBB11, a specific inhibitor of HSF1, effectively inhibits HSF1 activity, leading to cell cycle arrest in the G2/M phase, cell apoptosis, and inhibition of tumor cell proliferation [\u003ca href=\"#_ENREF_29\"\u003e29\u003c/a\u003e]. The Cells were seeded onto 24-well plates were treated with increasing concentrations of KRIBB11, and the IC\u003csub\u003e50\u003c/sub\u003e values of the two cell lines were measured (Fig. 4A). As we expected, KRIBB11 inhibited the growth of endometriosis cells (Fig. 4B and C). Cell-scratch tests indicated that KRIBB11 inhibited the migration of endometrial cells (Fig. 4D). Moreover, western blot showed that the PFKFB3 protein level was reduced after HSF1 inhibition by KRIBB11 (Fig. 4E). Thus, these data reveal that the HSF1-specific inhibitor KRIBB11 reduces the expression of the key glycolytic enzyme PFKFB3 by inhibiting HSF1 expression, and ultimately inhibits the proliferation of endometriosis cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKRIBB11 plays a therapeutic role in a mouse model of endometriosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether KRIBB11 regulates endometriosis \u003cem\u003ein vivo\u003c/em\u003e, the endometria of donor mice were cut up and intraperitoneally injected into recipient mice, and a mouse model of endometriosis was established after one week (Fig. 5A). Two days after the last injection, the mice were sacrificed, and the abdominal cavity was opened to observe the ectopic lesion. Interestingly, the endometriosis tissues were observed in all control mice, but only two in seven experimental mice (Fig. 5B). Ectopic lesions without KRIBB11 grew significantly faster than those in the experimental group, and the weight of ectopic lesions with KRIBB11 was substantially lower than that of ectopic lesions in the control group (Fig. 5C). We performed immunohistochemical staining of ectopic tissue collected from mice, and HSF1 expression was significantly lower in the mice with KRIBB11 (Fig. 5D). The above results indicate that the HSF1-specific inhibitor KRIBB11 plays a therapeutic role in the mouse model of endometriosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndometriosis is an age-related disease of the reproductive system, and its prevalence is up to 10% in premenopausal women worldwide [\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e]. The diagnosis of endometriosis is difficult, because experienced obstetricians and gynecologists are required to assess the clinical symptoms of the disease and assess the existence of ectopic endometrium in the abdominal cavity and pelvis [\u003ca href=\"#_ENREF_30\"\u003e30\u003c/a\u003e]. In recent years, more studies have been published on how to treat endometriosis. However, the treatment of endometriosis is still a challenge in clinical, which causes increased burdens to women of childbearing age. Moreover, endometriosis has the characteristics of invasion and metastasis, which is similar to tumor behavior. HSF1 is an oncogene to promote tumor progress, so we speculate that HSF1 plays a similar role in the development of endometriosis. Our hypothesis is supported by the finding that HSF1 promotes endometriosis development through a series of experiments, including glycolysis, cell counting, cloning, and cell scratching.\u003c/p\u003e\n\u003cp\u003eBecause endometriosis cells must get more energy to support rapid cell proliferation, glycolysis must be enhanced. As we expected, we find that HSF1 up-regulates PFKFB3 expression to promote glycolysis, thus accelerating the development of endometriosis. By increasing the expression of PFKFB3, the efficiency of glycolysis can be rapidly improved. Importantly, HSF1 regulates glucose metabolism through PFKFB3, ultimately influencing the development of endometriosis. Interestingly, these effects are abrogated using the HSF1-specific inhibitor KRIBB11, which bind to HSF1 to prevent HSF1-dependent recruitment of p-TEFb to HSP70 promoters [\u003ca href=\"#_ENREF_31\"\u003e31\u003c/a\u003e]. The effects of KRIBB11 are demonstrated \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Therefore, HSF1 is a potential target for the treatment of endometriosis. However, the regulatory role of HSF1 in endometriosis is still elusive. Other mechanisms may be studied in the future. Our findings provide some new insights into the functions of HSF1 in endometriosis, which identifies a new pathway to treat endometriosis (Fig. 5E).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe have verified the important roles of HSF1 in endometriosis, which will provide new ideas for the treatment of endometriosis in the future.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eHSF1: \u003c/strong\u003eHeat shock factor 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSR:\u003c/strong\u003e Heat shock response\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSP:\u003c/strong\u003e Heat shock protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR:\u003c/strong\u003e Polymerase chain reaction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR:\u003c/strong\u003e Quantitative real-time PCR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWB:\u003c/strong\u003e Western blot\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePFKFB3: \u003c/strong\u003e6-Phosphofructo-2-kinase/Fructose-2, 6-Biphosphatase 3\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDMEM/F-12:\u003c/strong\u003e Dulbecco\u0026rsquo;s Modified Eagle Medium/Ham\u0026rsquo;s F-12 50/50 Mix\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFBS:\u003c/strong\u003e Fetal bovine serum\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA: \u003c/strong\u003eRibonucleic acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNA: \u003c/strong\u003eSmall interfering RNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA:\u003c/strong\u003e Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ecDNA:\u003c/strong\u003e Complementary DNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBS:\u003c/strong\u003e Phosphate buffer saline\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePKM2:\u003c/strong\u003e pyruvate kinase 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHK2: \u003c/strong\u003eHexokinase 2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIC\u003csub\u003e50\u003c/sub\u003e: \u003c/strong\u003e50% inhibiting concentration\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ep-TEFb:\u003c/strong\u003e Positive transcription elongation factor b\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSEM:\u003c/strong\u003e Standard error of the means\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSD: \u003c/strong\u003eStandard devistion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003en.s.:\u003c/strong\u003e Not significant\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in this study involving were in accordance with the ethical standards of the institutional research committee of Weifang medical university.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by research grants from National Natural Science Foundation of China (Grant no. 81972489) and National Natural Science Foundation of Shandong Province (Grant no. ZR2020YQ58).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.Y. and C.R. designed research; Z.Y. and Y.W. wrote and revised the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, 261000, Shandong Province, P.R. China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYixin Wang, Jing Xiu, Tingting Yang, Chune Ren and Zhenhai Yu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Chune Ren and Zhenhai Yu.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMehedintu C, Plotogea MN, Ionescu S, Antonovici M: \u003cstrong\u003eEndometriosis still a challenge.\u003c/strong\u003e \u003cem\u003eJ Med Life \u003c/em\u003e2014, \u003cstrong\u003e7:\u003c/strong\u003e349-357.\u003c/li\u003e\n\u003cli\u003eGiudice LC: \u003cstrong\u003eClinical practice. 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challenges in modeling a multifactorial disease of unknown etiology.\u003c/strong\u003e \u003cem\u003eJ Transl Med \u003c/em\u003e2020, \u003cstrong\u003e18:\u003c/strong\u003e311.\u003c/li\u003e\n\u003cli\u003eKijima T, Prince T, Neckers L, Koga F, Fujii Y: \u003cstrong\u003eHeat shock factor 1 (HSF1)-targeted anticancer therapeutics: overview of current preclinical progress\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cem\u003eExpert Opin Ther Targets \u003c/em\u003e2019, \u003cstrong\u003e23:\u003c/strong\u003e369-377.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HSF1, KRIBB11, PFKFB3, Glycolysis, Endometriosis","lastPublishedDoi":"10.21203/rs.3.rs-134073/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-134073/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eEndometriosis is a chronic hormonal inflammatory disease characterized by the presence of endometrial tissue outside the uterus. Endometriosis often causes infertility, which affects the body and mind of patients and their families.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eWe examined the functions of heat shock factor 1 (HSF1) in endometriosis development through cell count, scratch and clone formation experiments. We used quantitative real-time PCR (qRT-PCR) and Western blot (WB) to detect the functions of HSF1 in endometriosis cells. Glucose and lactate levels were determined using a glucose (GO) assay kit and a lactate assay kit. Furthermore, we established a mouse model of endometriosis by using a HSF1 inhibitor-KRIBB11.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eOur study demonstrated that HSF1 was highly expressed in endometriosis, and promoted endometriosis development. Interestingly, we found that HSF1 promoted glycolysis in endometriosis cells. Further, HSF1 enhanced glycolysis by up-regulating PFKFB3 in endometriosis cells, which was a key enzyme in glucose metabolism. Moreover, the HSF1 inhibitor KRIBB11 could abrogate endometriosis progression \u003cem\u003ein vivo \u003c/em\u003eand \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eFindings indicate that HSF1 plays an important role in the development of endometriosis, which might become a new target for the treatment of endometriosis and provide a new idea for the clinical treatment of endometriosis.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eElectronic supplementary material \u003c/strong\u003eSupplementary data are available.\u003c/p\u003e","manuscriptTitle":"HSF1 promotes endometriosis development and glycolysis by up-regulating PFKFB3 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":"","date":"2021-04-22 00:00:00","doi":"","editorialEvents":[{"type":"decision","content":"Major 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