{"paper_id":"9a8032a0-bef6-4fdb-b29a-b1945fac6e57","body_text":"RESEARCH Open Access\n© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, \nsharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and \nthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included \nin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will \nneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The \nCreative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available \nin this article, unless otherwise stated in a credit line to the data.\nYang et al. BMC Molecular and Cell Biology           (2023) 24:12 \nhttps://doi.org/10.1186/s12860-023-00470-9\nBMC Molecular and Cell \nBiology\n*Correspondence:\nChunfan Jiang\n3667@hbuas.edu.cn\n1Department of Obstetrics and Gynecology, Xiangyang Central Hospital, \nAffiliated Hospital of Hubei University of Arts and Science,  \nXiangyang 441021, Hubei, China\n2Institute of Maternity disease, Xiangyang Central Hospital, Affiliated \nHospital of Hubei University of Arts and Science, Xiangyang, Hubei, China\n3Department of Obstetrics and Gynecology, Renmin Hospital of Wuhan \nUniversity, 238 Jiefang Road, Wuhan 430060, China\n4Department of Pathology, Xiangyang Central Hospital, Affiliated Hospital \nof Hubei University of Arts and Science, Xiangyang 441021, Hubei, China\nAbstract\nBackground Endometriosis cause decreases in life quality and pelvic pain in reproductive-age women. Methylation \nabnormalities played a functional role in the progression of endometriosis, this study aimed to explore the \nmechanisms mediated by abnormal methylation in the development of EMS.\nMaterials and methods Next-generation sequencing dataset and methylation profiling dataset were used to \nscreen out the key gene SFRP2.  Western bolt, Real-time PCR, Aza-2'deoxycytidine treatment, luciferase reporter assay, \nMethylation-specific PCR , Bisulfite sequencing PCR and lentivirus infection were carried out to detect the methylation \nstatus and signaling pathway with the primary epithelial cells. Transwell assay and wound scratch assay were \nimplemented to observe the differences of migration ability with the intervening with the expression of SFRP2.\nResults To define the role of the DNA methylation-regulated genes in the pathogenesis of EMS, we performed \nboth DNA methylomic and expression analyses of ectopic endometrium and ectopic endometrium epithelial \ncells(EEECs) and found that SFRP2 is demethylated/upregulated in ectopic endometrium and EEECs. The expression \nof lentivirus carrying SFRP2 cDNA up-regulates the activity of Wnt signaling and the protein expression of ß-catenin \nin EEECs. SFRP2 impact on the invasion and migration of ectopic endometrium by modulating the activities of the \nWnt/ß-catenin signaling pathway. The invasion and migration ability of EEECs were significantly strengthened after \ndemethylation treatment including 5-Aza and the knockdown of DNMT1.\nConclusion In summary, the increased SFRP2 expression-induced Wnt/ß-catenin signaling due to the demethylation \nof the SFRP2 promoter plays an important role in the pathogenesis of EMS, suggesting that SFRP2 might be a \ntherapeutic target for EMS treatment.\nKeywords SFRP2, Endometriosis, Wnt pathway, Demethylation\nThe DNA demethylation-regulated SFRP2 \ndictates the progression of endometriosis \nvia activation of the Wnt/β-catenin signaling \npathway\nMei Yang1,2, Lin Li1, Xiaojie Huang1, Hui Xing1,2, Li Hong3 and Chunfan Jiang4*\n\nPage 2 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nBackground\nAs a common disease, EMS affects about 5-10% of \nwomen of reproductive age, which causes decreases in \nlife quality and is accompanied by symptoms such as pel -\nvic pain and affects more than 10% of reproductive-age \nwomen[1, 2]. Several classical theories including Mül-\nLerianosis, retrograde menstruation, and coelomic meta -\nplasia have been proposed to elucidate the pathogenesis \nof EMS, but the molecular mechanism is still unknown[3, \n4].\nMany differences were found in gene expression pro -\nfiles between EMS samples and the normal endometrium \ntissue samples. Nowadays, microarray technology has \nbecome a mature and stable technology, and during the \nlast decade’s bioinformatics analysis has been widely used \nto identify general genetic of etiology and pathogenesis in \nmany malignant tumors  [ 7– 9], but there have been few \nreports of EMS.Some scholars found that the epigenetic \nmechanisms including DNA methylation and histone \nmodification closely related to the expression of estro -\ngen receptors and progesterone receptors in patients \nwith EMS[ 29]. Other scholars evaluated and compared \nthe methylation pattern of Human Homeobox clusters \nin normal, eutopic (endometrium in the uterine cavity \nof the EMS patients), and ectopic endometrial tissues, a \nconserved pattern of methylation alterations in EMS tis -\nsues was observed for most of the investigated genes (56 \nof 84) which indicating epigenetic changes in EMS[ 32]. \nAnd there are some other researches about aberrant \nendometrial DNA methylation in EMS[ 38, 39] [ 40, 41], \nso we need to clarify the mechanism of aberrant methy -\nlom in EMS.\nWnt signaling is an early event in some tissue carcino -\ngenesis, there is evidence that the Wnt signaling pathway \nalso plays a role in the etiology of EMS[ 20– 22]. Charac-\nterized histologically by dense fibrous tissue consisting, \nEMS is researched by many scholars and it was found \nthat treatment with Wingless mouse mamary umor virus \n(MMTV) integration site family member 3a (Wnt3a) sig -\nnificantly increased the proliferation and migration of \nendometrial cells in patients with EMS, and significantly \nenhanced the expression of fiber marker genes, such as \nα-smooth muscle actin, type I collagen, connective tis -\nsue growth factor and fibulin, which were closely related \nto the contraction of collagen gel[ 14, 15]. Some studies \nfocus on the effect of endometrial cells-mediated col -\nlagen gel contraction on EMS[ 16]. After treatment with \nWnt3a, the contraction of collagen gel I in the endome -\ntrial cells in normal endometrium was increased to a level \ncomparable to that in EMS patients[ 17– 19]. In different \ndiseases, SFRP proteins were reported correlating with \nthe Wnt pathway, and their expression was regulated by \nmethylation[24, 34]. For example, SFRP2 is reported to \nbe closely related to Wnt and regulated by methylation in \nnasopharyngeal carcinoma[35].\nThis study aimed to explore whether the wnt signal -\ning pathway are mediated by abnomal methylation in the \ndevelopment of EMS.\nMethods\nMicroarray data\nNext-generation sequencing dataset (GSE135485) and \nmethylation profiling dataset (GSE47359) were obtained \nfrom the GEO database. GSE135485 included 54 EMS \nsamples and 4 normal endometrium tissue samples, \nbased on GPL21290 Illumina Human HiSeq 3000 plat -\nform. GSE47359 consisted of 3 EMS samples and 6 nor -\nmal endometrium tissue samples, based on the GPL8490 \nIllumina Human Methylation 27 platform.\nOn data processing and identification of differentially \nexpressed genes (DEGs), R software (ver. 3.6.3, https://\nwww.rproject.org/) were used to identify DEGs and dif -\nferentially methylated genes(DMGs). The matrix file for \nGSE135485 was downloaded from https://www.ncbi.\nnlm.nih.gov/geo/query/acc.cgi?acc=GSE135485 and then \ngene IDs conversion was conducted with strawberry-\nPerl-5.30.0.1. The data normalization was done with the \nlimma package and then processed with the edge R pack -\nage to get DEGs. The cut off value of DEGs was set as \n｜log2FC｜> 4. P < 0.05 was considered to indicate a sta -\ntistically significant difference.\nDifferential methylation genes (DMGs) identification\nThe HumanMethylation 27 BeadChip array, covers \napproximately 27,578 CpG sites at different gene regions, \nembodying the upstream region of the transcriptional \nstart site, 5 ′untranslated region, exons, 3 ′untranslated \nregion. The matrix file for GSE47359 was downloaded \nfrom http://ftp.ncbi.nlm.nih.gov/geo/series/GSE47nnn/\nGSE47359/matrix/.\nThe Champ package of R was used for the identifica -\ntion of CpG sites and DMGs with the threshold P < 0.05 \nand ｜log2FC｜> 0.2. The Champ package is a highly \nintegrated methylation analysis tool, matching the cor -\nresponding DMGs with the most differentially methyl -\nated CpG sites. A Venn diagram was used to illustrate \nthe intersection between DEGs and DMGs. As a result, \nupregulated hypomethylated genes were listed.\nGO term and KEGG pathway enrichment\nOnline analysis tool DAVID was used to conduct Gene \nontology (GO) Enrichment Analysis of DEGs into the \nCell Components(CC), Molecular Functions(MF), and \nBiological Processes(BP). All p values < 0.05 were consid-\nered to be statistically significant.\n\nPage 3 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nPatient recruitment\nThis study was initiated on November 11th, 2019 and ter-\nminated on April 20th, 2021. All of the women recruited \nin this study were being at child-bearing age and under -\nwent laparoscopic surgery at the Department of Gyne -\ncology of Xiangyang Central Hospital. Five women with \nendometriosis were recruited before surgery. All these \nwomen had not received GnRH-a agonist or hormones \ntreatment for at least six months and were preoperative \ndiagnosed as an ovarian cyst. They were aged between \n24 and 39 years old, mean ± SD (32.12 ± 4.90) years; Each \ncase of endometriosis was staged during the operation \naccording to the revised American Fertility Society clas -\nsification of endometriosis (rAFS) and subsequently \nconfirmed by histology. Among them, two were in rAFS \nstaging III and the other three were in rAFS staging IV. \nAll these patients were in the secretory phase of the men-\nstrual cycle. Ectopic endometrium from the ovarian cyst \nof these 5 patients were obtained by laparoscopy.\nFive women undergoing tubal ligation for steriliza -\ntion were recruited as controls. All these five patients \nwere aged between 28 and 40 years old, mean ± SD \n(34.60 ± 4.38) years. No minimal endometriosis was \nfound in these control subjects and no hormones treat -\nment for at least six months. All these women were in the \nsecretory phase of the menstrual cycle. Normal endome -\ntrium were obtained by curettage during tubal ligation \noperation.\nCell culture\nAccording to our previous study[36], tissues were washed \nwith sterile Hank’s Balanced Salt Solution(HBSS, phenol-\nred-free) three times, then minced into pieces of approxi-\nmately 1 mm3 and digested in 10 ml of HBSS containing \n10 U/ml DNase I (Sigma) and type IV collagenase (0.03%; \nSigma, St. Louis, MO) for 40  min at 37  °C. The super -\nnatant was kept and epithelial cells and stromal cells in \nit were separated by differential centrifugation [ 21]. To \nrepurify the endometrial cells, the selective attachment \nwas carried out [ 22]. The endometrial cells were cultured \nin phenol-red-free DMEM/Ham’s F12 (Invitrogen, Carls-\nbad, CA) supplemented with 10% v/v fetal bovine serum \n(FBS; Invitrogen). Next, they were subjected to differ -\nential trypsinization and attachment for further puri -\nfication. Finally, the primary epithelial cells were plated \n(2 × 104 cells/ml) in dishes in a culture medium as men -\ntioned above. The detect the phenotypic characteriza -\ntion and ensure the purity of endometrial cell > 95%, the \nprimary epithelial cells were tested by dyeing of vimentin \nand PCK.\nWestern blot\nWestern blot was performed according to our previ -\nous study [ 36] using primary anti-bodies against human \nSFRP2 (rabbit polyclonal, #HPA002652, Sigma-Aldrich, \nMerck, USA), anti-β-catenin (#ab6302, Abcam), DNMT1 \n(#ab13537, Abcam), and mouse monoclonal anti-β-actin \n(#A5441, Sigma-Aldrich) antibodies. The intensities of \nthe protein bands were measured using the ImageJ (1.49 \nv) program.\n5Aza-2′deoxycytidine (aza) treatment of EEECs\nAs deoxycytidine analogs, 5-Aza-CdR can be irreversibly \nmixed into DNA for synthesis, thus reducing the abil -\nity of DNA to accept methyl under the action of meth -\nyltransferase (DNMT). Meanwhile, 5-Aza-CdR forms a \ncovalent complex with DNA methyltransferase (DNMT), \nreducing the activity of DNMT. And we want to decrease \nthe methylation rate of the promoter of SFRP2 by using \nthis drug. The EEECs were grown and treated with 1 μm \nof 5-Aza (Sigma-Aldrich #CAS 2353- 33-5) for 3 days for \nthe inhibition of DNA methyltransferase activity.\nReal-time RT PCR\nTotal RNA was isolated from EMS tissues and EEECs \nutilizing the TRIzol reagent (Invitrogen, Shang -\nhai, China), and all cRNA transcripts were gener -\nated using a primeScript™ RT kit (Qiagen, Hilden, \nJournal of Molecular Histology1 Germany). All prim -\ners (Sangon Biotechnology, China) were listed as fel -\nlows: SFRP2, 5 ′-TGGGGGAAACGGTCGCACTC-3′, \nand 5 ′-GGCCACGAGACCATGAAGGAGG-3′. \nβ-catenin, 5 ′-AAAGCGGCTGTTAGTCACTGG-3′ and \n5′-CGAGTCATTGCATACTGTCCAT-3′. The qPCR \nwas performed in triplicate to determine the relative \nlevels of the target mRNA using SYBR premix Ex Taq™ \nGreen II (Takara) in the CFX96 Touch sequence detec -\ntion system (Bio-Rad, Hercules, CA, USA). Quantitative \nreal-time PCR was conducted ABI 7500 Real-Time PCR \nSystem(Applied Biosystems/Life Tech).\nLuciferase reporter assay\nTo detect the Wnt/β-catenin activation in EEECs, TOP/\nFLASH and FOP/FLASH reporter gene system (GenePh -\narma Company, Shanghai) were selected to test the \nWnt signaling pathway and the Promega dual-luciferase \nreporter gene assay system was used to measure the \nreporter activity. TOP/FOP values were used to represent \nthe result. A higher value of TOP /FOP indicates a stron -\nger Wnt pathway activity.\nMethylation-specific PCR (MSP)\nGenomic DNA from 5 ectopic endometrium and 5 nor -\nmal endometrium was isolated using the DNA Extraction \nKit (Sangon Biotech, Shanghai, China). In the 50ul sys -\ntem, DNA (2–5 µg) was denatured by NaOH (final con -\ncentration 0.2 mol/L) at 37 ℃ for 10 Min. Add 30 µL of \n10 mmol /L hydroquinone and 40.5% sodium bisulfite to \n\nPage 4 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nmix well, then incubate for 16  h in the condition of air \nisolation and out of light. The modified DNA passed by \na DNA purification column and then eluted by water. At \nroom temperature, it was modified with NaOH (the final \nconcentration was 0.3  mol/L) for 5  min, and then pre -\ncipitated with ethanol. Dissolve the DNA in 20µL water, \nstored at -20℃. Two pairs of specific primers were used to \namplify the same nucleotide sequence of the tested gene \nusing methylated primer pairs (M) 5 ′-GGAGTTTTTC-\nGGAGTTGCGC-3′ and 5 ′-CTCTTCGCTAAATAC-\nGACTCG-3′, or unmethylated primer pairs (U) \n5′-GTTGGAGTTTTTTGGAGTTGTGT-3′ and \n5′-CTCTCTTCACTAAATACAACTCA-3′. The ampli -\nfied products were detected by DNA agarose gel electro -\nphoresis and analyzed by gel scanning.\nBisulfite sequencing PCR\nGenomic DNA from 5 ectopic endometrium and 5 nor -\nmal endometrium was isolated using the DNA Extrac -\ntion Kit (Sangon Biotech, Shanghai, China). According \nto the manufacture’s instruction, and bisulfite modifica -\ntion was performed with the EZ DNA Methylation Gold \nKit (Tianmo Technology, Beijing, China). Primer(Sangon \nBiotechnology, China) sequences for bisulfite sequenc -\ning were listed as follows: forward(M818-F)5 ′-\nTTTATGTTTGGTAATTTAGTAGAAATTT-3′ and \nreverse (M818-R) 5′-ATTTTACRTTAAAAATACCCCT-\nCAC-3′. This area was 302-bp fragments including 28 \nCpG dinucleotides. The PCR conditions were: pre-dena -\nturation at 95 °C for 3–5 min, denaturation at 94 °C for \n30s, 55–60 °C for 30s, and 72 °C for 30s, 35cycles totally. \nThen, the sequence containing the SFRP2 sequence was \nsequenced(Sangon Biotech, Shanghai, China).\nPlasmid construction and lentivirus production\nThe human SFRP2 lentiviral vectors were purchased \nfrom GenePharma and transfected EEECs according to \nstandard manufacturer protocols. Furthermore, lentiviral \nvectors to knockdown DNMT1 expression were gener -\nated by the GenePharma Company, (Shanghai), and the \ninterfering sequence is as follows: DNMT1-Homo-2664 \nGGAGCTGTTCTTGGTGGATGA. Three kinds of \ninfection sequence were tested in the preliminary experi -\nments, and one is useful as mentioned above. Post-\ninfected cells were cultured for one week consecutively \nand lentivirus infection condition of target cells were \ndetermined by observing the expression time and inten -\nsity of GFP . To screen the stably transfection clusters, at \nthe basis of transient infection, puromycin with mini -\nmum lethal concentration lasts for at least 4 days.\nImmunohistochemistry\nA cohort of 84 formalin-fixation paraffin-embed -\nded specimens (FFPE), including 28 EMS ectopic \nendometrium, 28 eutopic endometrium and 28 normal \nendometrium were retrieved from Xiangyang Central \nHospital from 2006 to 2020 with necessary clinical infor -\nmation. 28 eutopic endometrium and ectopic endome -\ntrium were get from 28 ovarian endometrial cyst patients \nwhich were aged between 25 and 43 years old, mean ± SD \n(35.05 ± 8.70) years; normal endometrium patients \nwere aged between 29 and 48 years old, mean ± SD \n(41.80 ± 6.22) years. All the cases were reviewed by two \nsenior pathologists separately again to ensure the diagno-\nsis accuracy.\nImmunohistochemical staining for SFRP2 was per -\nformed with 3-µm-thick sections using the Ventana \nBenchmark ULTRA automated staining system (Ventana \nMedical Systems, Tucson, AZ) according to the manufac-\nturer’s protocol. SFRP2 (Abcam), the primary antibodies \nwere added on the cell sections for two hours, Sections \nwere incubated with a secondary antibody and visualized \nwith 3, 3’-diaminobenzidine tetrahydro-chloride (DAB; \nGolden Bridge, Beijing, China). Sections were then sub -\njected to nuclear counterstaining (blue staining) with \nhematoxylin. Two investigators were asked to review and \nscore the anti-SFRP2 staining on the stained sections \nby adding the percentage score with the intensity score. \nStaining intensity was scored as 0 (negative), 1 (weak), \n2 (moderate) and 3 (strong), while staining percentage \nwas scored as 0 ( < 10% staining), 1 (11–25% staining), 2 \n(25–75% staining) and 3 ( ≥ 75% staining). And these two \nfractions were added together, score 0–3: low; 4–6: mod -\nerate; 7–9: high.\nTranswell assay\nBD matrigel and 1640 were diluted in a ratio of 1:3 and \n80ul was added to the upper chamber of the transwell \nchamber(8  μm; Millipore, Billerica, MA). EEECs were \ntreated with 5-Aza, sh-DNMT1 or lentivirus carrying \nSFRP2-cDNA. Cell suspensions were configured accord -\ning to the concentration of 200ul of serum-free medium \ncontaining 2.5 × 104 cells. 500  µl DMEM medium was \nadded to the subchamber wells of the Transwell plate \nand the chamber was placed into the plate with care not \nto produce bubbles. Celcultures were grown in 37 ℃ \nincubator containing 5%CO2 for 24  h. Assays were \nthen stopped by removing the non-invading cells in the \ntop chamber with swabs. The chamber was removed \nand the medium was washed with PBS and the cham -\nber was stained for 10 min; next the crystal violet of the \ncleaned chamber surface was washed with water, the \ncells in the upper chamber were wiped with a cotton \nswab and photographed under an inverted microscope. \nCells in five visual fields per insert were counted (400 × \nmagnification).\n\nPage 5 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nWound scratch assay\nLog-growth EEECs were digested with trypsin and cells \nwere evenly spread out into 6-well plates according to \nexperimental grouping. They were incubated in an incu -\nbator at 5%CO2 and 37 ℃. When the cells grows to 80 \n-90% confluence, a straight line was drawn in the well \nusing the appropriate pipette gun head along a steriliza -\ntion ruler. The shed cells were washed out three times \nwith PBS. In the presence of serum, untreated cells \nshould migrate and fill the scratch area after approxi -\nmately 48  h. Twenty-four hours after scratching, differ -\nent treatments displayed remarkable effects on cellular \nmigration in preliminary experiments, so this time point \nwas chosen to end the assay. EEECs were treated with \n5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA \nas described above. Pictures were taken at 0 and 24  h \nunder an inverted microscope. The relative migration \nlength in five random fields was measured with ImageJ \nfor further quantitative analysis.\nStatistical analysis\nAll the experiments were repeated at least three times. \nSPSS 13 software was used for statistical analysis of all \nexperimental data. The data were normally distributed. \nThe comparison between the two groups was estimated \nby Student’s t-test. A p-value < 0.05 was considered sig -\nnificant. Chi-square was used in the Statistical analysis of \nimmunohistochemistry data.\nResults\nIdentification of aberrantly methylated genes\nHeatmap clustering of the gene methylation status from \nGSE47359 in 3 EMS samples vs. 6 normal endometrium \ntissue samples was made. A total of 3215 CpG sites were \nfound and associated with the profile of differentially \nmethylated genes from a microarray analysis from the \nGEO database (Fig. 1). A total of 85 differentially methyl-\nated genes were identified after the screening, 27 genes \nwere hypermethylated and 58 genes were hypomethyl -\nated in EMS. After the GO analysis of the low methyla -\ntion expression, the functions of these hypomethylated \ngenes were explored in several important cell processes, \nincluding repressor, secreted, and signaling (Table 1).\nLegend Datas were reported as mean ± SD. All experi -\nments were carried out in three or more replicates, and \nrepeated at least twice. Statistically significant was dis -\nplayed as *P<0.05, **P<0.01, ***P<0.001.\nScreening for DEGs\nTo identify DEGs in EMS compared to healthy controls, \none next-generation sequencing dataset (GSE135485) \nwith the 54 EMS and 4 healthy controls has been ana -\nlyzed using the linear modeling approach. A total of \n134 DEGs were identified after the screening, of which \n48 genes were upregulated and 86 were downregulated \n(｜log2FC｜> 4, p < 0.05) (Fig.  2). The downregulation \ngenes were not the research focus, so we did not pres -\nent them here. 48 upregulation genes ( ｜log2FC｜> 5, \np < 0.05) were selected for subsequent bioinformatic \nanalysis. The significant terms of GO enrichment analy -\nsis performed by DAVID were illustrated in Table  2. The \nintersection of hypomethylated genes and overexpressed \ngenes in EMS were get and there are two upregulation \nand demethylation genes(Fig.  3). Among them, SFRP2 \nwas seldom described in EMS yet. Therefore, we further \nverified the expression of SFRP2 and study its function in \nthe development of EMS.\nIncreased SFRP2 expression in EMS tissues and EEECs\nThe immunohistochemistry experiments’ results of nor -\nmal endometrium(Fig.  4A), eutopic endometrium of \nEMS patients(Fig. 4B) and ectopic endometrium(Fig. 4C) \nwere presented in Fig.  4., Compared with the normal \nendometrium and eutopic endometrium, the protein \nexpression levels of SFRP2 were significantly increased in \nectopic endometrium (χ2 = 17.907, p < 0.001). The SFRP2 \nexpression in the eutopic endometrium of EMS patients \nshowing no differences compared with normal endome -\ntrium samples( χ2 = 2.9, 8,p = 0.087 > 0.05). Meanwhile, \nthrough the immunohistochemistry experiments, it was \nfound that SFRP2 was located in the cytoplasm of EEECs \nand was yellowy-brown(Fig. 4D).\nThe SFRP2 protein expression in EMS vs. normal endo-\nmetrium and EEECs vs. NEECs assessed using western \nblot were exhibited in Fig.  5A. All the experiments were \nrepeated three times, the results of statistical analysis \nwere shown in Fig.  5B and C. It was found that SFRP2 \nprotein levels were significantly upregulated in ectopic \nendometrium/EEECs, compared with normal endome -\ntrium/NEECs (p < 0.001. Next, through the RT-PCR, it \nwas found that compared with normal endometrium/\nNEECs, the mRNA expression levels of SFRP2 in ecto -\npic endometrium/EEECs were significantly increased \n(P < 0.001, Fig. 5D and E).\nDemethylation of the SFRP2 promoter in EMS\nFrom the MSP data, it was found that compared with \nthat in normal endometrium, the SFRP2 promoter region \nwas hypomethylated in EMS, (P < 0.0001, Fig.  5F). To \nfurther investigate whether the activation of SFRP2 is \nrelated to the methylation status of the promoter, Bisul -\nfite sequencing PCR was used in EEECs and NEECs. \nDirect sequencing analysis of a 302-bp fragment includ -\ning 28 CpG dinucleotides in the SFRP2 promoter was \nperformed. Differential methylation was observed in 28 \nCpG dinucleotides of the promoter in these two kinds of \ncells. We found that the percentage of methylated CpG \n\nPage 6 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nFig. 1 Heatmap clustering of the gene methylation status from GSE47359 in 3 EMS samples vs. 6 normal endometrium tissue samples\n \n\nPage 7 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \ndinucleotides in EEECs and NEECs was 41.8% and 77.6%, \nrespectively, P = 0.002 (Fig. 5G), This suggests that hypo -\nmethylation of the SFRP2 promoter in EMS.\nSFRP2 was upregulated due to the reduced methylation \nstatus of the promoter\nDNA methyltransferase(DNMT) is a pivotal isozyme for \nDNA methylation. To further understand the influence of \npromoter methylation on SFRP2 expression, the deple -\ntion of DNMT was performed in EEECs. There were sev -\neral DNMT, and through preliminary experiments, it was \nfound that the level of SFRP2 promoter methylation was \nsignificantly reduced in EEECs by DNMT1 knockout. So \nnext we choose lentiviral vectors to knockdown DNMT1 \nexpression in the following experiments. To detect the \nSFRP2 protein expression in EEECs, western-blot was \nused. After the treatment with 5-Aza-2 ′-deoxycytidine, \nit was found that the levels of SFRP2 protein were \nsignificantly increased, meanwhile, the levels of SFRP2 \nprotein were significantly increased after knockdown of \nDNMT1 (Fig.  6A-C, p < 0.001). After the treatment with \n5-Aza-2′-deoxycytidine, and the mRNA(p < 0.001) levels \nof SFRP2 were significantly increased, at the same time, \nfrom MSP data, it was found that the level of SFRP2 \npromoter methylation rate in EEECs was significantly \nreduced(P < 0.01) (Fig. 6D F) in EEECs. And after knock -\ndown of DNMT1 in EEECs, it was found that the mRNA \n(P < 0.0001) levels of SFRP2 were significantly increased \nand the level of SFRP2 promoter methylation rate was \nsignificantly reduced(Fig. 6E and G).\nThe regulation of the wnt signaling pathway after ectopic \nexpression of SFRP2 in EMS\nAfter the transfection of SFRP2 cDNA into EEECs, it was \nfound that SFRP2 protein and mRNA levels were upregu-\nlated significantly ( Fig.  7A, B, D and p < 0.001). The pro-\ntein and mRNA (Fig.  7A C, 7E, p < 0.001) expression of \nβ-catenin, the downstream target gene, were increased \nafter SFRP2 cDNA transfection. Meanwhile, from the \nLuciferase reporter assay, after the transfection of SFRP2 \ncDNA, the relative TCF/LEF luciferase activity was also \nincreased compared with the control group(p < 0.001, \nFig. 7F).\nDemethylation of SFRP2 promoter changed the invasion \nand migratory ability of EEECs\nTo observe the differences in the migration abilities of \nEEECs influenced by 5-Aza, sh-DNMT1 or lentivirus \ncarrying SFRP2-cDNA intervention, we performed tran -\nswell and wound scratch assays. For EEECs, the numbers \nof cells on the lower surface of the insert membrane of \nthe transwell were counted after each treatment includ -\ning 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-\ncDNA(Fig.  8). All these three intervention were clearly \nstrong impetus promoting the invasion ability of EEECs. \nSimilarly, EEECs had the stronger ability to migrate after \n5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA \nintervention and left smaller unfilled scratch area (Fig. 8). \nSignificant differences were observed between the \nuntreated cells and any of those three groups receiving \ndifferent treatment(p < 0.001).\nDiscussion\nBecause of the complexity of biological traits and high \nheterogeneity, inadequate knowledge about mechanisms \nat the molecular and cellular levels [ 11, 37], though \nmany differences were found in gene expression profiles \nbetween EMS samples and the normal endometrium tis -\nsue samples[4– 6]. [10– 13], the pathogenesis of EMS has \nyet to be fully elucidated.\nBeing considered as a heritable change in gene expres -\nsion, epigenetics covers abnormal DNA methylation[ 26], \nTable 1 GO analysis of the low methylation expression gene \nfrom the GSE47359 data\nCategory Term Count PValue\nGOTERM_CC_DIRECT GO:0000790 ~ nu-\nclear chromatin\n4 0.005055271\nGOTERM_BP_DIRECT GO:0042493 ~ re-\nsponse to drug\n3 0.011057299\nGOTERM_BP_DIRECT GO:0031668 ~ cel-\nlular response \nto extracellular \nstimulus\n2 0.029690877\nGOTERM_MF_DIRECT GO:0003713 ~ tran-\nscription coactiva-\ntor activity\n3 0.030570734\nGOTERM_BP_DIRECT GO:0048646 ~ ana-\ntomical structure \nformation involved \nin morphogenesis\n2 0.031939077\nGOTERM_BP_DIRECT GO:0045944 ~ posi-\ntive regulation of \ntranscription from \nRNA polymerase II \npromoter\n5 0.043516788\nGOTERM_CC_DIRECT GO:0005667 ~ tran-\nscription factor \ncomplex\n3 0.053312753\nGOTERM_CC_DIRECT GO:0090575 ~ RNA \npolymerase II \ntranscription factor \ncomplex\n2 0.058856003\nGOTERM_BP_DIRECT GO:0050680 ~ nega-\ntive regulation \nof epithelial cell \nproliferation\n2 0.099183706\nGOTERM_BP_DIRECT GO:0035914 ~ skel-\netal muscle cell \ndifferentiation\n2 0.099183706\nAfter the GO analysis of the low methylation expression, the functions of these \nhypomethylated genes were explored in several important cell processes, \nincluding repressor, secreted, and signaling (Table  1)\n\nPage 8 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nabnormal non-coding RNA, Histone modification \nchanges after translation, etc., among which abnormal \nDNA methylation is most widely studied[ 27, 28]. Being \nas heritable changes in gene expression, epigenetics espe-\ncially methylation of key regulators plays a critical role \nin carcinogenesis without alteration in DNA sequence. \nFor example, effects of certain genes with aberrant DNA \nmethylation on HCC and mammary stem cells have \nbeen extensively reported[ 26]. Aberrant DNA methyla -\ntion could influence some tumor suppressor genes which \nwere key genes involved in the carcinogenesis of HCC \nand mammary stem cells. Epigenetic modifications were \nreported to play a role in the pathogenesis of EMS in \nrecent years[29– 33], such as epigenetics of estrogen and \nprogesterone receptors and DNA methylation analysis of \nHOX genes, however, the regulatory mechanism is still \nunclear. Interestingly, in this study, bioinformatics analy -\nsis of DMG microarray and related experiments showed \nthat SFRP2 was significantly demethylated in EMS. From \nthe MSP data, it was found that compared with that in \nnormal endometrium, the SFRP2 promoter region was \nhypomethylated in EMS. Furthermore, direct sequenc -\ning analysis of a 302-bp fragment including 28 CpG \ndinucleotides in the SFRP2 promoter was performed in \nEEECs and NEECs. Significant difference was observed \nin the methylation rate of 28 CpG dinucleotides of the \npromoter in these two kinds of cells. This suggests that \nhypomethylation of the SFRP2 promoter in EMS. Meth -\nylation of SFRPs was frequently detected in cancers, such \nas nasopharyngeal carcinoma[ 35], however, the methyla-\ntion status of SFRPs has not been reported in EMS yet.\nFig. 2 Heatmap clustering of differentially expressed genes in mRNA expression profiling datasets (GSE135485), which includes 54 EMS samples and 4 \nnormal endometrium tissue samples\n \n\nPage 9 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nSFRP2 is a member of the various secreted frizzled-\nrelated protein (SFRP) family proteins, which are main \nregulator proteins members of the Wnt pathway. And \nin different tissues, it could have the opposite activity. \nStudies have shown that SFRP2 can act as an agonist or \nantagonist for Wnt signaling[ 23, 24]. Scholars found \nthat secreted frizzled-related proteins (SFRPs) and some \nother secreted proteins can competitively displace cer -\ntain WNT ligands in some cancer models, and increase \nin SFRP levels attenuates cancer growth, particularly in \nbreast cancer cells[20, 42]. But in the researches of pros -\ntate cancer cells in vitro, the overexpression of SFRP1 \npromotes the growth of BPH1, whereas over-expression \nof SFRP4 or SFRP3 decreases the proliferation of human \nPC3 cells[42].\nOnly two studies concern the experession of SFRPs in \nEMS. Heinosalo et al. found that after SFRP2 knockout, \ncell proliferation, and β-catenin protein expression in pri-\nmary cultured cells with EMS significantly reduced, sug -\ngesting that in EMS, SFRP2 acts as an agonist for the Wnt \nsignaling pathway and stimulates lesion growth[ 24, 25]. \nThe scholars found the increased SFRP2 expression in the \nEMS lesion, too. Meanwhile, they also found β-catenin \nand SFRP2 showed similar expression patterns, suggest -\ning that overexpression of SFRP2 promotes the activity \nof Wnt signal and the growth of EMS lesions[ 24]. In our \nstudy, it was found that compared with the normal endo -\nmetrium/NEECs, the protein expression levels of SFRP2 \nwere significantly increased in ectopic endometrium and \nEEECs. And in our research, after the up-regulation of \nSFRP2 caused by the lentivirus, the up-regulation of the \nTable 2 GO analysis of the upregulation genes from the GSE135485 data\nCategory Term Count PValue\nGOTERM_BP_DIRECT GO:0008584 ~ male gonad development 7 1.35E-07\nGOTERM_MF_DIRECT GO:0000980 ~ RNA polymerase II distal enhancer sequence-specific DNA binding 4 5.23E-04\nGOTERM_BP_DIRECT GO:0008585 ~ female gonad development 3 6.58E-04\nGOTERM_MF_DIRECT GO:0043565 ~ sequence-specific DNA binding 7 0.001463497\nGOTERM_MF_DIRECT GO:0001077 ~ transcriptional activator activity, RNA polymerase II core promoter proxi-\nmal region sequence-specific binding\n5 0.002513506\nGOTERM_BP_DIRECT GO:0006366 ~ transcription from RNA polymerase II promoter 6 0.009651672\nGOTERM_BP_DIRECT GO:0045944 ~ positive regulation of transcription from RNA polymerase II promoter 8 0.01158106\nGOTERM_MF_DIRECT GO:0016491 ~ oxidoreductase activity 4 0.012548298\nGOTERM_BP_DIRECT GO:0007584 ~ response to nutrient 3 0.015396063\nGOTERM_BP_DIRECT GO:0050810 ~ regulation of steroid biosynthetic process 2 0.020307471\nGOTERM_MF_DIRECT GO:0047498 ~ calcium-dependent phospholipase A2 activity 2 0.021652815\nGOTERM_BP_DIRECT GO:0036149 ~ phosphatidylinositol acyl-chain remodeling 2 0.040211948\nGOTERM_BP_DIRECT GO:0036148 ~ phosphatidylglycerol acyl-chain remodeling 2 0.045126018\nGOTERM_BP_DIRECT GO:0036150 ~ phosphatidylserine acyl-chain remodeling 2 0.045126018\nGOTERM_BP_DIRECT GO:0036152 ~ phosphatidylethanolamine acyl-chain remodeling 2 0.059721243\nGOTERM_BP_DIRECT GO:0050482 ~ arachidonic acid secretion 2 0.059721243\nGOTERM_MF_DIRECT GO:0008270 ~ zinc ion binding 7 0.0613717\nGOTERM_BP_DIRECT GO:0036151 ~ phosphatidylcholine acyl-chain remodeling 2 0.066936914\nGOTERM_MF_DIRECT GO:0003682 ~ chromatin binding 4 0.068885928\nGOTERM_BP_DIRECT GO:0009755 ~ hormone-mediated signaling pathway 2 0.069330097\nGOTERM_MF_DIRECT GO:0004623 ~ phospholipase A2 activity 2 0.072674345\nGOTERM_MF_DIRECT GO:0017147 ~ Wnt-protein binding 2 0.072674345\nGOTERM_BP_DIRECT GO:0070374 ~ positive regulation of ERK1 and ERK2 cascade 3 0.073862462\nGOTERM_CC_DIRECT GO:0090575 ~ RNA polymerase II transcription factor complex 2 0.07673751\nGOTERM_BP_DIRECT GO:0050873 ~ brown fat cell differentiation 2 0.078843017\nGOTERM_MF_DIRECT GO:0003700 ~ transcription factor activity, sequence-specific DNA binding 6 0.081625912\nGOTERM_MF_DIRECT GO:0004879 ~ RNA polymerase II transcription factor activity, ligand-activated sequence-\nspecific DNA binding\n2 0.083902905\nGOTERM_BP_DIRECT GO:0006654 ~ phosphatidic acid biosynthetic process 2 0.085915314\nGOTERM_CC_DIRECT GO:0005576 ~ extracellular region 8 0.08929768\nGOTERM_BP_DIRECT GO:0035094 ~ response to nicotine 2 0.090600685\nGOTERM_BP_DIRECT GO:0030522 ~ intracellular receptor signaling pathway 2 0.092934565\nGOTERM_BP_DIRECT GO:0010811 ~ positive regulation of cell-substrate adhesion 2 0.092934565\nGOTERM_BP_DIRECT GO:0048468 ~ cell development 2 0.097584789\nThe significant terms of GO enrichment analysis performed by DAVID from the GSE135485 data\n\nPage 10 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nprotein expression of β-catenin and activity of Wnt sig -\nnaling in EEECs were observed, further confirming that \nSFRP2 may be an important factor in the up-regulation \nof Wnt signaling in EMS tissues. Our conclusions are \nconsistent with other scholars[24].\nThe classical Wnt signaling pathway requires β-catenin \nto enter the nucleus and then bind to the transcription \nfactor TCF/LEF to form a complex, which initiates the \ntranscription of downstream regulatory genes. Scholars \nfound that in the proliferative progenitor cells of colon \ncrypts, the activation of a specific subset of the TCF/\nLEF family regulate the expression of many target genes \nthat are normally associated with tumorigenesis[ 42]. \nSome researches have found that the aberrant activa -\ntion of Wnt/β-catenin signaling significantly correlated \nwith the pathophysiology of EMS. Some studies found \nthat being a subunit of the cell surface cadherin protein \ncomplex, β-catenin act as an intercellular signal trans -\nducer in the Wnt signaling pathway and involve in the \nprogress of EMS[ 20]. Other scholars found that under \nthe regulation of E2, the promotion of MMP9 by Wnt \nsignaling pathway may contribute to the metastasis, \ndetachment, invasion, and implantation of EMS[21]. And \nthere are still researches found that defective endometrial \nFig. 5 Increased SFRP2 expression in EEECs and endometriosis tissues. A Western blot analysis of SFRP2 protein level in ectopic endometrium vs. \nNE(Normal endometrium) and protein level in EEECs vs. NEECs. Full-length blots/gels are presented in Supplementary Figure 1. B Western blot data of \nSFRP2 protein level in ectopic endometrium vs. NE C Western blot data on SFRP2 protein level. in EEECs vs. NEECs. D Real-time RT PCR. The mRNA expres-\nsion levels of SFRP2 in ectopic endometrium vs Normal endometrium. E Real-time RT PCR. The mRNA expression levels of SFRP2 in EEECs vs NEECs. F MSP . \nThe methylation rates of normal endometrium and ectopic endometrium endmetrium were detected by MSP respectively. G Bisulfite sequencing PCR \non SFRP2 promoter in EEECs and NEECs.\n \nFig. 4 Immunohistochemical staining of SFRP2 protein in different endometrium tissues. A Normal endometrium. B Eutopic endometrium (endome -\ntrium in the uterine cavity of the EMS patients). C ectopic endometrium. D Through the immunohistochemistry staining, it was found that SFRP2 was \nclearly located in the cytoplasm and was yellowy-brown\n \nFig. 3  Identification of aberrantly methylated-differentially expressed \ngenes in mRNA expression profiling datasets (GSE135485) and gene meth-\nylation profiling datasets (GSE47359).\n \n\nPage 11 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nstromal fibroblasts (EMSFs) contribute to EMS, but \nbefore implantation, the activation of β-catenin was \nessential for the key differentiation step of EMSFs[ 22]. \nAll these studies indicates that WNT signaling in EMS \ncannot be targeted using the same strategy of cancer, \nincreasingly detailed understanding of WNT signaling in \nEMS will help us to make clinical decision. In our study, \nto determine how SFRP2 regulated the Wnt/β-catenin \nsignaling pathway, the expression of downstream target \nwere detected after using transfection of SFRP2-cDNA \non EEECs We found that after the up-regulation of \nSFRP2 caused by the lentivirus, the up-regulation of the \nprotein expression of β-catenin and activity of Wnt sig -\nnaling in EEECs were observed, further confirming that \nSFRP2 may be an important factor in the up-regulation \nof Wnt signaling in EMS tissues. To confirm that the \nmigration of EEECs could be affected by the regulation \nof the demethylation of SFRP2 promoter, we performed \nTranswell and wound scratch assays with different treat -\nments. We found that the number of cells on the lower \nsurface of the membrane and the scratch area left unfilled \nvaried significantly after 5-Aza, sh-DNMT1 or lentivirus \ncarrying SFRP2-cDNA intervention. Our data suggest \neither demethylation of SFRP2 promoter or upregulation \nof SFRP2 intervention could significantly promote the \ninvasion and migration of EEECs.\nThere are great clinical relevance in our study. Abnor -\nmal activation of the Wnt/β-catenin signaling pathway \nmay be involved in the aggressive phenotype of EMS \nFig. 7 Increase of Wnt signaling gene expression and activity after ectopic \nSFRP2 expression in EEECs. A Western blot analysis. EEECs were transfected \nwith SFRP2 cDNA or lv-control and subjected to Western blot analysis. Full-\nlength blots/gels are presented in Supplementary Figure 1. B Western blot \ndata of SFRP2 protein level. The protein expression levels of SFRP2 in EEECs \ntransfected with SFRP2 cDNAvs control group. C Western blot data of \nbeta-Catenin protein level. The protein expression levels of beta-Catenin \nin EEECs transfected with SFRP2 cDNAvs control group. D Real-time RT \nPCR. The mRNA expression levels of SFRP2 in EEECs transfected with SFRP2 \ncDNA vs control group. E Real-time RT PCR. The mRNA expression levels \nof beta-Catenin in EEECs transfected with SFRP2 cDNA vs control group. \nF Luciferase reporter assay. The relative TCF/LEF luciferase activity in EEECs \ntransfected with SFRP2 cDNA vs control group\n \nFig. 6 SFRP2 was upregulated due to the reduced methylation status of the promoter. A Western blot analysis of SFRP2 protein level in EEECs under \ntreatment of 5-Aza vs DMSO or in EEECs under transfection with DNMT1 shRNAs vs control group. Full-length blots/gels are presented in Supplementary \nFigure 1. B Western blot data of SFRP2 protein level. The protein expression levels of SFRP2 in EEECs treated with 5-Aza vs DMSO group. C Western blot \ndata of SFRP2 protein level. The protein expression levels of SFRP2 in EEECs transfected with DNMT1 shRNAs vs control group. D Real-time RT PCR. The \nmRNA expression levels of SFRP2 in EEECs treated with 5-Aza vs control group. E Real-time RT PCR. The mRNA expression levels of SFRP2 in EEECs trans-\nfected with DNMT1 shRNAs vs control group. F MSP . The methylation rate of SFRP2 promoter in EEECs treated with 5-Aza vs DMSO group. G MSP . The \nmethylation rate of SFRP2 promoter in EEECs transfected with DNMT1 shRNAs vs control group\n \n\nPage 12 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \ncells[23]. Pain is a major clinical problem in patients with \nEMS. Wnt3a and β-catenin are upregulated in various \nmouse pain models, activating Wnt signaling and pos -\nsibly contributing to central spinal cord conduction[ 37]. \nHowever, only one literature has reported the regulatory \neffect of SFRP on the Wnt pathway in EMS, and no litera-\nture has been found about the role of methylation on the \nSFRP expression in EMS. Therefore, the study of SFRP2 \nin EMS can provide more profound information for the \ndevelopment of EMS and provide new strategies for the \nclinical control of EMS in the future.\nThere are obvious strengths in the present study. To \ndate, bioinformatics analysis was rarely used in EMS \nand there have been few reports regarding the role of \nSFRP2 in the development of EMS. In this study, next-\ngeneration sequencing dataset and methylation profiling \ndataset were used together and differentially expressed \nand abnormally methylated genes were found in EMS. \nFurther more, in this study, primary endometrial cells \nwere isolated and cultured, and cell models of transfec -\ntion were build. This is the first study which clarified the \nmechanism of SFRP2 demethylation and its interaction \nwith Wnt pathway in the pathogenesis of EMS.\nHowever, there are limitations in the present study: \nOne is that the microarray data were not generated by \nthe authors but from the GEO database. The second limi-\ntation of the study is the sample size was relatively small.\nConclusions\nIn summary, the increased SFRP2 expression-induced \nWnt/β-catenin signaling due to the demethylation of the \nSFRP2 promoter plays an important role in the pathogen-\nesis of EMS, suggesting that SFRP2 might be a novel reg -\nulatory gene and therapeutic target for EMS treatment. \nThis study confirmed that SFRP2 is activated in EMS due \nto promoter demethylation. Our study could provide new \nclues to the underlying biological mechanisms.\nAbbreviations\nEMS  endometriosis\nEEECs  ectopic endometrium epithelial cells\nNEECs  normal endometrial epithelial cells\nDEGs  differentially expressed genes\nSFRP2  secreted frizzled-related protein 2\nDMGs  Differential methylation genes\nMSP  Methylation-specific PCR\nDNMT  DNA methyltransferase\nFig. 8 Demethylation of SFRP2 promoter changed the invasion and migratory ability of EEECs. A Transwell assays. EEECs were treated by 5-Aza, sh-DNMT1 \nor lentivirus carrying SFRP2-cDNA respectively and subjected to transwell assays. B The numbers of EEECs on the lower surface of the insert membrane \nof the transwell were counted after each treatment. C Wound scratch assays. EEECs were treated with 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA \nrespectively and subjected to wound scratch assays. D The relative migration length in five random fields was measured after each treatment\n \n\nPage 13 of 14\nYang et al. BMC Molecular and Cell Biology            (2023) 24:12 \nSupplementary Information\nThe online version contains supplementary material available at https://doi.\norg/10.1186/s12860-023-00470-9.\nSupplementary Material 1\nAcknowledgements\nNot applicable.\nAuthor contributions\nDesigned project: JCF, HL, XH. Collected samples: YM, JCF, LL, HXJ. Analyzed \ndata: YM, JCF. Generated figures and tables: YM, JCF. Wrote manuscript: YM. All \nauthors read and approved the final manuscript.\nFunding\nNational Natural Science Foundation of China (No. 81972449), Foundation \nof Hubei University of Arts and Science(XK2019046), Funder:Hui Xing, the \ncorresponding author; Science and technology project of Xiangyang Central \nHospital(2021C12), Funder: Xiaojie Huang, the author.\nData Availability\nAll data generated or analysed during this study are included in this published \narticle.\nDeclarations\nEthics approval and consent to participate\nThis study received the verification of Medicine Ethics Committee of \nXiangyang Central Hospital (Approved Code: 2017-004) and was carried out \nin accordance with the Declaration of Helsinki. Written informed consent was \nobtained from individual or guardian participants.\nConsent to publish\nNot applicable\nCompeting interests\nThe authors declare that no conflict of interest exists.\nReceived: 5 June 2022 / Accepted: 1 March 2023\nReferences\n1. 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Nat Rev Cancer. 2013;13(1):11–26.\nPublisher’s Note\nSpringer Nature remains neutral with regard to jurisdictional claims in \npublished maps and institutional affiliations.","source_license":"CC0","license_restricted":false}