The DNA demethylation-regulated SFRP2 dictates the progression of endometriosis via activation of the Wnt/β-catenin signaling pathway

Background Endometriosis cause decreases in life quality and pelvic pain in reproductive-age women. Methylation abnormalities played a functional role in the progression of endometriosis, this study aimed to explore the mechanisms mediated by abnormal methylation in the development of EMS.

Next-generation sequencing dataset and methylation profiling dataset were used to screen out the key gene SFRP2. Western bolt, Real-time PCR, Aza-2'deoxycytidine treatment, luciferase reporter assay, Methylation-specific PCR , Bisulfite sequencing PCR and lentivirus infection were carried out to detect the methylation status and signaling pathway with the primary epithelial cells. Transwell assay and wound scratch assay were implemented to observe the differences of migration ability with the intervening with the expression of SFRP2.

To define the role of the DNA methylation-regulated genes in the pathogenesis of EMS, we performed both DNA methylomic and expression analyses of ectopic endometrium and ectopic endometrium epithelial cells(EEECs) and found that SFRP2 is demethylated/upregulated in ectopic endometrium and EEECs. The expression of lentivirus carrying SFRP2 cDNA up-regulates the activity of Wnt signaling and the protein expression of ß-catenin in EEECs. SFRP2 impact on the invasion and migration of ectopic endometrium by modulating the activities of the Wnt/ß-catenin signaling pathway. The invasion and migration ability of EEECs were significantly strengthened after demethylation treatment including 5-Aza and the knockdown of DNMT1.

In summary, the increased SFRP2 expression-induced Wnt/ß-catenin signaling due to the demethylation of the SFRP2 promoter plays an important role in the pathogenesis of EMS, suggesting that SFRP2 might be a therapeutic target for EMS treatment.

SFRP2, Endometriosis, Wnt pathway, Demethylation The DNA demethylation-regulated SFRP2 dictates the progression of endometriosis via activation of the Wnt/β-catenin signaling pathway Mei Yang1,2, Lin Li1, Xiaojie Huang1, Hui Xing1,2, Li Hong3 and Chunfan Jiang4* Page 2 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12

As a common disease, EMS affects about 5-10% of women of reproductive age, which causes decreases in life quality and is accompanied by symptoms such as pel - vic pain and affects more than 10% of reproductive-age women[1, 2]. Several classical theories including Mül- Lerianosis, retrograde menstruation, and coelomic meta - plasia have been proposed to elucidate the pathogenesis of EMS, but the molecular mechanism is still unknown[3, 4]. Many differences were found in gene expression pro - files between EMS samples and the normal endometrium tissue samples. Nowadays, microarray technology has become a mature and stable technology, and during the last decade’s bioinformatics analysis has been widely used to identify general genetic of etiology and pathogenesis in many malignant tumors  [ 7– 9], but there have been few reports of EMS.Some scholars found that the epigenetic mechanisms including DNA methylation and histone modification closely related to the expression of estro - gen receptors and progesterone receptors in patients with EMS[ 29]. Other scholars evaluated and compared the methylation pattern of Human Homeobox clusters in normal, eutopic (endometrium in the uterine cavity of the EMS patients), and ectopic endometrial tissues, a conserved pattern of methylation alterations in EMS tis - sues was observed for most of the investigated genes (56 of 84) which indicating epigenetic changes in EMS[ 32]. And there are some other researches about aberrant endometrial DNA methylation in EMS[ 38, 39] [ 40, 41], so we need to clarify the mechanism of aberrant methy - lom in EMS. Wnt signaling is an early event in some tissue carcino - genesis, there is evidence that the Wnt signaling pathway also plays a role in the etiology of EMS[ 20– 22]. Charac- terized histologically by dense fibrous tissue consisting, EMS is researched by many scholars and it was found that treatment with Wingless mouse mamary umor virus (MMTV) integration site family member 3a (Wnt3a) sig - nificantly increased the proliferation and migration of endometrial cells in patients with EMS, and significantly enhanced the expression of fiber marker genes, such as α-smooth muscle actin, type I collagen, connective tis - sue growth factor and fibulin, which were closely related to the contraction of collagen gel[ 14, 15]. Some studies focus on the effect of endometrial cells-mediated col - lagen gel contraction on EMS[ 16]. After treatment with Wnt3a, the contraction of collagen gel I in the endome - trial cells in normal endometrium was increased to a level comparable to that in EMS patients[ 17– 19]. In different diseases, SFRP proteins were reported correlating with the Wnt pathway, and their expression was regulated by methylation[24, 34]. For example, SFRP2 is reported to be closely related to Wnt and regulated by methylation in nasopharyngeal carcinoma[35]. This study aimed to explore whether the wnt signal - ing pathway are mediated by abnomal methylation in the development of EMS.

Microarray data Next-generation sequencing dataset (GSE135485) and methylation profiling dataset (GSE47359) were obtained from the GEO database. GSE135485 included 54 EMS samples and 4 normal endometrium tissue samples, based on GPL21290 Illumina Human HiSeq 3000 plat - form. GSE47359 consisted of 3 EMS samples and 6 nor - mal endometrium tissue samples, based on the GPL8490 Illumina Human Methylation 27 platform. On data processing and identification of differentially expressed genes (DEGs), R software (ver. 3.6.3, https:// www.rproject.org/) were used to identify DEGs and dif - ferentially methylated genes(DMGs). The matrix file for GSE135485 was downloaded from https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE135485 and then gene IDs conversion was conducted with strawberry- Perl-5.30.0.1. The data normalization was done with the limma package and then processed with the edge R pack - age to get DEGs. The cut off value of DEGs was set as ｜log2FC｜> 4. P < 0.05 was considered to indicate a sta - tistically significant difference. Differential methylation genes (DMGs) identification The HumanMethylation 27 BeadChip array, covers approximately 27,578 CpG sites at different gene regions, embodying the upstream region of the transcriptional start site, 5 ′untranslated region, exons, 3 ′untranslated region. The matrix file for GSE47359 was downloaded from http://ftp.ncbi.nlm.nih.gov/geo/series/GSE47nnn/ GSE47359/matrix/. The Champ package of R was used for the identifica - tion of CpG sites and DMGs with the threshold P 0.2. The Champ package is a highly integrated methylation analysis tool, matching the cor - responding DMGs with the most differentially methyl - ated CpG sites. A Venn diagram was used to illustrate the intersection between DEGs and DMGs. As a result, upregulated hypomethylated genes were listed. GO term and KEGG pathway enrichment Online analysis tool DAVID was used to conduct Gene ontology (GO) Enrichment Analysis of DEGs into the Cell Components(CC), Molecular Functions(MF), and Biological Processes(BP). All p values < 0.05 were consid- ered to be statistically significant. Page 3 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 Patient recruitment This study was initiated on November 11th, 2019 and ter- minated on April 20th, 2021. All of the women recruited in this study were being at child-bearing age and under - went laparoscopic surgery at the Department of Gyne - cology of Xiangyang Central Hospital. Five women with endometriosis were recruited before surgery. All these women had not received GnRH-a agonist or hormones treatment for at least six months and were preoperative diagnosed as an ovarian cyst. They were aged between 24 and 39 years old, mean ± SD (32.12 ± 4.90) years; Each case of endometriosis was staged during the operation according to the revised American Fertility Society clas - sification of endometriosis (rAFS) and subsequently confirmed by histology. Among them, two were in rAFS staging III and the other three were in rAFS staging IV. All these patients were in the secretory phase of the men- strual cycle. Ectopic endometrium from the ovarian cyst of these 5 patients were obtained by laparoscopy. Five women undergoing tubal ligation for steriliza - tion were recruited as controls. All these five patients were aged between 28 and 40 years old, mean ± SD (34.60 ± 4.38) years. No minimal endometriosis was found in these control subjects and no hormones treat - ment for at least six months. All these women were in the secretory phase of the menstrual cycle. Normal endome - trium were obtained by curettage during tubal ligation operation. Cell culture According to our previous study[36], tissues were washed with sterile Hank’s Balanced Salt Solution(HBSS, phenol- red-free) three times, then minced into pieces of approxi- mately 1 mm3 and digested in 10 ml of HBSS containing 10 U/ml DNase I (Sigma) and type IV collagenase (0.03%; Sigma, St. Louis, MO) for 40  min at 37  °C. The super - natant was kept and epithelial cells and stromal cells in it were separated by differential centrifugation [ 21]. To repurify the endometrial cells, the selective attachment was carried out [ 22]. The endometrial cells were cultured in phenol-red-free DMEM/Ham’s F12 (Invitrogen, Carls- bad, CA) supplemented with 10% v/v fetal bovine serum (FBS; Invitrogen). Next, they were subjected to differ - ential trypsinization and attachment for further puri - fication. Finally, the primary epithelial cells were plated (2 × 104 cells/ml) in dishes in a culture medium as men - tioned above. The detect the phenotypic characteriza - tion and ensure the purity of endometrial cell > 95%, the primary epithelial cells were tested by dyeing of vimentin and PCK. Western blot Western blot was performed according to our previ - ous study [ 36] using primary anti-bodies against human SFRP2 (rabbit polyclonal, #HPA002652, Sigma-Aldrich, Merck, USA), anti-β-catenin (#ab6302, Abcam), DNMT1 (#ab13537, Abcam), and mouse monoclonal anti-β-actin (#A5441, Sigma-Aldrich) antibodies. The intensities of the protein bands were measured using the ImageJ (1.49 v) program. 5Aza-2′deoxycytidine (aza) treatment of EEECs As deoxycytidine analogs, 5-Aza-CdR can be irreversibly mixed into DNA for synthesis, thus reducing the abil - ity of DNA to accept methyl under the action of meth - yltransferase (DNMT). Meanwhile, 5-Aza-CdR forms a covalent complex with DNA methyltransferase (DNMT), reducing the activity of DNMT. And we want to decrease the methylation rate of the promoter of SFRP2 by using this drug. The EEECs were grown and treated with 1 μm of 5-Aza (Sigma-Aldrich #CAS 2353- 33-5) for 3 days for the inhibition of DNA methyltransferase activity. Real-time RT PCR Total RNA was isolated from EMS tissues and EEECs utilizing the TRIzol reagent (Invitrogen, Shang - hai, China), and all cRNA transcripts were gener - ated using a primeScript™ RT kit (Qiagen, Hilden, Journal of Molecular Histology1 Germany). All prim - ers (Sangon Biotechnology, China) were listed as fel - lows: SFRP2, 5 ′-TGGGGGAAACGGTCGCACTC-3′, and 5 ′-GGCCACGAGACCATGAAGGAGG-3′. β-catenin, 5 ′-AAAGCGGCTGTTAGTCACTGG-3′ and 5′-CGAGTCATTGCATACTGTCCAT-3′. The qPCR was performed in triplicate to determine the relative levels of the target mRNA using SYBR premix Ex Taq™ Green II (Takara) in the CFX96 Touch sequence detec - tion system (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR was conducted ABI 7500 Real-Time PCR System(Applied Biosystems/Life Tech). Luciferase reporter assay To detect the Wnt/β-catenin activation in EEECs, TOP/ FLASH and FOP/FLASH reporter gene system (GenePh - arma Company, Shanghai) were selected to test the Wnt signaling pathway and the Promega dual-luciferase reporter gene assay system was used to measure the reporter activity. TOP/FOP values were used to represent the result. A higher value of TOP /FOP indicates a stron - ger Wnt pathway activity. Methylation-specific PCR (MSP) Genomic DNA from 5 ectopic endometrium and 5 nor - mal endometrium was isolated using the DNA Extraction Kit (Sangon Biotech, Shanghai, China). In the 50ul sys - tem, DNA (2–5 µg) was denatured by NaOH (final con - centration 0.2 mol/L) at 37 ℃ for 10 Min. Add 30 µL of 10 mmol /L hydroquinone and 40.5% sodium bisulfite to Page 4 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 mix well, then incubate for 16  h in the condition of air isolation and out of light. The modified DNA passed by a DNA purification column and then eluted by water. At room temperature, it was modified with NaOH (the final concentration was 0.3  mol/L) for 5  min, and then pre - cipitated with ethanol. Dissolve the DNA in 20µL water, stored at -20℃. Two pairs of specific primers were used to amplify the same nucleotide sequence of the tested gene using methylated primer pairs (M) 5 ′-GGAGTTTTTC- GGAGTTGCGC-3′ and 5 ′-CTCTTCGCTAAATAC- GACTCG-3′, or unmethylated primer pairs (U) 5′-GTTGGAGTTTTTTGGAGTTGTGT-3′ and 5′-CTCTCTTCACTAAATACAACTCA-3′. The ampli - fied products were detected by DNA agarose gel electro - phoresis and analyzed by gel scanning. Bisulfite sequencing PCR Genomic DNA from 5 ectopic endometrium and 5 nor - mal endometrium was isolated using the DNA Extrac - tion Kit (Sangon Biotech, Shanghai, China). According to the manufacture’s instruction, and bisulfite modifica - tion was performed with the EZ DNA Methylation Gold Kit (Tianmo Technology, Beijing, China). Primer(Sangon Biotechnology, China) sequences for bisulfite sequenc - ing were listed as follows: forward(M818-F)5 ′- TTTATGTTTGGTAATTTAGTAGAAATTT-3′ and reverse (M818-R) 5′-ATTTTACRTTAAAAATACCCCT- CAC-3′. This area was 302-bp fragments including 28 CpG dinucleotides. The PCR conditions were: pre-dena - turation at 95 °C for 3–5 min, denaturation at 94 °C for 30s, 55–60 °C for 30s, and 72 °C for 30s, 35cycles totally. Then, the sequence containing the SFRP2 sequence was sequenced(Sangon Biotech, Shanghai, China). Plasmid construction and lentivirus production The human SFRP2 lentiviral vectors were purchased from GenePharma and transfected EEECs according to standard manufacturer protocols. Furthermore, lentiviral vectors to knockdown DNMT1 expression were gener - ated by the GenePharma Company, (Shanghai), and the interfering sequence is as follows: DNMT1-Homo-2664 GGAGCTGTTCTTGGTGGATGA. Three kinds of infection sequence were tested in the preliminary experi - ments, and one is useful as mentioned above. Post- infected cells were cultured for one week consecutively and lentivirus infection condition of target cells were determined by observing the expression time and inten - sity of GFP . To screen the stably transfection clusters, at the basis of transient infection, puromycin with mini - mum lethal concentration lasts for at least 4 days. Immunohistochemistry A cohort of 84 formalin-fixation paraffin-embed - ded specimens (FFPE), including 28 EMS ectopic endometrium, 28 eutopic endometrium and 28 normal endometrium were retrieved from Xiangyang Central Hospital from 2006 to 2020 with necessary clinical infor - mation. 28 eutopic endometrium and ectopic endome - trium were get from 28 ovarian endometrial cyst patients which were aged between 25 and 43 years old, mean ± SD (35.05 ± 8.70) years; normal endometrium patients were aged between 29 and 48 years old, mean ± SD (41.80 ± 6.22) years. All the cases were reviewed by two senior pathologists separately again to ensure the diagno- sis accuracy. Immunohistochemical staining for SFRP2 was per - formed with 3-µm-thick sections using the Ventana Benchmark ULTRA automated staining system (Ventana Medical Systems, Tucson, AZ) according to the manufac- turer’s protocol. SFRP2 (Abcam), the primary antibodies were added on the cell sections for two hours, Sections were incubated with a secondary antibody and visualized with 3, 3’-diaminobenzidine tetrahydro-chloride (DAB; Golden Bridge, Beijing, China). Sections were then sub - jected to nuclear counterstaining (blue staining) with hematoxylin. Two investigators were asked to review and score the anti-SFRP2 staining on the stained sections by adding the percentage score with the intensity score. Staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong), while staining percentage was scored as 0 ( < 10% staining), 1 (11–25% staining), 2 (25–75% staining) and 3 ( ≥ 75% staining). And these two fractions were added together, score 0–3: low; 4–6: mod - erate; 7–9: high. Transwell assay BD matrigel and 1640 were diluted in a ratio of 1:3 and 80ul was added to the upper chamber of the transwell chamber(8  μm; Millipore, Billerica, MA). EEECs were treated with 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA. Cell suspensions were configured accord - ing to the concentration of 200ul of serum-free medium containing 2.5 × 104 cells. 500  µl DMEM medium was added to the subchamber wells of the Transwell plate and the chamber was placed into the plate with care not to produce bubbles. Celcultures were grown in 37 ℃ incubator containing 5%CO2 for 24  h. Assays were then stopped by removing the non-invading cells in the top chamber with swabs. The chamber was removed and the medium was washed with PBS and the cham - ber was stained for 10 min; next the crystal violet of the cleaned chamber surface was washed with water, the cells in the upper chamber were wiped with a cotton swab and photographed under an inverted microscope. Cells in five visual fields per insert were counted (400 × magnification). Page 5 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 Wound scratch assay Log-growth EEECs were digested with trypsin and cells were evenly spread out into 6-well plates according to experimental grouping. They were incubated in an incu - bator at 5%CO2 and 37 ℃. When the cells grows to 80 -90% confluence, a straight line was drawn in the well using the appropriate pipette gun head along a steriliza - tion ruler. The shed cells were washed out three times with PBS. In the presence of serum, untreated cells should migrate and fill the scratch area after approxi - mately 48  h. Twenty-four hours after scratching, differ - ent treatments displayed remarkable effects on cellular migration in preliminary experiments, so this time point was chosen to end the assay. EEECs were treated with 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA as described above. Pictures were taken at 0 and 24  h under an inverted microscope. The relative migration length in five random fields was measured with ImageJ for further quantitative analysis. Statistical analysis All the experiments were repeated at least three times. SPSS 13 software was used for statistical analysis of all experimental data. The data were normally distributed. The comparison between the two groups was estimated by Student’s t-test. A p-value < 0.05 was considered sig - nificant. Chi-square was used in the Statistical analysis of immunohistochemistry data.

Identification of aberrantly methylated genes Heatmap clustering of the gene methylation status from GSE47359 in 3 EMS samples vs. 6 normal endometrium tissue samples was made. A total of 3215 CpG sites were found and associated with the profile of differentially methylated genes from a microarray analysis from the GEO database (Fig. 1). A total of 85 differentially methyl- ated genes were identified after the screening, 27 genes were hypermethylated and 58 genes were hypomethyl - ated in EMS. After the GO analysis of the low methyla - tion expression, the functions of these hypomethylated genes were explored in several important cell processes, including repressor, secreted, and signaling (Table 1). Legend Datas were reported as mean ± SD. All experi - ments were carried out in three or more replicates, and repeated at least twice. Statistically significant was dis - played as *P<0.05, **P<0.01, ***P<0.001. Screening for DEGs To identify DEGs in EMS compared to healthy controls, one next-generation sequencing dataset (GSE135485) with the 54 EMS and 4 healthy controls has been ana - lyzed using the linear modeling approach. A total of 134 DEGs were identified after the screening, of which 48 genes were upregulated and 86 were downregulated (｜log2FC｜> 4, p 5, p < 0.05) were selected for subsequent bioinformatic analysis. The significant terms of GO enrichment analy - sis performed by DAVID were illustrated in Table  2. The intersection of hypomethylated genes and overexpressed genes in EMS were get and there are two upregulation and demethylation genes(Fig.  3). Among them, SFRP2 was seldom described in EMS yet. Therefore, we further verified the expression of SFRP2 and study its function in the development of EMS. Increased SFRP2 expression in EMS tissues and EEECs The immunohistochemistry experiments’ results of nor - mal endometrium(Fig.  4A), eutopic endometrium of EMS patients(Fig. 4B) and ectopic endometrium(Fig. 4C) were presented in Fig.  4., Compared with the normal endometrium and eutopic endometrium, the protein expression levels of SFRP2 were significantly increased in ectopic endometrium (χ2 = 17.907, p 0.05). Meanwhile, through the immunohistochemistry experiments, it was found that SFRP2 was located in the cytoplasm of EEECs and was yellowy-brown(Fig. 4D). The SFRP2 protein expression in EMS vs. normal endo- metrium and EEECs vs. NEECs assessed using western blot were exhibited in Fig.  5A. All the experiments were repeated three times, the results of statistical analysis were shown in Fig.  5B and C. It was found that SFRP2 protein levels were significantly upregulated in ectopic endometrium/EEECs, compared with normal endome - trium/NEECs (p < 0.001. Next, through the RT-PCR, it was found that compared with normal endometrium/ NEECs, the mRNA expression levels of SFRP2 in ecto - pic endometrium/EEECs were significantly increased (P < 0.001, Fig. 5D and E). Demethylation of the SFRP2 promoter in EMS From the MSP data, it was found that compared with that in normal endometrium, the SFRP2 promoter region was hypomethylated in EMS, (P < 0.0001, Fig.  5F). To further investigate whether the activation of SFRP2 is related to the methylation status of the promoter, Bisul - fite sequencing PCR was used in EEECs and NEECs. Direct sequencing analysis of a 302-bp fragment includ - ing 28 CpG dinucleotides in the SFRP2 promoter was performed. Differential methylation was observed in 28 CpG dinucleotides of the promoter in these two kinds of cells. We found that the percentage of methylated CpG Page 6 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 Fig. 1 Heatmap clustering of the gene methylation status from GSE47359 in 3 EMS samples vs. 6 normal endometrium tissue samples Page 7 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 dinucleotides in EEECs and NEECs was 41.8% and 77.6%, respectively, P = 0.002 (Fig. 5G), This suggests that hypo - methylation of the SFRP2 promoter in EMS. SFRP2 was upregulated due to the reduced methylation status of the promoter DNA methyltransferase(DNMT) is a pivotal isozyme for DNA methylation. To further understand the influence of promoter methylation on SFRP2 expression, the deple - tion of DNMT was performed in EEECs. There were sev - eral DNMT, and through preliminary experiments, it was found that the level of SFRP2 promoter methylation was significantly reduced in EEECs by DNMT1 knockout. So next we choose lentiviral vectors to knockdown DNMT1 expression in the following experiments. To detect the SFRP2 protein expression in EEECs, western-blot was used. After the treatment with 5-Aza-2 ′-deoxycytidine, it was found that the levels of SFRP2 protein were significantly increased, meanwhile, the levels of SFRP2 protein were significantly increased after knockdown of DNMT1 (Fig.  6A-C, p < 0.001). After the treatment with 5-Aza-2′-deoxycytidine, and the mRNA(p < 0.001) levels of SFRP2 were significantly increased, at the same time, from MSP data, it was found that the level of SFRP2 promoter methylation rate in EEECs was significantly reduced(P < 0.01) (Fig. 6D F) in EEECs. And after knock - down of DNMT1 in EEECs, it was found that the mRNA (P < 0.0001) levels of SFRP2 were significantly increased and the level of SFRP2 promoter methylation rate was significantly reduced(Fig. 6E and G). The regulation of the wnt signaling pathway after ectopic expression of SFRP2 in EMS After the transfection of SFRP2 cDNA into EEECs, it was found that SFRP2 protein and mRNA levels were upregu- lated significantly ( Fig.  7A, B, D and p < 0.001). The pro- tein and mRNA (Fig.  7A C, 7E, p < 0.001) expression of β-catenin, the downstream target gene, were increased after SFRP2 cDNA transfection. Meanwhile, from the Luciferase reporter assay, after the transfection of SFRP2 cDNA, the relative TCF/LEF luciferase activity was also increased compared with the control group(p < 0.001, Fig. 7F). Demethylation of SFRP2 promoter changed the invasion and migratory ability of EEECs To observe the differences in the migration abilities of EEECs influenced by 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA intervention, we performed tran - swell and wound scratch assays. For EEECs, the numbers of cells on the lower surface of the insert membrane of the transwell were counted after each treatment includ - ing 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2- cDNA(Fig.  8). All these three intervention were clearly strong impetus promoting the invasion ability of EEECs. Similarly, EEECs had the stronger ability to migrate after 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA intervention and left smaller unfilled scratch area (Fig. 8). Significant differences were observed between the untreated cells and any of those three groups receiving different treatment(p < 0.001).

Because of the complexity of biological traits and high heterogeneity, inadequate knowledge about mechanisms at the molecular and cellular levels [ 11, 37], though many differences were found in gene expression profiles between EMS samples and the normal endometrium tis - sue samples[4– 6]. [10– 13], the pathogenesis of EMS has yet to be fully elucidated. Being considered as a heritable change in gene expres - sion, epigenetics covers abnormal DNA methylation[ 26], Table 1 GO analysis of the low methylation expression gene from the GSE47359 data Category Term Count PValue GOTERM_CC_DIRECT GO:0000790 ~ nu- clear chromatin 4 0.005055271 GOTERM_BP_DIRECT GO:0042493 ~ re- sponse to drug 3 0.011057299 GOTERM_BP_DIRECT GO:0031668 ~ cel- lular response to extracellular stimulus 2 0.029690877 GOTERM_MF_DIRECT GO:0003713 ~ tran- scription coactiva- tor activity 3 0.030570734 GOTERM_BP_DIRECT GO:0048646 ~ ana- tomical structure formation involved in morphogenesis 2 0.031939077 GOTERM_BP_DIRECT GO:0045944 ~ posi- tive regulation of transcription from RNA polymerase II promoter 5 0.043516788 GOTERM_CC_DIRECT GO:0005667 ~ tran- scription factor complex 3 0.053312753 GOTERM_CC_DIRECT GO:0090575 ~ RNA polymerase II transcription factor complex 2 0.058856003 GOTERM_BP_DIRECT GO:0050680 ~ nega- tive regulation of epithelial cell proliferation 2 0.099183706 GOTERM_BP_DIRECT GO:0035914 ~ skel- etal muscle cell differentiation 2 0.099183706 After the GO analysis of the low methylation expression, the functions of these hypomethylated genes were explored in several important cell processes, including repressor, secreted, and signaling (Table  1) Page 8 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 abnormal non-coding RNA, Histone modification changes after translation, etc., among which abnormal DNA methylation is most widely studied[ 27, 28]. Being as heritable changes in gene expression, epigenetics espe- cially methylation of key regulators plays a critical role in carcinogenesis without alteration in DNA sequence. For example, effects of certain genes with aberrant DNA methylation on HCC and mammary stem cells have been extensively reported[ 26]. Aberrant DNA methyla - tion could influence some tumor suppressor genes which were key genes involved in the carcinogenesis of HCC and mammary stem cells. Epigenetic modifications were reported to play a role in the pathogenesis of EMS in recent years[29– 33], such as epigenetics of estrogen and progesterone receptors and DNA methylation analysis of HOX genes, however, the regulatory mechanism is still unclear. Interestingly, in this study, bioinformatics analy - sis of DMG microarray and related experiments showed that SFRP2 was significantly demethylated in EMS. From the MSP data, it was found that compared with that in normal endometrium, the SFRP2 promoter region was hypomethylated in EMS. Furthermore, direct sequenc - ing analysis of a 302-bp fragment including 28 CpG dinucleotides in the SFRP2 promoter was performed in EEECs and NEECs. Significant difference was observed in the methylation rate of 28 CpG dinucleotides of the promoter in these two kinds of cells. This suggests that hypomethylation of the SFRP2 promoter in EMS. Meth - ylation of SFRPs was frequently detected in cancers, such as nasopharyngeal carcinoma[ 35], however, the methyla- tion status of SFRPs has not been reported in EMS yet. Fig. 2 Heatmap clustering of differentially expressed genes in mRNA expression profiling datasets (GSE135485), which includes 54 EMS samples and 4 normal endometrium tissue samples Page 9 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 SFRP2 is a member of the various secreted frizzled- related protein (SFRP) family proteins, which are main regulator proteins members of the Wnt pathway. And in different tissues, it could have the opposite activity. Studies have shown that SFRP2 can act as an agonist or antagonist for Wnt signaling[ 23, 24]. Scholars found that secreted frizzled-related proteins (SFRPs) and some other secreted proteins can competitively displace cer - tain WNT ligands in some cancer models, and increase in SFRP levels attenuates cancer growth, particularly in breast cancer cells[20, 42]. But in the researches of pros - tate cancer cells in vitro, the overexpression of SFRP1 promotes the growth of BPH1, whereas over-expression of SFRP4 or SFRP3 decreases the proliferation of human PC3 cells[42]. Only two studies concern the experession of SFRPs in EMS. Heinosalo et al. found that after SFRP2 knockout, cell proliferation, and β-catenin protein expression in pri- mary cultured cells with EMS significantly reduced, sug - gesting that in EMS, SFRP2 acts as an agonist for the Wnt signaling pathway and stimulates lesion growth[ 24, 25]. The scholars found the increased SFRP2 expression in the EMS lesion, too. Meanwhile, they also found β-catenin and SFRP2 showed similar expression patterns, suggest - ing that overexpression of SFRP2 promotes the activity of Wnt signal and the growth of EMS lesions[ 24]. In our study, it was found that compared with the normal endo - metrium/NEECs, the protein expression levels of SFRP2 were significantly increased in ectopic endometrium and EEECs. And in our research, after the up-regulation of SFRP2 caused by the lentivirus, the up-regulation of the Table 2 GO analysis of the upregulation genes from the GSE135485 data Category Term Count PValue GOTERM_BP_DIRECT GO:0008584 ~ male gonad development 7 1.35E-07 GOTERM_MF_DIRECT GO:0000980 ~ RNA polymerase II distal enhancer sequence-specific DNA binding 4 5.23E-04 GOTERM_BP_DIRECT GO:0008585 ~ female gonad development 3 6.58E-04 GOTERM_MF_DIRECT GO:0043565 ~ sequence-specific DNA binding 7 0.001463497 GOTERM_MF_DIRECT GO:0001077 ~ transcriptional activator activity, RNA polymerase II core promoter proxi- mal region sequence-specific binding 5 0.002513506 GOTERM_BP_DIRECT GO:0006366 ~ transcription from RNA polymerase II promoter 6 0.009651672 GOTERM_BP_DIRECT GO:0045944 ~ positive regulation of transcription from RNA polymerase II promoter 8 0.01158106 GOTERM_MF_DIRECT GO:0016491 ~ oxidoreductase activity 4 0.012548298 GOTERM_BP_DIRECT GO:0007584 ~ response to nutrient 3 0.015396063 GOTERM_BP_DIRECT GO:0050810 ~ regulation of steroid biosynthetic process 2 0.020307471 GOTERM_MF_DIRECT GO:0047498 ~ calcium-dependent phospholipase A2 activity 2 0.021652815 GOTERM_BP_DIRECT GO:0036149 ~ phosphatidylinositol acyl-chain remodeling 2 0.040211948 GOTERM_BP_DIRECT GO:0036148 ~ phosphatidylglycerol acyl-chain remodeling 2 0.045126018 GOTERM_BP_DIRECT GO:0036150 ~ phosphatidylserine acyl-chain remodeling 2 0.045126018 GOTERM_BP_DIRECT GO:0036152 ~ phosphatidylethanolamine acyl-chain remodeling 2 0.059721243 GOTERM_BP_DIRECT GO:0050482 ~ arachidonic acid secretion 2 0.059721243 GOTERM_MF_DIRECT GO:0008270 ~ zinc ion binding 7 0.0613717 GOTERM_BP_DIRECT GO:0036151 ~ phosphatidylcholine acyl-chain remodeling 2 0.066936914 GOTERM_MF_DIRECT GO:0003682 ~ chromatin binding 4 0.068885928 GOTERM_BP_DIRECT GO:0009755 ~ hormone-mediated signaling pathway 2 0.069330097 GOTERM_MF_DIRECT GO:0004623 ~ phospholipase A2 activity 2 0.072674345 GOTERM_MF_DIRECT GO:0017147 ~ Wnt-protein binding 2 0.072674345 GOTERM_BP_DIRECT GO:0070374 ~ positive regulation of ERK1 and ERK2 cascade 3 0.073862462 GOTERM_CC_DIRECT GO:0090575 ~ RNA polymerase II transcription factor complex 2 0.07673751 GOTERM_BP_DIRECT GO:0050873 ~ brown fat cell differentiation 2 0.078843017 GOTERM_MF_DIRECT GO:0003700 ~ transcription factor activity, sequence-specific DNA binding 6 0.081625912 GOTERM_MF_DIRECT GO:0004879 ~ RNA polymerase II transcription factor activity, ligand-activated sequence- specific DNA binding 2 0.083902905 GOTERM_BP_DIRECT GO:0006654 ~ phosphatidic acid biosynthetic process 2 0.085915314 GOTERM_CC_DIRECT GO:0005576 ~ extracellular region 8 0.08929768 GOTERM_BP_DIRECT GO:0035094 ~ response to nicotine 2 0.090600685 GOTERM_BP_DIRECT GO:0030522 ~ intracellular receptor signaling pathway 2 0.092934565 GOTERM_BP_DIRECT GO:0010811 ~ positive regulation of cell-substrate adhesion 2 0.092934565 GOTERM_BP_DIRECT GO:0048468 ~ cell development 2 0.097584789 The significant terms of GO enrichment analysis performed by DAVID from the GSE135485 data Page 10 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 protein expression of β-catenin and activity of Wnt sig - naling in EEECs were observed, further confirming that SFRP2 may be an important factor in the up-regulation of Wnt signaling in EMS tissues. Our conclusions are consistent with other scholars[24]. The classical Wnt signaling pathway requires β-catenin to enter the nucleus and then bind to the transcription factor TCF/LEF to form a complex, which initiates the transcription of downstream regulatory genes. Scholars found that in the proliferative progenitor cells of colon crypts, the activation of a specific subset of the TCF/ LEF family regulate the expression of many target genes that are normally associated with tumorigenesis[ 42]. Some researches have found that the aberrant activa - tion of Wnt/β-catenin signaling significantly correlated with the pathophysiology of EMS. Some studies found that being a subunit of the cell surface cadherin protein complex, β-catenin act as an intercellular signal trans - ducer in the Wnt signaling pathway and involve in the progress of EMS[ 20]. Other scholars found that under the regulation of E2, the promotion of MMP9 by Wnt signaling pathway may contribute to the metastasis, detachment, invasion, and implantation of EMS[21]. And there are still researches found that defective endometrial Fig. 5 Increased SFRP2 expression in EEECs and endometriosis tissues. A Western blot analysis of SFRP2 protein level in ectopic endometrium vs. NE(Normal endometrium) and protein level in EEECs vs. NEECs. Full-length blots/gels are presented in Supplementary Figure 1. B Western blot data of SFRP2 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- sion 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 . The methylation rates of normal endometrium and ectopic endometrium endmetrium were detected by MSP respectively. G Bisulfite sequencing PCR on SFRP2 promoter in EEECs and NEECs. Fig. 4 Immunohistochemical staining of SFRP2 protein in different endometrium tissues. A Normal endometrium. B Eutopic endometrium (endome - trium in the uterine cavity of the EMS patients). C ectopic endometrium. D Through the immunohistochemistry staining, it was found that SFRP2 was clearly located in the cytoplasm and was yellowy-brown Fig. 3 Identification of aberrantly methylated-differentially expressed genes in mRNA expression profiling datasets (GSE135485) and gene meth- ylation profiling datasets (GSE47359). Page 11 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 stromal fibroblasts (EMSFs) contribute to EMS, but before implantation, the activation of β-catenin was essential for the key differentiation step of EMSFs[ 22]. All these studies indicates that WNT signaling in EMS cannot be targeted using the same strategy of cancer, increasingly detailed understanding of WNT signaling in EMS will help us to make clinical decision. In our study, to determine how SFRP2 regulated the Wnt/β-catenin signaling pathway, the expression of downstream target were detected after using transfection of SFRP2-cDNA on EEECs We found that after the up-regulation of SFRP2 caused by the lentivirus, the up-regulation of the protein expression of β-catenin and activity of Wnt sig - naling in EEECs were observed, further confirming that SFRP2 may be an important factor in the up-regulation of Wnt signaling in EMS tissues. To confirm that the migration of EEECs could be affected by the regulation of the demethylation of SFRP2 promoter, we performed Transwell and wound scratch assays with different treat - ments. We found that the number of cells on the lower surface of the membrane and the scratch area left unfilled varied significantly after 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA intervention. Our data suggest either demethylation of SFRP2 promoter or upregulation of SFRP2 intervention could significantly promote the invasion and migration of EEECs. There are great clinical relevance in our study. Abnor - mal activation of the Wnt/β-catenin signaling pathway may be involved in the aggressive phenotype of EMS Fig. 7 Increase of Wnt signaling gene expression and activity after ectopic SFRP2 expression in EEECs. A Western blot analysis. EEECs were transfected with SFRP2 cDNA or lv-control and subjected to Western blot analysis. Full- length blots/gels are presented in Supplementary Figure 1. B Western blot data of SFRP2 protein level. The protein expression levels of SFRP2 in EEECs transfected with SFRP2 cDNAvs control group. C Western blot data of beta-Catenin protein level. The protein expression levels of beta-Catenin in EEECs transfected with SFRP2 cDNAvs control group. D Real-time RT PCR. The mRNA expression levels of SFRP2 in EEECs transfected with SFRP2 cDNA vs control group. E Real-time RT PCR. The mRNA expression levels of beta-Catenin in EEECs transfected with SFRP2 cDNA vs control group. F Luciferase reporter assay. The relative TCF/LEF luciferase activity in EEECs transfected with SFRP2 cDNA vs control group Fig. 6 SFRP2 was upregulated due to the reduced methylation status of the promoter. A Western blot analysis of SFRP2 protein level in EEECs under treatment of 5-Aza vs DMSO or in EEECs under transfection with DNMT1 shRNAs vs control group. Full-length blots/gels are presented in Supplementary Figure 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 data 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 mRNA 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- fected 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 methylation rate of SFRP2 promoter in EEECs transfected with DNMT1 shRNAs vs control group Page 12 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 cells[23]. Pain is a major clinical problem in patients with EMS. Wnt3a and β-catenin are upregulated in various mouse pain models, activating Wnt signaling and pos - sibly contributing to central spinal cord conduction[ 37]. However, only one literature has reported the regulatory effect of SFRP on the Wnt pathway in EMS, and no litera- ture has been found about the role of methylation on the SFRP expression in EMS. Therefore, the study of SFRP2 in EMS can provide more profound information for the development of EMS and provide new strategies for the clinical control of EMS in the future. There are obvious strengths in the present study. To date, bioinformatics analysis was rarely used in EMS and there have been few reports regarding the role of SFRP2 in the development of EMS. In this study, next- generation sequencing dataset and methylation profiling dataset were used together and differentially expressed and abnormally methylated genes were found in EMS. Further more, in this study, primary endometrial cells were isolated and cultured, and cell models of transfec - tion were build. This is the first study which clarified the mechanism of SFRP2 demethylation and its interaction with Wnt pathway in the pathogenesis of EMS. However, there are limitations in the present study: One is that the microarray data were not generated by the authors but from the GEO database. The second limi- tation of the study is the sample size was relatively small.

In summary, the increased SFRP2 expression-induced Wnt/β-catenin signaling due to the demethylation of the SFRP2 promoter plays an important role in the pathogen- esis of EMS, suggesting that SFRP2 might be a novel reg - ulatory gene and therapeutic target for EMS treatment. This study confirmed that SFRP2 is activated in EMS due to promoter demethylation. Our study could provide new clues to the underlying biological mechanisms. Abbreviations EMS endometriosis EEECs ectopic endometrium epithelial cells NEECs normal endometrial epithelial cells DEGs differentially expressed genes SFRP2 secreted frizzled-related protein 2 DMGs Differential methylation genes MSP Methylation-specific PCR DNMT DNA methyltransferase Fig. 8 Demethylation of SFRP2 promoter changed the invasion and migratory ability of EEECs. A Transwell assays. EEECs were treated by 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA respectively and subjected to transwell assays. B The numbers of EEECs on the lower surface of the insert membrane of the transwell were counted after each treatment. C Wound scratch assays. EEECs were treated with 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2-cDNA respectively and subjected to wound scratch assays. D The relative migration length in five random fields was measured after each treatment Page 13 of 14 Yang et al. BMC Molecular and Cell Biology (2023) 24:12 Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12860-023-00470-9. Supplementary Material 1

Not applicable. Author contributions Designed project: JCF, HL, XH. Collected samples: YM, JCF, LL, HXJ. Analyzed data: YM, JCF. Generated figures and tables: YM, JCF. Wrote manuscript: YM. All authors read and approved the final manuscript. Funding National Natural Science Foundation of China (No. 81972449), Foundation of Hubei University of Arts and Science(XK2019046), Funder:Hui Xing, the corresponding author; Science and technology project of Xiangyang Central Hospital(2021C12), Funder: Xiaojie Huang, the author. Data Availability All data generated or analysed during this study are included in this published article. Declarations Ethics approval and consent to participate This study received the verification of Medicine Ethics Committee of Xiangyang Central Hospital (Approved Code: 2017-004) and was carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from individual or guardian participants. Consent to publish Not applicable Competing interests The authors declare that no conflict of interest exists. Received: 5 June 2022 / Accepted: 1 March 2023

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