BMC Molecular and Cell
Biology

Yang et al. BMC Molecular and Cell Biology(2023) 24:12
https://doi.org/10.1186/s12860-023-00470-9Open Access

RESEARCH

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*
Abstract
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 themechanisms mediated by abnormal methylation in the development of EMS.
Materials and methods Next-generation sequencing dataset and methylation profiling dataset were used toscreen 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.
Results 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 expressionof lentivirus carrying SFRP2 cDNA up-regulates the activity of Wnt signaling and the protein expression of ß-cateninin 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.
Conclusion In summary, the increased SFRP2 expression-induced Wnt/ß-catenin signaling due to the demethylationof the SFRP2 promoter plays an important role in the pathogenesis of EMS, suggesting that SFRP2 might be atherapeutic target for EMS treatment.
Keywords SFRP2, Endometriosis, Wnt pathway, Demethylation*Correspondence:
Chunfan Jiang
3667@hbuas.edu.cn
1
Department of Obstetrics and Gynecology, Xiangyang Central Hospital,
Affiliated Hospital of Hubei University of Arts and Science,
Xiangyang 441021, Hubei, China

2

Institute of Maternity disease, Xiangyang Central Hospital, AffiliatedHospital of Hubei University of Arts and Science, Xiangyang, Hubei, China
3
Department of Obstetrics and Gynecology, Renmin Hospital of Wuhan
University, 238 Jiefang Road, Wuhan 430060, China
4
Department of Pathology, Xiangyang Central Hospital, Affiliated Hospitalof Hubei University of Arts and Science, Xiangyang 441021, Hubei, China© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Yang et al. BMC Molecular and Cell Biology(2023) 24:12

BackgroundAs a common disease, EMS affects about 5-10% of
women of reproductive age, which causes decreases inlife quality and is accompanied by symptoms such as pelvic pain and affects more than 10% of reproductive-age
women[1, 2]. Several classical theories including MülLerianosis, retrograde menstruation, and coelomic metaplasia have been proposed to elucidate the pathogenesisof EMS, but the molecular mechanism is still unknown[3,4].
Many differences were found in gene expression profiles between EMS samples and the normal endometrium
tissue samples. Nowadays, microarray technology has
become a mature and stable technology, and during thelast decade’s bioinformatics analysis has been widely used
to identify general genetic of etiology and pathogenesis inmany 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 estrogen receptors and progesterone receptors in patientswith EMS[29]. Other scholars evaluated and compared
the methylation pattern of Human Homeobox clusters
in normal, eutopic (endometrium in the uterine cavityof the EMS patients), and ectopic endometrial tissues, aconserved pattern of methylation alterations in EMS tissues was observed for most of the investigated genes (56of 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 methylom in EMS.
Wnt signaling is an early event in some tissue carcinogenesis, there is evidence that the Wnt signaling pathway
also plays a role in the etiology of EMS[20–22]. Characterized histologically by dense fibrous tissue consisting,
EMS is researched by many scholars and it was foundthat treatment with Wingless mouse mamary umor virus(MMTV) integration site family member 3a (Wnt3a) significantly increased the proliferation and migration ofendometrial cells in patients with EMS, and significantly
enhanced the expression of fiber marker genes, such as
α-smooth muscle actin, type I collagen, connective tissue 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 collagen gel contraction on EMS[16]. After treatment with
Wnt3a, the contraction of collagen gel I in the endometrial cells in normal endometrium was increased to a levelcomparable to that in EMS patients[17–19]. In different
diseases, SFRP proteins were reported correlating with
the Wnt pathway, and their expression was regulated bymethylation[24, 34]. For example, SFRP2 is reported toPage 2 of 14

be closely related to Wnt and regulated by methylation innasopharyngeal carcinoma[35].
This study aimed to explore whether the wnt signaling pathway are mediated by abnomal methylation in thedevelopment of EMS.

Methods
Microarray data

Next-generation sequencing dataset (GSE135485) andmethylation 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 platform. GSE47359 consisted of 3 EMS samples and 6 normal endometrium tissue samples, based on the GPL8490
Illumina Human Methylation 27 platform.
On data processing and identification of differentiallyexpressed genes (DEGs), R software (ver. 3.6.3, https://www.rproject.org/) were used to identify DEGs and differentially methylated genes(DMGs). The matrix file for
GSE135485 was downloaded from https://www.ncbi.
nlm.nih.gov/geo/query/acc.cgi?acc=GSE135485 and thengene IDs conversion was conducted with strawberryPerl-5.30.0.1. The data normalization was done with thelimma package and then processed with the edge R package to get DEGs. The cut off value of DEGs was set as｜log2FC｜> 4. P < 0.05 was considered to indicate a statistically 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 identification of CpG sites and DMGs with the threshold P < 0.05
and ｜log2FC｜> 0.2. The Champ package is a highly
integrated methylation analysis tool, matching the corresponding DMGs with the most differentially methylated 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), andBiological Processes(BP). All p values < 0.05 were considered to be statistically significant.

Yang et al. BMC Molecular and Cell Biology(2023) 24:12

Patient recruitment

This study was initiated on November 11th, 2019 and terminated on April 20th, 2021. All of the women recruitedin this study were being at child-bearing age and underwent laparoscopic surgery at the Department of Gynecology of Xiangyang Central Hospital. Five women withendometriosis 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; Eachcase of endometriosis was staged during the operationaccording to the revised American Fertility Society classification 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 menstrual cycle. Ectopic endometrium from the ovarian cystof these 5 patients were obtained by laparoscopy.
Five women undergoing tubal ligation for sterilization were recruited as controls. All these five patientswere aged between 28 and 40 years old, mean ± SD
(34.60 ± 4.38) years. No minimal endometriosis wasfound in these control subjects and no hormones treatment for at least six months. All these women were in thesecretory phase of the menstrual cycle. Normal endometrium were obtained by curettage during tubal ligation
operation.
Cell culture

According to our previous study[36], tissues were washedwith sterile Hank’s Balanced Salt Solution(HBSS, phenolred-free) three times, then minced into pieces of approximately 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 supernatant 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, Carlsbad, CA) supplemented with 10% v/v fetal bovine serum(FBS; Invitrogen). Next, they were subjected to differential trypsinization and attachment for further purification. Finally, the primary epithelial cells were plated(2 × 104 cells/ml) in dishes in a culture medium as mentioned above. The detect the phenotypic characterization and ensure the purity of endometrial cell > 95%, theprimary epithelial cells were tested by dyeing of vimentin
and PCK.
Western blot

Western blot was performed according to our previous study [36] using primary anti-bodies against humanPage 3 of 14

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 ofthe protein bands were measured using the ImageJ (1.49v) 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 ability of DNA to accept methyl under the action of methyltransferase (DNMT). Meanwhile, 5-Aza-CdR forms acovalent 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 forthe inhibition of DNA methyltransferase activity.
Real-time RT PCR

Total RNA was isolated from EMS tissues and EEECs
utilizing the TRIzol reagent (Invitrogen, Shanghai, China), and all cRNA transcripts were generated using a primeScript™ RT kit (Qiagen, Hilden,
Journal of Molecular Histology1 Germany). All primers (Sangon Biotechnology, China) were listed as fellows: SFRP2, 5′-TGGGGGAAACGGTCGCACTC-3′,
and5′-GGCCACGAGACCATGAAGGAGG-3′.
β-catenin, 5′-AAAGCGGCTGTTAGTCACTGG-3′ and5′-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 detection 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 (GenePharma Company, Shanghai) were selected to test theWnt signaling pathway and the Promega dual-luciferase
reporter gene assay system was used to measure thereporter activity. TOP/FOP values were used to represent
the result. A higher value of TOP /FOP indicates a stronger Wnt pathway activity.
Methylation-specific PCR (MSP)

Genomic DNA from 5 ectopic endometrium and 5 normal endometrium was isolated using the DNA Extraction
Kit (Sangon Biotech, Shanghai, China). In the 50ul system, DNA (2–5 µg) was denatured by NaOH (final concentration 0.2 mol/L) at 37℃ for 10 Min. Add 30 µL of
10 mmol /L hydroquinone and 40.5% sodium bisulfite to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 precipitated with ethanol. Dissolve the DNA in 20µL water,
stored at -20℃. Two pairs of specific primers were used toamplify the same nucleotide sequence of the tested geneusing methylated primer pairs (M) 5′-GGAGTTTTTCGGAGTTGCGC-3′ and 5′-CTCTTCGCTAAATACGACTCG-3′, or unmethylated primer pairs (U)5′-GTTGGAGTTTTTTGGAGTTGTGT-3′
and5′-CTCTCTTCACTAAATACAACTCA-3′. The amplified products were detected by DNA agarose gel electrophoresis and analyzed by gel scanning.
Bisulfite sequencing PCR

Genomic DNA from 5 ectopic endometrium and 5 normal endometrium was isolated using the DNA Extraction Kit (Sangon Biotech, Shanghai, China). According
to the manufacture’s instruction, and bisulfite modification was performed with the EZ DNA Methylation GoldKit (Tianmo Technology, Beijing, China). Primer(Sangon
Biotechnology, China) sequences for bisulfite sequencing were listed as follows: forward(M818-F)5′TTTATGTTTGGTAATTTAGTAGAAATTT-3′
andreverse (M818-R) 5′-ATTTTACRTTAAAAATACCCCTCAC-3′. This area was 302-bp fragments including 28
CpG dinucleotides. The PCR conditions were: pre-denaturation 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 wassequenced(Sangon Biotech, Shanghai, China).
Plasmid construction and lentivirus production

The human SFRP2 lentiviral vectors were purchasedfrom GenePharma and transfected EEECs according tostandard manufacturer protocols. Furthermore, lentiviral
vectors to knockdown DNMT1 expression were generated by the GenePharma Company, (Shanghai), and theinterfering sequence is as follows: DNMT1-Homo-2664
GGAGCTGTTCTTGGTGGATGA. Three kinds ofinfection sequence were tested in the preliminary experiments, and one is useful as mentioned above. Postinfected cells were cultured for one week consecutively
and lentivirus infection condition of target cells were
determined by observing the expression time and intensity of GFP. To screen the stably transfection clusters, atthe basis of transient infection, puromycin with minimum lethal concentration lasts for at least 4 days.
Immunohistochemistry

A cohort of 84 formalin-fixation paraffin-embedded specimens (FFPE), including 28 EMS ectopic

Page 4 of 14

endometrium, 28 eutopic endometrium and 28 normal
endometrium were retrieved from Xiangyang Central
Hospital from 2006 to 2020 with necessary clinical information. 28 eutopic endometrium and ectopic endometrium 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 patientswere aged between 29 and 48 years old, mean ± SD
(41.80 ± 6.22) years. All the cases were reviewed by twosenior pathologists separately again to ensure the diagnosis accuracy.
Immunohistochemical staining for SFRP2 was performed with 3-µm-thick sections using the Ventana
Benchmark ULTRA automated staining system (Ventana
Medical Systems, Tucson, AZ) according to the manufacturer’s protocol. SFRP2 (Abcam), the primary antibodies
were added on the cell sections for two hours, Sections
were incubated with a secondary antibody and visualizedwith 3, 3’-diaminobenzidine tetrahydro-chloride (DAB;
Golden Bridge, Beijing, China). Sections were then subjected to nuclear counterstaining (blue staining) withhematoxylin. Two investigators were asked to review andscore 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: moderate; 7–9: high.
Transwell assay

BD matrigel and 1640 were diluted in a ratio of 1:3 and80ul was added to the upper chamber of the transwellchamber(8 μm; Millipore, Billerica, MA). EEECs weretreated with 5-Aza, sh-DNMT1 or lentivirus carrying
SFRP2-cDNA. Cell suspensions were configured according to the concentration of 200ul of serum-free medium
containing 2.5 × 104 cells. 500 µl DMEM medium wasadded to the subchamber wells of the Transwell plate
and the chamber was placed into the plate with care notto produce bubbles. Celcultures were grown in 37℃
incubator containing 5%CO2 for 24 h. Assays werethen 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 chamber was stained for 10 min; next the crystal violet of thecleaned chamber surface was washed with water, thecells in the upper chamber were wiped with a cottonswab and photographed under an inverted microscope.
Cells in five visual fields per insert were counted (400×magnification).

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 toexperimental grouping. They were incubated in an incubator at 5%CO2 and 37℃. When the cells grows to 80-90% confluence, a straight line was drawn in the wellusing the appropriate pipette gun head along a sterilization 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 approximately 48 h. Twenty-four hours after scratching, different treatments displayed remarkable effects on cellularmigration 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 hunder an inverted microscope. The relative migration
length in five random fields was measured with ImageJfor 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 estimatedby Student’s t-test. A p-value < 0.05 was considered significant. Chi-square was used in the Statistical analysis of
immunohistochemistry data.

Results
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 differentiallymethylated genes from a microarray analysis from the
GEO database (Fig. 1). A total of 85 differentially methylated genes were identified after the screening, 27 genes
were hypermethylated and 58 genes were hypomethylated in EMS. 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).
Legend Datas were reported as mean ± SD. All experiments were carried out in three or more replicates, andrepeated at least twice. Statistically significant was displayed 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 analyzed using the linear modeling approach. A total ofPage 5 of 14

134 DEGs were identified after the screening, of which
48 genes were upregulated and 86 were downregulated(｜log2FC｜> 4, p < 0.05) (Fig. 2). The downregulation
genes were not the research focus, so we did not present them here. 48 upregulation genes (｜log2FC｜> 5,
p < 0.05) were selected for subsequent bioinformatic
analysis. The significant terms of GO enrichment analysis performed by DAVID were illustrated in Table 2. Theintersection of hypomethylated genes and overexpressed
genes in EMS were get and there are two upregulationand 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 normal 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 inectopic endometrium (χ2 = 17.907, p < 0.001). The SFRP2
expression in the eutopic endometrium of EMS patients
showing no differences compared with normal endometrium samples( χ2 = 2.9, 8,p = 0.087 > 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 endometrium and EEECs vs. NEECs assessed using western
blot were exhibited in Fig. 5A. All the experiments wererepeated 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 endometrium/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 ectopic 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 withthat in normal endometrium, the SFRP2 promoter region
was hypomethylated in EMS, (P < 0.0001, Fig. 5F). To
further investigate whether the activation of SFRP2 isrelated to the methylation status of the promoter, Bisulfite sequencing PCR was used in EEECs and NEECs.
Direct sequencing analysis of a 302-bp fragment including 28 CpG dinucleotides in the SFRP2 promoter was
performed. Differential methylation was observed in 28
CpG dinucleotides of the promoter in these two kinds ofcells. We found that the percentage of methylated CpG

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 6 of 14

Yang et al. BMC Molecular and Cell Biology(2023) 24:12

Table 1 GO analysis of the low methylation expression genefrom the GSE47359 data
Category
GOTERM_CC_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT

GOTERM_MF_DIRECT

GOTERM_BP_DIRECT

GOTERM_BP_DIRECT

GOTERM_CC_DIRECT

GOTERM_CC_DIRECT

GOTERM_BP_DIRECT

GOTERM_BP_DIRECT

Term
GO:0000790 ~ nuclear chromatin
GO:0042493 ~ response to drug
GO:0031668 ~ cellular response
to extracellular
stimulus
GO:0003713 ~ transcription coactivator activity
GO:0048646 ~ anatomical structureformation involved
in morphogenesis
GO:0045944 ~ positive regulation oftranscription fromRNA polymerase IIpromoter
GO:0005667 ~ transcription factor
complex
GO:0090575 ~ RNApolymerase IItranscription factor
complex
GO:0050680 ~ negative regulationof epithelial cellproliferation
GO:0035914 ~ skeletal muscle celldifferentiation

Count
4

PValue0.005055271

30.011057299

20.029690877

30.030570734

20.031939077

50.043516788

30.053312753

20.058856003

20.099183706

20.099183706

After the GO analysis of the low methylation expression, the functions of thesehypomethylated genes were explored in several important cell processes,
including repressor, secreted, and signaling (Table 1)

dinucleotides in EEECs and NEECs was 41.8% and 77.6%,
respectively, P = 0.002 (Fig. 5G), This suggests that hypomethylation 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 ofpromoter methylation on SFRP2 expression, the depletion of DNMT was performed in EEECs. There were several 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 wasused. After the treatment with 5-Aza-2′-deoxycytidine,
it was found that the levels of SFRP2 protein were

Page 7 of 14

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 knockdown 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 wassignificantly 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 upregulated significantly ( Fig. 7A, B, D and p < 0.001). The protein 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 transwell and wound scratch assays. For EEECs, the numbersof cells on the lower surface of the insert membrane of
the transwell were counted after each treatment including 5-Aza, sh-DNMT1 or lentivirus carrying SFRP2cDNA(Fig. 8). All these three intervention were clearlystrong 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 theuntreated cells and any of those three groups receiving
different treatment(p < 0.001).

Discussion
Because of the complexity of biological traits and highheterogeneity, inadequate knowledge about mechanisms
at the molecular and cellular levels [11, 37], thoughmany differences were found in gene expression profiles
between EMS samples and the normal endometrium tissue samples[4–6]. [10–13], the pathogenesis of EMS has
yet to be fully elucidated.
Being considered as a heritable change in gene expression, epigenetics covers abnormal DNA methylation[26],Yang et al. BMC Molecular and Cell Biology(2023) 24:12

Page 8 of 14

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

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 especially methylation of key regulators plays a critical rolein carcinogenesis without alteration in DNA sequence.
For example, effects of certain genes with aberrant DNA
methylation on HCC and mammary stem cells havebeen extensively reported[26]. Aberrant DNA methylation could influence some tumor suppressor genes whichwere key genes involved in the carcinogenesis of HCC
and mammary stem cells. Epigenetic modifications werereported to play a role in the pathogenesis of EMS in
recent years[29–33], such as epigenetics of estrogen andprogesterone receptors and DNA methylation analysis of
HOX genes, however, the regulatory mechanism is still

unclear. Interestingly, in this study, bioinformatics analysis of DMG microarray and related experiments showedthat SFRP2 was significantly demethylated in EMS. From
the MSP data, it was found that compared with that innormal endometrium, the SFRP2 promoter region was
hypomethylated in EMS. Furthermore, direct sequencing analysis of a 302-bp fragment including 28 CpG
dinucleotides in the SFRP2 promoter was performed in
EEECs and NEECs. Significant difference was observedin the methylation rate of 28 CpG dinucleotides of thepromoter in these two kinds of cells. This suggests thathypomethylation of the SFRP2 promoter in EMS. Methylation of SFRPs was frequently detected in cancers, suchas nasopharyngeal carcinoma[35], however, the methylation status of SFRPs has not been reported in EMS yet.

Yang et al. BMC Molecular and Cell Biology(2023) 24:12

Page 9 of 14

Table 2 GO analysis of the upregulation genes from the GSE135485 data
Category
GOTERM_BP_DIRECT
GOTERM_MF_DIRECT
GOTERM_BP_DIRECT
GOTERM_MF_DIRECT
GOTERM_MF_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT
GOTERM_MF_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT
GOTERM_MF_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT
GOTERM_MF_DIRECT
GOTERM_BP_DIRECT
GOTERM_MF_DIRECT
GOTERM_BP_DIRECT
GOTERM_MF_DIRECT
GOTERM_MF_DIRECT
GOTERM_BP_DIRECT
GOTERM_CC_DIRECT
GOTERM_BP_DIRECT
GOTERM_MF_DIRECT
GOTERM_MF_DIRECT
GOTERM_BP_DIRECT
GOTERM_CC_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT
GOTERM_BP_DIRECT

Term
GO:0008584 ~ male gonad development
GO:0000980 ~ RNA polymerase II distal enhancer sequence-specific DNA binding
GO:0008585 ~ female gonad development
GO:0043565 ~ sequence-specific DNA binding
GO:0001077 ~ transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding
GO:0006366 ~ transcription from RNA polymerase II promoter
GO:0045944 ~ positive regulation of transcription from RNA polymerase II promoter
GO:0016491 ~ oxidoreductase activity
GO:0007584 ~ response to nutrient
GO:0050810 ~ regulation of steroid biosynthetic process
GO:0047498 ~ calcium-dependent phospholipase A2 activity
GO:0036149 ~ phosphatidylinositol acyl-chain remodeling
GO:0036148 ~ phosphatidylglycerol acyl-chain remodeling
GO:0036150 ~ phosphatidylserine acyl-chain remodeling
GO:0036152 ~ phosphatidylethanolamine acyl-chain remodeling
GO:0050482 ~ arachidonic acid secretion
GO:0008270 ~ zinc ion binding
GO:0036151 ~ phosphatidylcholine acyl-chain remodeling
GO:0003682 ~ chromatin binding
GO:0009755 ~ hormone-mediated signaling pathway
GO:0004623 ~ phospholipase A2 activity
GO:0017147 ~ Wnt-protein binding
GO:0070374 ~ positive regulation of ERK1 and ERK2 cascade
GO:0090575 ~ RNA polymerase II transcription factor complex
GO:0050873 ~ brown fat cell differentiation
GO:0003700 ~ transcription factor activity, sequence-specific DNA binding
GO:0004879 ~ RNA polymerase II transcription factor activity, ligand-activated sequencespecific DNA binding
GO:0006654 ~ phosphatidic acid biosynthetic process
GO:0005576 ~ extracellular region
GO:0035094 ~ response to nicotine
GO:0030522 ~ intracellular receptor signaling pathway
GO:0010811 ~ positive regulation of cell-substrate adhesion
GO:0048468 ~ cell development

Count
7
4
3
7
5

PValue
1.35E-07
5.23E-04
6.58E-04
0.001463497
0.002513506

6
8
4
3
2
2
2
2
2
2
2
7
2
4
2
2
2
3
2
2
6
2

0.009651672
0.01158106
0.012548298
0.015396063
0.020307471
0.021652815
0.040211948
0.045126018
0.045126018
0.059721243
0.059721243
0.0613717
0.066936914
0.068885928
0.069330097
0.072674345
0.072674345
0.073862462
0.07673751
0.078843017
0.081625912
0.083902905

2
8
2
2
2
2

0.085915314
0.08929768
0.090600685
0.092934565
0.092934565
0.097584789

The significant terms of GO enrichment analysis performed by DAVID from the GSE135485 data

SFRP2 is a member of the various secreted frizzledrelated 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 foundthat secreted frizzled-related proteins (SFRPs) and someother secreted proteins can competitively displace certain WNT ligands in some cancer models, and increase
in SFRP levels attenuates cancer growth, particularly inbreast cancer cells[20, 42]. But in the researches of prostate cancer cells in vitro, the overexpression of SFRP1
promotes the growth of BPH1, whereas over-expressionof SFRP4 or SFRP3 decreases the proliferation of humanPC3 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 primary cultured cells with EMS significantly reduced, suggesting 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, suggesting that overexpression of SFRP2 promotes the activityof Wnt signal and the growth of EMS lesions[24]. In ourstudy, it was found that compared with the normal endometrium/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Yang et al. BMC Molecular and Cell Biology(2023) 24:12

Fig. 3 Identification of aberrantly methylated-differentially expressed
genes in mRNA expression profiling datasets (GSE135485) and gene methylation profiling datasets (GSE47359).

protein expression of β-catenin and activity of Wnt signaling in EEECs were observed, further confirming that
SFRP2 may be an important factor in the up-regulation

Page 10 of 14

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 thetranscription 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 genesthat are normally associated with tumorigenesis[42].
Some researches have found that the aberrant activation of Wnt/β-catenin signaling significantly correlatedwith the pathophysiology of EMS. Some studies foundthat being a subunit of the cell surface cadherin protein
complex, β-catenin act as an intercellular signal transducer 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. 4 Immunohistochemical staining of SFRP2 protein in different endometrium tissues. A Normal endometrium. B Eutopic endometrium (endometrium in the uterine cavity of the EMS patients). C ectopic endometrium. D Through the immunohistochemistry staining, it was found that SFRP2 wasclearly located in the cytoplasm and was yellowy-brown

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 expression 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.

Yang et al. BMC Molecular and Cell Biology(2023) 24:12

Page 11 of 14

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 transfected 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

Fig. 7 Increase of Wnt signaling gene expression and activity after ectopic
SFRP2 expression in EEECs. A Western blot analysis. EEECs were transfectedwith SFRP2 cDNA or lv-control and subjected to Western blot analysis. Fulllength 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 ofbeta-Catenin protein level. The protein expression levels of beta-Cateninin 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 levelsof 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

stromal fibroblasts (EMSFs) contribute to EMS, but
before implantation, the activation of β-catenin wasessential 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-cateninsignaling 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 theprotein expression of β-catenin and activity of Wnt signaling in EEECs were observed, further confirming that
SFRP2 may be an important factor in the up-regulationof Wnt signaling in EMS tissues. To confirm that the
migration of EEECs could be affected by the regulationof the demethylation of SFRP2 promoter, we performed
Transwell and wound scratch assays with different treatments. We found that the number of cells on the lower
surface of the membrane and the scratch area left unfilledvaried significantly after 5-Aza, sh-DNMT1 or lentivirus
carrying SFRP2-cDNA intervention. Our data suggest
either demethylation of SFRP2 promoter or upregulationof SFRP2 intervention could significantly promote theinvasion and migration of EEECs.
There are great clinical relevance in our study. Abnormal activation of the Wnt/β-catenin signaling pathway
may be involved in the aggressive phenotype of EMS

Yang et al. BMC Molecular and Cell Biology(2023) 24:12

Page 12 of 14

Fig. 8 Demethylation of SFRP2 promoter changed the invasion and migratory ability of EEECs. A Transwell assays. EEECs were treated by 5-Aza, sh-DNMT1or lentivirus carrying SFRP2-cDNA respectively and subjected to transwell assays. B The numbers of EEECs on the lower surface of the insert membraneof 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

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 possibly 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 literature 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 thedevelopment of EMS and provide new strategies for theclinical 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, nextgeneration sequencing dataset and methylation profilingdataset 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 transfection were build. This is the first study which clarified themechanism 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 limitation of the study is the sample size was relatively small.

Conclusions
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 pathogenesis of EMS, suggesting that SFRP2 might be a novel regulatory gene and therapeutic target for EMS treatment.
This study confirmed that SFRP2 is activated in EMS due
to promoter demethylation. Our study could provide newclues 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

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
Acknowledgements
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.
FundingNational Natural Science Foundation of China (No. 81972449), Foundationof Hubei University of Arts and Science(XK2019046), Funder:Hui Xing, thecorresponding 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 publishedarticle.

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 applicableCompeting interests
The authors declare that no conflict of interest exists.
Received: 5 June 2022 / Accepted: 1 March 2023

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