Abnormal activation of the Wnt3a/β-catenin signaling pathway promotes the expression of TBX3 and the EMT pathway to mediate the occurrence of adenomyosis

Abnormal activation of the Wnt3a/β-catenin signaling pathway promotes the expression of TBX3 and the EMT pathway to mediate the occurrence of adenomyosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Abnormal activation of the Wnt3a/β-catenin signaling pathway promotes the expression of TBX3 and the EMT pathway to mediate the occurrence of adenomyosis Mengqi Li, Ting Li, Tingting Jin, Yi Chen, Lan Cheng, Qiheng Liang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2803345/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Oct, 2023 Read the published version in Molecular Biology Reports → Version 1 posted 4 You are reading this latest preprint version Abstract Background TBX3 is a transcription factor that can regulate cell proliferation, apoptosis, invasion, and migration in different tumor cells; however, its role in adenomyosis (ADM) has not been previously studied. Some of ADM’s pathophysiological characteristics are similar to those of malignant tumors (e.g., abnormal proliferation, migration, and invasion). Methods and results we hypothesized that TBX3 might have a role in ADM. We used tamoxifen-induced ICR mice to establish ADM disease model. The study procedure included western blotting and immunohistochemistry to analyze protein levels; additionally, we used intraperitoneal injection of Wnt/β-catenin pathway inhibitor XAV-939 to study the relationship between TBX3 and Wnt/β-catenin pathway as well as PCNA and TUNEL to detect cell proliferation and apoptosis, respectively. TBX3 overexpression and epithelial-to-mesenchymal transition (EMT) in ADM mice was found to be associated with activation of the Wnt3a/β-catenin pathway. Treatment with XAV-939 in ADM mice led to the inhibition of both TBX3 and EMT; moreover, abnormal cell proliferation was suppressed, the depth of invasion of endometrium cells was limited and the expression of ERα was suppressed. Thus, the use of XAV-939 effectively inhibited further invasion of endometrial cells. Conclusion These findings suggest that TBX3 may play an important role in the development of ADM. The expression of TBX3 in ADM was regulated by the Wnt3a/β-catenin pathway. The activation of the Wnt3a/β-catenin pathway in ADM promoted TBX3 and ERα expression and induced the occurrence of EMT, thus promoting cell proliferation and inhibiting apoptosis, ultimately accelerating the development of ADM. The study provides a reference for the diagnosis of ADM Adenomyosis (ADM) Epithelial-to-mesenchymal transition (EMT) T-box transcription factor3.Wnt3a/β-catenin signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Adenomyosis (ADM), a benign gynecological disease[ 1 ], is pathologically characterized as endometrium/gland invasion of the myometrium, along with hyperplasia and hypertrophy of the myometrium[ 2, 3]. Clinical symptoms include uterine diffusion and enlargement, pelvic pain, abnormal uterine bleeding, and hypofertility [ 4 – 7 ], and this disease may seriously impact the patients’ quality of life. Notably, as no effective drug has been developed for the treatment of ADM, surgery remains the only treatment option at present [ 8 ]. Thus, further research into the specific mechanisms underlying the occurrence and development of ADM may aid the development of alternative treatment options. T-box transcription factor 3 (TBX3), a member of the T-box transcription factor family of proteins, may play an essential regulatory role in numerous organs, including the heart, mammary glands and genitalia[ 9 ]. TBX3 may also regulate cell proliferation and differentiation, tissue integrity, and epithelial-to-mesenchymal transition (EMT) through acting as a transcriptional inhibitor or activator [ 10 – 13 ]. Previous studies have indicated that associated overexpression of TBX3, as a tumor promoter or tumor suppressor, promoted tumorigenesis in several cancers. Specifically, overexpression of TBX3 has been with head and neck squamous cell carcinoma, gastric cancer, breast cancer, rhabdomyoma, cervical cancer, pancreatic cancer, papillary thyroid cancer, lung cancer and chondrosarcoma [ 14 – 17 ]; however, the specific function in ADM is yet to be determined. Although ADM is a benign disease, certain pathophysiological features are comparable with those of malignant tumors; for example, abnormal proliferation, migration and invasion [ 18 ]. Therefore, the present study aimed to determine the association between TBX3 expression and the occurrence of ADM. Numerous functional proteins or signaling pathways, such as (wingless-related MMTV integration site, transforming growth factor-β), bone morphogenetic protein, fibroblast growth factor and Hippo, regulate cell proliferation, apoptosis and migration [ 19 – 22 ]. Specifically, the Wnt signaling pathway plays a critical role in complex cellular behaviors, such as tissue development, regeneration and cell homeostasis, in addition to mediating cell proliferation, polarity, differentiation, cell motility and stem cell activity [ 23 ]. Numerus previous studies have also suggested the existence of an association between the Wnt pathway and TBX3, as TBX3 may be regulated by, and act as a downstream target of, the Wnt signaling pathway in certain diseases [ 24–26]. Thus, determining whether TBX3 may also be regulated by the Wnt signaling pathway in ADM may provide useful insights into the underlying mechanisms of ADM. EMT plays a crucial role in the occurrence and development of ADM. Results of a previous study demonstrated that estrogen-induced EMT of endometrial epithelial cells may enhance invasion, thereby contributing to the development of ADM [ 27]. Notably, EMT is characterized by the upregulation of N-cadherin, vimentin and α-smooth muscle actin (α-SMA), and the downregulation of E-cadherin. This is regulated by transcription factors Slug, Snail and Twist [ 28 ]. The occurrence of EMT in tissues may result in acquired mesenchymal phenotypes, and to the enhancement of cellular migratory capacity [ 29 ]. In addition, EMT is regulated by the Wnt signaling pathway. TBX3 directly induces the occurrence of EMT through promotion of Slug expression in breast cancer; thus, promoting the proliferation of breast cancer cells [ 30 ]. Therefore, treatment with a Wnt pathway inhibitor, such as XAV-939, may provide valuable insight into whether EMT is reversed in ADM. The present study aimed to investigate the expression of TBX3 in ADM mice, and determine the association between TBX3 and the Wnt/β-catenin signaling pathway in ADM. Using tamoxifen-induced ICR mice to establish an ADM disease model, numerous methods were carried out to analyze protein levels, and determine the association between the Wnt/β-catenin signaling pathway and TBX3, EMT, cell proliferation and apoptosis. The present study provides novel insights into the diagnosis and treatment of ADM. Materials and methods Chemicals. Tamoxifen citrate (TAM) was purchased from Yangtze River Pharmaceutical Co. Ltd. and Wnt signaling pathway inhibitor, XAV-939, was purchased from Selleck Chemicals. Corn Oil was purchased from MedChemExpress and DMSO was purchased from Beijing Solarbio Science & Technology Co., Ltd. Animal experiments All animal experiments were conducted according to ethical guidelines and procedures approved by the Institutional Animal Care and Use Committee of Jinan University (ethics approval number, IACUC-20200905-01). The animal disease model, which followed the protocol established by Shen et al. [ 31 ], consisted of four female and four male ICR mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.). The mice were housed under controlled conditions (12 h light/12 h dark cycle and a constant temperature of 23°C), with food and water available ad libitum . After the female mice gave birth, each dam was housed in the same cage as the pups. Newborn female mice were used in the experiments that were subsequently conducted. A total of 40 neonatal female ICR mice were randomly divided into two groups: i) TAM or ii) control. From Days 2–5 after birth, the TAM group mice were fed with 1 mg/kg TAM suspended in a mixture of peanut oil, lecithin and condensed milk (volume, 2:0.2:3) at a dose of 5 µl/g body weight. Moreover, the control group mice were fed the same amount of solution without TAM during the same period. Newborn mice were breastfed. Upon reaching 21 days of age, the female mice were weaned and separated from the dams and were fed until Day 60, when three mice were randomly selected from the two respective groups. The uteri of the sacrificed mice were used to prepare paraffin sections. Hematoxylin and eosin (H&E) staining was used to determine whether the ADM disease model had been successfully constructed. Following successful construction of the model, five new mice from the respective TAM and control groups were randomly selected to be in ADM or control groups for the next phase of the study. Moreover, the remaining 24 mice were randomly divided into three groups: i) Control (n = 8); ii) ADM (n = 8); and iii) XAV-939 groups (n = 8). The XAV-939 group mice received an intraperitoneal injection of 3 mg/kg Wnt/β-catenin signaling pathway inhibitor, XAV-939 (4% DMSO + corn oil + XAV-939), while mice from both the Control (n = 8) and ADM (n = 8) groups received intraperitoneal injections with the same dosage of solution (4% DMSO + corn oil). All mice were injected once per day for seven consecutive days, and the samples were sacrificed one day after the completion of the injection period. Tissue preparation. The left uteri were used to create paraffin sections. Once obtained from mice, the uteri were immediately fixed for 24 h in 4% paraformaldehyde. Subsequently, the uteri were rinsed with water, and successively dehydrated with 75, 85, 90, 95 and 100% ethanol, prior to submersion in mixtures of xylene and ethanol, and xylene I and xylene II, to obtain improved transparency of sections. Tissues were immersed in molten paraffin, embedded and cut into 4-µm sections. H&E staining For the histological examinations of tissues for the pathological diagnosis of ADM, three randomly selected paraffin-embedded sections from each mouse uterus was stained using H&E. Briefly, slices were heated for 1 h at 60℃, followed by xylene I and II deparaffinization, and successive ethanol hydration at 100, 95, 90 and 80%. Slides were successively stained with H&E, and sealed with neutral gum prior to being photographed under a light microscope (Nikon Corporation). Immunohistochemistry (IHC) analyses Paraffin-embedded sections were deparaffinized and hydrated as previously described, followed by a 15 min submersion period in citrate buffer solution (pH, 6.0) for antigen retrieval. Endogenous peroxidase activity was eliminated using 3% hydrogen peroxide. In addition, Triton X-100 (Beyotime Institute of Biotechnology) was added to tissue sections to increase the permeability of cells, and non-specific antigens were blocked with 10% goat serum for 1 h at room temperature. Tissue sections were subsequently incubated with the following antibodies: anti-TBX3 (1:200; cat. no. ab99302; Abcam), anti-E-cadherin (1:200; cat. no. 3195; Cell Signaling Technology, Inc.), anti-N-cadherin (1:200; cat. no. 22018-1-AP; ProteinTech Group, Inc.), anti-Twist1 (1:200; cat. no. 25465-1-AP; ProteinTech Group, Inc.), anti-α-SMA (1:200; cat. no. 19245; Cell Signaling Technology, Inc.), anti-vimentin (1:200; cat. no. ab137321; Abcam), anti-Wnt3a (1:200; cat. no. ab219412; Abcam) and anti-proliferation cell nuclear antigen (PCNA; 1:200; cat. no. 10205-2-AP; ProteinTech Group, Inc.) overnight at 4℃. In addition, PBS was used to replace the primary antibody as a negative control. Following primary incubation, tissues were incubated with the HRP-conjugated secondary antibody (1:200; cat. no. 7074; Cell Signaling Technology, Inc.) for 2 h at room temperature. Immunoreactive proteins were visualized using 3, 3-N-Diaminobenzidine tetrahydrochloride (Sangon Biotech Co., Ltd.), and nuclei were counterstained with hematoxylin. Sections were fixed with neutral resins and observed under a light microscope (Nikon Corporation). Semi-quantitative immunoreactivity was determined using ImageJ (National Institutes of Health), as previously described [ 31 , 32 ]. A series of five images (magnification, x200) were randomly selected from multiple sections per tissue sample to obtain the mean. Staining was carried out using a color mask and assessed using color intensity. The mask was equally applied to all images, and measurement readings were recorded. Immunohistochemical parameters included: i) Integrated optical density (IOD); ii) total stained area (S), and iii) the mean optical density (MOD). The latter was defined as MOD = IOD/S, and was equivalent to the mean intensity of staining across all evaluated areas, including the glands and stroma. The immunostaining level (MOD) for all slides was computed for statistical analysis. In situ cell proliferation C ell proliferation of the uterine tissues was assessed using IHC with a proliferating cell marker, PCNA, as previously described. In situ TUNEL analysis Apoptosis in mouse uterine tissues was assessed using a One-step TUNEL Apoptosis Assay kit (Green FITC-Labeled Fluorescence Assay; Universal Imaging, Inc.) following the manufacturer’s protocol (Nanjing KeyGen Biotech Co., Ltd.). Briefly, following deparaffinization and rehydration, protease K was used to increase the permeability of cells. Tissue sections were incubated with a 45:1:4 TdT reaction mixture (Equilibration buffer: biotin-11-dUTP: TdT enzyme) for 1 h at 37℃ in a humidified chamber. Following washing with PBS, tissue sections were incubated with a 1:9 Streptavidin-Fluoresce solution (Streptavidin-Fluoresce solution: diluted labeling buffer) for 30 min at 37℃ in the dark. Sections were subsequently counterstained with DAPI for 10 min prior to sample analysis using fluorescence microscopy (Leica Microsystems GmbH). Western blotting The right sides of the mice uteri were homogenized and ultrasonicated following the addition of RIPA lysis buffer (containing 10% PMSF; Beyotime Institute of Technology). Ice lysis was performed for 30 min, and the lysate was centrifuged at 14,000 g for 15 min at 4℃. Protein concentration was determined using a BCA detection kit (Beyotime Institute of Biotechnology). Following electrophoresis on a 10% SDS-PAGE gel, proteins were transferred to PVDF membranes (MilliporeSigma) and blocked with 5% skimmed milk (BD Biosciences) for 1 h. Membranes were washed and incubated overnight at 4℃ with the following primary antibodies: Anti-TBX3 (1:1,000; cat. no. ab99302; Abcam), anti-Wnt3a (1:1,000; cat. no. ab219412; Abcam), anti-bcl2 (1:2,000; cat. no. ab182858; Abcam), anti-bax (1:8,000; cat. no. ab32503; Abcam), anti-β-catenin (1:10,000; cat. no. 51067-2-AP; ProteinTech Group, Inc.), anti-Slug (1:1,000; cat. no. 9585; Cell Signaling Technology, Inc.), anti-N-cadherin (1:6,000; cat. no. 22018-1-AP; ProteinTech Group, Inc.), anti-β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) and anti-GAPDH (1:2500; cat. no. ab245355; Abcam). Following primary incubation, membranes were incubated with HRP-conjugated goat-anti-rabbit IgG (1:8,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were visualized using ECL chemiluminescence reagent (MilliporeSigma), according to the manufacturer’s instructions. Statistical analysis Data are expressed as the mean ± standard deviation. An unpaired two-tailed Student’s t-test was used to evaluate the differences between two groups, and one-way ANOVA followed by Tukey’s post hoc test was used to compare three groups. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc.). P < 0.05 was considered to indicate a statistically significant difference. Results Establishment of an ADM disease model in mice Newborn female mice were fed until Day 60, after which, the uteri were removed and stained using H&E for the diagnosis of ADM. As shown in Fig. 1 A, the myometrium of the control group was arrayed in bundles and was well-spaced from the endometrium, whereas the endometrium stroma from the TAM group were immersed into the muscular layer, that was divided into multiple bundles by the stroma (Fig. 1 B). In addition, in the samples from the TAM group, the smooth muscle structure of the muscular layer was notably disordered, and the endometrium boundary appeared unclear. Through IHC staining of the α-SMA protein, results of the present study further demonstrated that endometrial stromal cells and glands invaded the myometrium (Fig. 1 D-E). These findings illustrated that the establishment of ADM was successful. In addition, the depth of endometrium focal myometrial infiltration was evaluated as follows: Grade 0, no endometrium infiltration into the myometrium; Grade 1, penetration of the endometrium focal into the superficial myometrium; Grade 2, penetration into the mid-myometrium; and Grade 3, penetration beyond the mid-myometrium. As illustrated in Fig. 1 C, the majority of infiltration depth scores for ADM mice were classified as Grade 2. Expression of TBX3 in the uterine tissues of ADM mice The association between TBX3 and ADM was investigated, and protein expression levels of TBX3 were determined in the uterine tissues of control and ADM mice. As indicated in Fig. 2 A, IHC analysis revealed the increased protein expression of TBX3 in the uterine tissues of the ADM group, compared with the control group. Moreover, results of the western blot analysis revealed that TBX3 protein expression levels were increased in the uterine tissues of ADM mice (Fig. 2 B). These findings suggested a potential association between the expression of TBX3 and ADM. Activation of Wnt/β-catenin and EMT pathways in the uterine tissues of ADM mice Results of previous studies demonstrated that TBX3 expression may be regulated by the Wnt signaling pathway in various cancers, which also regulates cell proliferation and migration[ 24, 26]. Therefore, changes in the Wnt3a/β-catenin pathway and the expression of EMT-related proteins were determined in the uterine tissues of ADM mice. Results of the western blot analysis demonstrated that the expression of nuclear β-catenin was significantly higher in the ADM group than in the control group (Fig. 3 A). In addition, levels of Wnt3a, a member of the Wnt family, were increased (Fig. 3 B). These results demonstrated abnormal activation of the Wnt3a/β-catenin pathway in the uterine tissues of ADM mice. The expression of EMT-related proteins in mouse uterine tissues was detected, and changes in EMT were investigated following activation of the Wnt3a/β-catenin pathway. Results of the western blot analysis demonstrated an increase in Slug and N-cadherin protein expression levels in the ADM group (Fig. 3 C). Results of the IHC assay further demonstrated the decreased protein expression of EMT marker, E-cadherin, in the uterine tissues of ADM mice, compared with the control group. In addition, results of the present study demonstrated that Twist1 protein expression levels were increased (Fig. 3 D). As shown in Fig. 3 D, protein expression levels of α-SMA and vimentin were also significantly increased in the uterine tissues of ADM mice. Thus, results of the present study suggested that global activation of the Wnt/β-catenin pathway and EMT occurred in ADM mice following the increased expression of TBX3. Thus, increased TBX3 expression in ADM mice may be associated with activation of the Wnt/β-catenin pathway and EMT. Abnormal proliferation and apoptosis in the uterine tissues of ADM mice Activation of Wnt/β-catenin and increased TBX3 expression impacted cell proliferation and apoptosis in the uterine tissues of ADM mice. Positive expression of PCNA was used to identify in situ cell proliferation (Fig. 4 A). Results of the present study demonstrated that PCNA expression was more prevalent in the nuclei of endometrial glands and stromal cells of uterine tissues than in smooth muscle cells. Additionally, PCNA positive staining levels were significantly higher in the uterine tissues of ADM mice than in control mice. Results of the western blot analysis demonstrated upregulation of bcl2 protein in the uterine tissues of ADM mice (Fig. 4 C), while protein expression of bax was reduced. These findings further indicated that cell proliferation was significantly increased when TBX3 expression was increased, and Wnt/β-catenin was activated in the uterine tissues of ADM mice. Moreover, results of the present study revealed that TUNEL-positive expression was reduced in the ADM group, compared with the control group. These results indicated that cell apoptosis was decreased in ADM mice when cell proliferation increased (Fig. 4 B). Collectively, these results suggested that cell proliferation increased and apoptosis decreased when the Wnt3a/β-catenin signaling pathway was activated, and TBX3 was highly expressed in ADM mice. TBX3 expression is regulated by the Wnt3a/β-catenin signaling pathway in the uterine tissues of ADM mice. To investigate the regulatory association between TBX3 and the Wnt3a/β-catenin signaling pathway in ADM mice, mice were injected with the Wnt signaling pathway inhibitor, XAV-939. Results of the western blot analysis demonstrated that Wnt3a and nuclear β-catenin protein expression levels were reduced in the XAV-939 group, compared with the ADM group (Fig. 5 B). In addition, Wnt3a and nuclear β-catenin protein expression levels were increased in the ADM group, compared with the control group. These results suggested that XAV-939 inhibited the Wnt3a/β-catenin signaling pathway in ADM mice. In addition, TBX3 protein expression levels were decreased in the XAV-939 group, compared with the ADM group (Fig. 5 A), which was consistent with results of the IHC analysis (Fig. 5 C). These results indicated that expression of TBX3 in the uterine tissues of ADM mice may be regulated by the Wnt3a/β-catenin signaling pathway. Wnt signaling pathway inhibitor, XAV-939, causes the reversal of EMT, and inhibition of endometrium cell invasion and proliferation. Analyses of the effects of XAV-939 revealed an expression change in EMT-related proteins post-injection. Results of the western blot analysis revealed reduced Slug protein expression levels in the XAV-939 group, compared with the ADM group (Fig. 6 B). Moreover, N-cadherin protein expression levels were also decreased in the XAV-939 group, demonstrated using western blotting (Fig. 6 B) and IHC analysis (Fig. 6 A). In addition, Twist1 and α-SMA protein expression was reduced, and E-cadherin protein expression was increased (Fig. 6 C). Results of the present study demonstrated that activation of EMT in ADM mice may be reversed following treatment with XAV-939. Moreover, regulation and inhibition of EMT by Wnt3a/β-catenin may impede further development of ADM. Histopathological changes in mouse uterine tissues were detected following inhibition of the Wnt3a/β-catenin pathway. As displayed in Fig. 7 A, H&E staining revealed that intrusion of the endometrium into the muscular layer of mice was limited following treatment with XAV-939, compared with the ADM group. Moreover, the infiltration depth score of ADM mice was decreased (Fig. 7 B), indicating that treatment with XAV-939 inhibited the invasion of endometrium cells in ADM mice. These results were comparable with those obtained following investigation of cell proliferation in the endometrium. Results of the western blot analysis revealed that protein expression levels of bcl2 were lower in the XAV-939 group, compared with the ADM group (Fig. 7 C). In addition, results of the IHC assay demonstrated that positive staining of PCNA was lower in the XAV-939 group than in the ADM group, which, in turn, was higher than that of the control group (Fig. 7 D). These results indicated that abnormal cell proliferation was inhibited in the uterine tissues of mice with ADM, following treatment with XAV-939. By contrast, positive TUNEL expression was higher in the XAV-939 group than in the ADM group, which, in turn, was lower than that of the control group (Fig. 7 E). These results suggested that cell apoptosis was increased in ADM mice following treatment with XAV-939. Results of the present study demonstrated that when Wnt3a/β-catenin signaling was inhibited, the protein expression of TBX3 was suppressed, and EMT was inhibited. Moreover, the Wnt signaling pathway inhibitor, XAV-939, reversed EMT and inhibited endometrial cell invasion and proliferation; thus, further suppressing the development of ADM. Therefore, the occurrence and development of ADM may be regulated by TBX3, EMT and the Wnt3a/β-catenin signaling pathway. Overexpression of TBX3 and the occurrence of EMT is regulated by Wnt3a/β-catenin signaling in ADM mice. Discussion The pathogenesis of ADM, a gynecological disease, is yet to be fully elucidated. The most widely accepted theory postulates that ADM lesions, originating from the basal layer of the endometrium, deeply invaginate into the myometrium [ 33 ]. Although some aspects remain unclear, numerous studies have investigated the molecular mechanisms of ADM. In addition, ADM is accompanied by abnormal cell proliferation, apoptosis and migration; which are similar characteristics to those of cancer cells. Therefore, the present study aimed to investigate the molecular mechanisms of ADM, using TBX3 as a target protein. As a regulatory transcription factor, TBX3 regulates cell proliferation, differentiation, tissue integrity and the process of EMT. Moreover, TBX3 regulates organ growth and development, and is also involved in the regulation of a variety of diseases [ 34 ]. Results of a previous study revealed that TBX3 is regulated by Wnt/β-catenin, and TBX3 overexpression may promote human colorectal cancer [ 35 ]. Results of our previous study demonstrated that TBX3, along with TBX2, are typical effectors of Wnt signaling in ureteral mesenchymal cells, and are regulated by the Wnt pathway when activated in the uterus [ 26 ]. In addition, results of the present study demonstrated that overexpression of TBX3 in the uterine tissues of mice was associated with ADM development. Increased protein expression of TBX3 in uterine tissues and stromal cells may be associated with endometrium invasion of the myometrium. The present study also aimed to determine whether the expression of TBX3 in ADM mice was regulated by the Wnt3a/β-catenin pathway. Results of the present study demonstrated that TBX3 protein expression was suppressed by XAV-939 in ADM mice, suggesting that the expression of TBX3 in ADM may be regulated by the Wnt3a/β-catenin pathway. Results of a previous study revealed that Wnt/β-catenin signaling may regulate the development of ADM [ 36 ]; however, results of the present study also demonstrated an increase in cell proliferation and a decrease in apoptosis, following TBX3 overexpression. Moreover, when TBX3 was inhibited by XAV-939, results of the present study demonstrated a reduction in cell proliferation and increased apoptosis. Collectively, these findings suggested that TBX3 may be situated downstream of the Wnt3a/β-catenin pathway. EMT also plays an important role in the development of ADM, and results of the present study illustrated the occurrence of EMT in ADM. Specifically, a loss of E-cadherin expression and an increase in the abundance of mesenchymal markers, such as vimentin, α-SMA and N-cadherin, were observed. Numerous previous studies demonstrated that functional proteins promote the occurrence of EMT by activating the Wnt/β-catenin pathway [ 37 – 40 ]. Inducers of EMT, such as Snail, Slug, Zeb1 and Twist, serve as target genes of the Wnt pathway [ 41 – 43 ]. Notably, activation of the Wnt/β-catenin pathway may promote EMT. Moreover, treatment with XAV-939 in ADM mice subsequently increased the expression of E-cadherin in the uterus, and reduced the levels of Twist1 and α-SMA. Thus, results of the present study revealed that EMT in ADM was regulated by the Wnt3a/β-catenin pathway. In addition, the occurrence of EMT in the uterus induced uterine epithelial cells to lose their polarity in acquiring a mesenchymal phenotype and migrate into the myometrium. Thus, ADM development was promoted. Results of the present study also confirmed that XAV-939 reversed EMT in ADM. Changes in cell proliferation and apoptosis are distinct phenotypes in the development of ADM regulated by the Wnt3a/β-catenin pathway. Results of the present study demonstrated an increase in cell proliferation and inhibition of apoptosis in ADM mice, following treatment with XAV-939. Thus, we hypothesized that abnormal activation of the Wnt3a/β-catenin pathway may promote cell proliferation and inhibit apoptosis which, in turn, will accelerate the development of ADM. Notably, TBX3 may directly upregulate Slug expression to promote the occurrence of EMT in breast cancer [ 30 ], and TBX3 may have a regulatory function in organ development and cancer development [ 9 ]. Results of a previous study demonstrated an association between TBX3 function, and cell proliferation and apoptosis, tumor formation, metastasis, cell survival, angiogenesis, invasion, and cancer stem cell expansion [ 33 ]. In addition, results of previous studies demonstrated that development of ADM involves immunization and adhesion factors, angiogenesis, inflammation, cell invasion, and migration [ 44 , 45 ]. Thus, further investigations into the function of TBX3 in cell survival, angiogenesis, fibrosis, inflammation, cell invasion and migration in ADM are required. Conclusion In conclusion, results of the present study provide a theoretical basis for the role of TBX3 in ADM, and further investigations into the regulatory association between TBX3 and relevant pathways, such as the Wnt and Hippo pathways, are required. Thus, the present study provides a reference for the potential treatment of ADM. Abbreviations TBX3 T-box transcription factor 3 ADM Adenomyosis ICR Institute of Cancer research EMT Epithelial-to-mesenchymal transition α-SMA α-smooth muscle actin TAM Tamoxifen citrate DMSO Dimethyl sulfoxide H&E Hematoxylin and eosin IHC Immunohistochemistry GAPDH Glyceraldehyde-3-phosphate dehydrogenase PCNA Anti-proliferation cell nuclear antigen ECL Enhanced chemiluminescence PBS Phosphate buffered saline PMSF Phenylmethanesulfonyl fluoride BCA Bicinchoninic Acid RIPA Radio immunoprecipitation assay PVDF Polyvinylidene fluoride IOD Integrated optical density MOD Mean optical density Declarations Acknowledgments The authors would like to thank the Laboratory Animal Center of Jinan University for caring for the animals in the present study. The authors would also like to thank the Central Laboratory of the School of Medicine for providing the experimental platform. Funding The present study was supported by the Guangdong Provincial Hospital of Chinese Medicine - Weixian Li famous doctor studio (grant no. E43719)and National Famous Traditional Chinese Medicine Expert inheritance Studio - Jianling Huang(project no.0102016205) Authors’ contributions WQC and QZR conceived and designed the study and revised the manuscript. MQL and TL performed the experiments and wrote the manuscript. TTJ ,YC,and SMY analyzed the data. LC and QHL provided clinical guidance. TTL provided guidance in HE experiments. All authors read and approved the final manuscript. Ethics approval and consent to participate All animal experiments were approved by the Laboratory Animal Review Committee of Jinan University (ethics approval number, IACUC-20200905-01). Patient consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. 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Cell 117(7):927-939. https://https://doi.org/10.1016/j.cell.2004.06.006 Sánchez-Tilló E, de Barrios O, Siles L, Cuatrecasas M, Castells A and Postigo A (2011) Β-catenin/tcf4 complex induces the epithelial-to-mesenchymal transition (emt)-activator zeb1 to regulate tumor invasiveness. Proc Natl Acad Sci U S A 108(48):19204-19209. https://doi:10.1073/pnas.1108977108 Benagiano G, Brosens I and Habiba M (2013) Structural and molecular features of the endomyometrium in endometriosis and adenomyosis. Hum Reprod Update 20(3):386-402. https://10.1093/humupd/dmt052 Vannuccini S, Tosti C, Carmona F, Huang SJ, Chapron C, Guo SW and Petraglia F (2017) Pathogenesis of adenomyosis: An update on molecular mechanisms. Reprod Biomed Online 35(5):592-601. https://https://doi.org/10.1016/j.rbmo.2017.06.016 Supplementary Files GraphicalAbstract.png Cite Share Download PDF Status: Published Journal Publication published 25 Oct, 2023 Read the published version in Molecular Biology Reports → Version 1 posted Reviewers agreed at journal 03 May, 2023 Reviewers invited by journal 02 May, 2023 Editor assigned by journal 14 Apr, 2023 First submitted to journal 12 Apr, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2803345","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":196812441,"identity":"bce91330-f1fb-421f-8cc4-8fd7a7766f6c","order_by":0,"name":"Mengqi Li","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Mengqi","middleName":"","lastName":"Li","suffix":""},{"id":196812442,"identity":"69e5bccc-4eaa-4393-8f12-95a627785da8","order_by":1,"name":"Ting Li","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Li","suffix":""},{"id":196812443,"identity":"45fa7b02-7259-4e8c-9b10-9b4797afb899","order_by":2,"name":"Tingting Jin","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Jin","suffix":""},{"id":196812444,"identity":"def2e5f6-296d-4a99-970f-fd453320021e","order_by":3,"name":"Yi Chen","email":"","orcid":"","institution":"Guangdong Provincial Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Chen","suffix":""},{"id":196812445,"identity":"c05fd3cb-6ce1-4d39-a650-5aa2efa48f27","order_by":4,"name":"Lan Cheng","email":"","orcid":"","institution":"Guangdong Provincial Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lan","middleName":"","lastName":"Cheng","suffix":""},{"id":196812446,"identity":"bd7a7a3a-02f2-48ca-b9fd-db1090eff16d","order_by":5,"name":"Qiheng Liang","email":"","orcid":"","institution":"Guangdong Provincial Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qiheng","middleName":"","lastName":"Liang","suffix":""},{"id":196812447,"identity":"27e2ece3-8d2f-4a1e-b503-58014a2ce65c","order_by":6,"name":"Simiao Yan","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Simiao","middleName":"","lastName":"Yan","suffix":""},{"id":196812448,"identity":"b4932d7c-5581-41ee-81da-ea51dc93ae94","order_by":7,"name":"Tingting Li","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Li","suffix":""},{"id":196812449,"identity":"993e5c13-41b1-461d-9311-47ea9b49429a","order_by":8,"name":"Wanqun Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYFACHjApx9gAotiI0gDRYky6lkSwDqK02LOfPSbxc4d1evPsHgOGD2WHGfhnNxCwhScvTbL3THpu45wzBowzzh1mkLhzgIAWCR6zG7xth3MbZ+QYMAMZDAYSCYS13PzbdjidEaTlL7FabgMNTwBrYSRKy5kc89+ybemGjTPSCg72nEvnkbhBQAt7+xljw7dt1vKGM5I3PvhRZi3HP4OAFihgZjBsYGA4wABLDERpkSdW6SgYBaNgFIw8AADSNT49b34cNgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4474-9040","institution":"暨南大学医学院","correspondingAuthor":true,"prefix":"","firstName":"Wanqun","middleName":"","lastName":"Chen","suffix":""},{"id":196812450,"identity":"f05e1d0e-ec1e-4de8-8235-082dbf41d98f","order_by":9,"name":"Qingzhen Ran","email":"","orcid":"","institution":"Guangdong Provincial Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qingzhen","middleName":"","lastName":"Ran","suffix":""}],"badges":[],"createdAt":"2023-04-12 05:58:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2803345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2803345/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-023-08870-y","type":"published","date":"2023-10-25T15:02:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":36659302,"identity":"cad0a245-acef-4b3a-8ae6-72e720d9cb37","added_by":"auto","created_at":"2023-05-05 21:33:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1170666,"visible":true,"origin":"","legend":"\u003cp\u003eSuccessful establishment of an ADM model in mice. (A) Uterine tissues of the control group. Arrayed bundles in the myometrium, well-spaced from the endometrium. (B) Uterine tissues of the TAM group. Endometrial stroma invaded the myometrium, and the muscular layer is divided into multiple bundles by the stroma, indicated by a black arrow. (C) Infiltration depth score of uterine tissues (n= 6). \u003csup\u003e****\u003c/sup\u003eP\u0026lt;0.0001. (D and E) Protein expression of α-SMA in mouse uterine tissues. ADM, adenomyosis; TAM, tamoxifen; α-SMA, α-smooth muscle actin\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2803345/v1/40b59d40df6388915568c31e.png"},{"id":36658421,"identity":"2ceace84-cc0f-446f-9b2e-ef9fdb628f4c","added_by":"auto","created_at":"2023-05-05 21:17:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1224126,"visible":true,"origin":"","legend":"\u003cp\u003eUpregulated expression of TBX3 in the \u003ca href=\"javascript:;\"\u003euteri\u003c/a\u003ene tissues of mice with ADM. (A) Protein expression of TBX3 in mouse \u003ca href=\"javascript:;\"\u003euteri\u003c/a\u003ene tissues (n=6). \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. (B) Protein expression of TBX3 in mouse uterine tissues (n=6). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05. TBX3, T-box transcription factor 3; ADM, adenomyosis\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2803345/v1/f1b9f8d839164ada25a9e430.png"},{"id":36659968,"identity":"45700d88-0ba1-47c3-b74e-b6249cb34928","added_by":"auto","created_at":"2023-05-05 21:49:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1502959,"visible":true,"origin":"","legend":"\u003cp\u003eActivated β-catenin and EMT pathways in the uterine tissues of ADM mice. (A and C) Protein expression levels of nuclear β-catenin, N-cadherin and Slug (n= 6). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. (B and D) Protein expression levels of Wnt3a and EMT-related in mouse uterine tissues (n= 6). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01, ***P\u0026lt;0.001. EMT, epithelial-to-mesenchymal transition; ADM, adenomyosis\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2803345/v1/dabff3c51c40afc8297ed467.png"},{"id":36658879,"identity":"663d99b0-8c08-45a8-960c-9e6529434600","added_by":"auto","created_at":"2023-05-05 21:25:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1570268,"visible":true,"origin":"","legend":"\u003cp\u003eUterine cells of ADM mice with increased proliferation and decreased apoptosis. (A) PCNA protein expression (n=6). \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. (B) Cell apoptosis in the uterine tissues of ADM mice (n=6). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;\u0026nbsp;0.05. (C) Expression levels of bcl2 and bax in the uterine tissues of ADM mice (n=6). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05. ADM, adenomyosis; PCNA, proliferation cell nuclear antigen\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2803345/v1/6b70c3dee34ab5c18dab17c9.png"},{"id":36658424,"identity":"9b152df8-8b12-4654-86f8-40764394c344","added_by":"auto","created_at":"2023-05-05 21:17:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1583181,"visible":true,"origin":"","legend":"\u003cp\u003eInhibited expression of TBX3 in uterine tissues of ADM mice following treatment with XAV-939. (A and B) Western blot analysis (n=8). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. (C) IHC detection (n=8). \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001. TBX3, T-box transcription factor 3; ADM, adenomyosis; IHC, immunohistochemistry\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2803345/v1/78a76d2fa4d9b7c00bd36df2.png"},{"id":36658418,"identity":"cc98ce4e-1836-4e01-8d7a-1e8df6ccebc7","added_by":"auto","created_at":"2023-05-05 21:17:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1367258,"visible":true,"origin":"","legend":"\u003cp\u003eXAV-939 treatment inhibited EMT due to Wnt3a/β-catenin pathway suppression. (A) Expression of N-cadherin detected using IHC. (B) Western blot analysis of N-cadherin and Slug proteins in the uterus (n=8). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05. (C) IHC analysis of E-cadherin, α-SMA and Twist1 following XAV-939 treatment in ADM mice (n=8). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003eP\u0026lt;0.0001. EMT, epithelial-to-mesenchymal transition; IHC, immunohistochemistry; α-SMA, α-smooth muscle actin; ADM, adenomyosis\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-2803345/v1/63fcc4d0fb181e2148fc1a89.png"},{"id":36658877,"identity":"3954afe7-f412-444c-afb5-cabf512a3245","added_by":"auto","created_at":"2023-05-05 21:25:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1668032,"visible":true,"origin":"","legend":"\u003cp\u003eProliferation and apoptosis in the uterine tissues of ADM mice following treatment with XAV-939. (A) H\u0026amp;E staining of tissues following XAV-939 treatment. (B) Infiltration depth score of uterine tissues with or without XAV-939 treatment (n=8). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e****\u003c/sup\u003eP\u0026lt;0.0001. (C) Western blot analysis of bcl2 in the uterine tissues of ADM mice following treatment with XAV-939 (n=8). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05. (D) PCNA analysis of the uterine tissues of ADM mice following treatment with XAV-939 (n=8). \u003csup\u003e*\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026lt;0.01. (E) TUNEL analysis of the uterine tissues of ADM mice following treatment with XAV-939 (n=8). \u003csup\u003e***\u003c/sup\u003eP\u0026lt;0.001. ADM, adenomyosis; PCNA, proliferation cell nuclear antigen\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-2803345/v1/7a60349727ef528456fd9108.png"},{"id":45453917,"identity":"4f01e152-a369-4f2d-b60e-e7115ea4b1ad","added_by":"auto","created_at":"2023-10-30 15:07:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8132944,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2803345/v1/879ed92a-138d-4f69-ac3c-ec7a92652930.pdf"},{"id":36659687,"identity":"0d31d543-4e1c-41fa-8837-df8c1b0dd3f7","added_by":"auto","created_at":"2023-05-05 21:41:02","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":259876,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-2803345/v1/5bc916eb482dd850c7d1cfa7.png"}],"financialInterests":"","formattedTitle":"Abnormal activation of the Wnt3a/β-catenin signaling pathway promotes the expression of TBX3 and the EMT pathway to mediate the occurrence of adenomyosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdenomyosis (ADM), a benign gynecological disease[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], is pathologically characterized as endometrium/gland invasion of the myometrium, along with hyperplasia and hypertrophy of the myometrium[ 2, 3]. Clinical symptoms include uterine diffusion and enlargement, pelvic pain, abnormal uterine bleeding, and hypofertility [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and this disease may seriously impact the patients\u0026rsquo; quality of life. Notably, as no effective drug has been developed for the treatment of ADM, surgery remains the only treatment option at present [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, further research into the specific mechanisms underlying the occurrence and development of ADM may aid the development of alternative treatment options.\u003c/p\u003e \u003cp\u003eT-box transcription factor 3 (TBX3), a member of the T-box transcription factor family of proteins, may play an essential regulatory role in numerous organs, including the heart, mammary glands and genitalia[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. TBX3 may also regulate cell proliferation and differentiation, tissue integrity, and epithelial-to-mesenchymal transition (EMT) through acting as a transcriptional inhibitor or activator [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous studies have indicated that associated overexpression of TBX3, as a tumor promoter or tumor suppressor, promoted tumorigenesis in several cancers. Specifically, overexpression of TBX3 has been with head and neck squamous cell carcinoma, gastric cancer, breast cancer, rhabdomyoma, cervical cancer, pancreatic cancer, papillary thyroid cancer, lung cancer and chondrosarcoma [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]; however, the specific function in ADM is yet to be determined. Although ADM is a benign disease, certain pathophysiological features are comparable with those of malignant tumors; for example, abnormal proliferation, migration and invasion [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, the present study aimed to determine the association between TBX3 expression and the occurrence of ADM.\u003c/p\u003e \u003cp\u003eNumerous functional proteins or signaling pathways, such as (wingless-related MMTV integration site, transforming growth factor-β), bone morphogenetic protein, fibroblast growth factor and Hippo, regulate cell proliferation, apoptosis and migration [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Specifically, the Wnt signaling pathway plays a critical role in complex cellular behaviors, such as tissue development, regeneration and cell homeostasis, in addition to mediating cell proliferation, polarity, differentiation, cell motility and stem cell activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Numerus previous studies have also suggested the existence of an association between the Wnt pathway and TBX3, as TBX3 may be regulated by, and act as a downstream target of, the Wnt signaling pathway in certain diseases [ 24\u0026ndash;26]. Thus, determining whether TBX3 may also be regulated by the Wnt signaling pathway in ADM may provide useful insights into the underlying mechanisms of ADM.\u003c/p\u003e \u003cp\u003eEMT plays a crucial role in the occurrence and development of ADM. Results of a previous study demonstrated that estrogen-induced EMT of endometrial epithelial cells may enhance invasion, thereby contributing to the development of ADM [ 27]. Notably, EMT is characterized by the upregulation of N-cadherin, vimentin and α-smooth muscle actin (α-SMA), and the downregulation of E-cadherin. This is regulated by transcription factors Slug, Snail and Twist [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The occurrence of EMT in tissues may result in acquired mesenchymal phenotypes, and to the enhancement of cellular migratory capacity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In addition, EMT is regulated by the Wnt signaling pathway. TBX3 directly induces the occurrence of EMT through promotion of Slug expression in breast cancer; thus, promoting the proliferation of breast cancer cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, treatment with a Wnt pathway inhibitor, such as XAV-939, may provide valuable insight into whether EMT is reversed in ADM.\u003c/p\u003e \u003cp\u003eThe present study aimed to investigate the expression of TBX3 in ADM mice, and determine the association between TBX3 and the Wnt/β-catenin signaling pathway in ADM. Using tamoxifen-induced ICR mice to establish an ADM disease model, numerous methods were carried out to analyze protein levels, and determine the association between the Wnt/β-catenin signaling pathway and TBX3, EMT, cell proliferation and apoptosis. The present study provides novel insights into the diagnosis and treatment of ADM.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eChemicals.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTamoxifen citrate (TAM) was purchased from Yangtze River Pharmaceutical Co. Ltd. and Wnt signaling pathway inhibitor, XAV-939, was purchased from Selleck Chemicals. Corn Oil was purchased from MedChemExpress and DMSO was purchased from Beijing Solarbio Science \u0026amp; Technology Co., Ltd.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted according to ethical guidelines and procedures approved by the Institutional Animal Care and Use Committee of Jinan University (ethics approval number, IACUC-20200905-01).\u003c/p\u003e \u003cp\u003eThe animal disease model, which followed the protocol established by Shen \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], consisted of four female and four male ICR mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.). The mice were housed under controlled conditions (12 h light/12 h dark cycle and a constant temperature of 23\u0026deg;C), with food and water available \u003cem\u003ead libitum\u003c/em\u003e. After the female mice gave birth, each dam was housed in the same cage as the pups. Newborn female mice were used in the experiments that were subsequently conducted.\u003c/p\u003e \u003cp\u003eA total of 40 neonatal female ICR mice were randomly divided into two groups: i) TAM or ii) control. From Days 2\u0026ndash;5 after birth, the TAM group mice were fed with 1 mg/kg TAM suspended in a mixture of peanut oil, lecithin and condensed milk (volume, 2:0.2:3) at a dose of 5 \u0026micro;l/g body weight. Moreover, the control group mice were fed the same amount of solution without TAM during the same period. Newborn mice were breastfed. Upon reaching 21 days of age, the female mice were weaned and separated from the dams and were fed until Day 60, when three mice were randomly selected from the two respective groups. The uteri of the sacrificed mice were used to prepare paraffin sections. Hematoxylin and eosin (H\u0026amp;E) staining was used to determine whether the ADM disease model had been successfully constructed.\u003c/p\u003e \u003cp\u003eFollowing successful construction of the model, five new mice from the respective TAM and control groups were randomly selected to be in ADM or control groups for the next phase of the study. Moreover, the remaining 24 mice were randomly divided into three groups: i) Control (n\u0026thinsp;=\u0026thinsp;8); ii) ADM (n\u0026thinsp;=\u0026thinsp;8); and iii) XAV-939 groups (n\u0026thinsp;=\u0026thinsp;8). The XAV-939 group mice received an intraperitoneal injection of 3 mg/kg Wnt/β-catenin signaling pathway inhibitor, XAV-939 (4% DMSO\u0026thinsp;+\u0026thinsp;corn oil\u0026thinsp;+\u0026thinsp;XAV-939), while mice from both the Control (n\u0026thinsp;=\u0026thinsp;8) and ADM (n\u0026thinsp;=\u0026thinsp;8) groups received intraperitoneal injections with the same dosage of solution (4% DMSO\u0026thinsp;+\u0026thinsp;corn oil). All mice were injected once per day for seven consecutive days, and the samples were sacrificed one day after the completion of the injection period.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTissue preparation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe left uteri were used to create paraffin sections. Once obtained from mice, the uteri were immediately fixed for 24 h in 4% paraformaldehyde. Subsequently, the uteri were rinsed with water, and successively dehydrated with 75, 85, 90, 95 and 100% ethanol, prior to submersion in mixtures of xylene and ethanol, and xylene I and xylene II, to obtain improved transparency of sections. Tissues were immersed in molten paraffin, embedded and cut into 4-\u0026micro;m sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E staining\u003c/h2\u003e \u003cp\u003eFor the histological examinations of tissues for the pathological diagnosis of ADM, three randomly selected paraffin-embedded sections from each mouse uterus was stained using H\u0026amp;E. Briefly, slices were heated for 1 h at 60℃, followed by xylene I and II deparaffinization, and successive ethanol hydration at 100, 95, 90 and 80%. Slides were successively stained with H\u0026amp;E, and sealed with neutral gum prior to being photographed under a light microscope (Nikon Corporation).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC) analyses\u003c/h2\u003e \u003cp\u003eParaffin-embedded sections were deparaffinized and hydrated as previously described, followed by a 15 min submersion period in citrate buffer solution (pH, 6.0) for antigen retrieval. Endogenous peroxidase activity was eliminated using 3% hydrogen peroxide. In addition, Triton X-100 (Beyotime Institute of Biotechnology) was added to tissue sections to increase the permeability of cells, and non-specific antigens were blocked with 10% goat serum for 1 h at room temperature. Tissue sections were subsequently incubated with the following antibodies: anti-TBX3 (1:200; cat. no. ab99302; Abcam), anti-E-cadherin (1:200; cat. no. 3195; Cell Signaling Technology, Inc.), anti-N-cadherin (1:200; cat. no. 22018-1-AP; ProteinTech Group, Inc.), anti-Twist1 (1:200; cat. no. 25465-1-AP; ProteinTech Group, Inc.), anti-α-SMA (1:200; cat. no. 19245; Cell Signaling Technology, Inc.), anti-vimentin (1:200; cat. no. ab137321; Abcam), anti-Wnt3a (1:200; cat. no. ab219412; Abcam) and anti-proliferation cell nuclear antigen (PCNA; 1:200; cat. no. 10205-2-AP; ProteinTech Group, Inc.) overnight at 4℃. In addition, PBS was used to replace the primary antibody as a negative control. Following primary incubation, tissues were incubated with the HRP-conjugated secondary antibody (1:200; cat. no. 7074; Cell Signaling Technology, Inc.) for 2 h at room temperature. Immunoreactive proteins were visualized using 3, 3-N-Diaminobenzidine tetrahydrochloride (Sangon Biotech Co., Ltd.), and nuclei were counterstained with hematoxylin. Sections were fixed with neutral resins and observed under a light microscope (Nikon Corporation).\u003c/p\u003e \u003cp\u003eSemi-quantitative immunoreactivity was determined using ImageJ (National Institutes of Health), as previously described [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A series of five images (magnification, x200) were randomly selected from multiple sections per tissue sample to obtain the mean. Staining was carried out using a color mask and assessed using color intensity. The mask was equally applied to all images, and measurement readings were recorded. Immunohistochemical parameters included: i) Integrated optical density (IOD); ii) total stained area (S), and iii) the mean optical density (MOD). The latter was defined as MOD\u0026thinsp;=\u0026thinsp;IOD/S, and was equivalent to the mean intensity of staining across all evaluated areas, including the glands and stroma. The immunostaining level (MOD) for all slides was computed for statistical analysis.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eIn situ cell proliferation\u003c/h2\u003e \u003cp\u003e \u003cb\u003eC\u003c/b\u003eell proliferation of the uterine tissues was assessed using IHC with a proliferating cell marker, PCNA, as previously described.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIn situ TUNEL analysis\u003c/h2\u003e \u003cp\u003eApoptosis in mouse uterine tissues was assessed using a One-step TUNEL Apoptosis Assay kit (Green FITC-Labeled Fluorescence Assay; Universal Imaging, Inc.) following the manufacturer\u0026rsquo;s protocol (Nanjing KeyGen Biotech Co., Ltd.). Briefly, following deparaffinization and rehydration, protease K was used to increase the permeability of cells. Tissue sections were incubated with a 45:1:4 TdT reaction mixture (Equilibration buffer: biotin-11-dUTP: TdT enzyme) for 1 h at 37℃ in a humidified chamber. Following washing with PBS, tissue sections were incubated with a 1:9 Streptavidin-Fluoresce solution (Streptavidin-Fluoresce solution: diluted labeling buffer) for 30 min at 37℃ in the dark. Sections were subsequently counterstained with DAPI for 10 min prior to sample analysis using fluorescence microscopy (Leica Microsystems GmbH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eThe right sides of the mice uteri were homogenized and ultrasonicated following the addition of RIPA lysis buffer (containing 10% PMSF; Beyotime Institute of Technology). Ice lysis was performed for 30 min, and the lysate was centrifuged at 14,000 g for 15 min at 4℃. Protein concentration was determined using a BCA detection kit (Beyotime Institute of Biotechnology). Following electrophoresis on a 10% SDS-PAGE gel, proteins were transferred to PVDF membranes (MilliporeSigma) and blocked with 5% skimmed milk (BD Biosciences) for 1 h. Membranes were washed and incubated overnight at 4℃ with the following primary antibodies: Anti-TBX3 (1:1,000; cat. no. ab99302; Abcam), anti-Wnt3a (1:1,000; cat. no. ab219412; Abcam), anti-bcl2 (1:2,000; cat. no. ab182858; Abcam), anti-bax (1:8,000; cat. no. ab32503; Abcam), anti-β-catenin (1:10,000; cat. no. 51067-2-AP; ProteinTech Group, Inc.), anti-Slug (1:1,000; cat. no. 9585; Cell Signaling Technology, Inc.), anti-N-cadherin (1:6,000; cat. no. 22018-1-AP; ProteinTech Group, Inc.), anti-β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) and anti-GAPDH (1:2500; cat. no. ab245355; Abcam). Following primary incubation, membranes were incubated with HRP-conjugated goat-anti-rabbit IgG (1:8,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were visualized using ECL chemiluminescence reagent (MilliporeSigma), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. An unpaired two-tailed Student\u0026rsquo;s t-test was used to evaluate the differences between two groups, and one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was used to compare three groups. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc.). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of an ADM disease model in mice\u003c/h2\u003e \u003cp\u003eNewborn female mice were fed until Day 60, after which, the uteri were removed and stained using H\u0026amp;E for the diagnosis of ADM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the myometrium of the control group was arrayed in bundles and was well-spaced from the endometrium, whereas the endometrium stroma from the TAM group were immersed into the muscular layer, that was divided into multiple bundles by the stroma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, in the samples from the TAM group, the smooth muscle structure of the muscular layer was notably disordered, and the endometrium boundary appeared unclear. Through IHC staining of the α-SMA protein, results of the present study further demonstrated that endometrial stromal cells and glands invaded the myometrium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). These findings illustrated that the establishment of ADM was successful. In addition, the depth of endometrium focal myometrial infiltration was evaluated as follows: Grade 0, no endometrium infiltration into the myometrium; Grade 1, penetration of the endometrium focal into the superficial myometrium; Grade 2, penetration into the mid-myometrium; and Grade 3, penetration beyond the mid-myometrium. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the majority of infiltration depth scores for ADM mice were classified as Grade 2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExpression of TBX3 in the uterine tissues of ADM mice\u003c/h2\u003e \u003cp\u003eThe association between TBX3 and ADM was investigated, and protein expression levels of TBX3 were determined in the uterine tissues of control and ADM mice. As indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, IHC analysis revealed the increased protein expression of TBX3 in the uterine tissues of the ADM group, compared with the control group. Moreover, results of the western blot analysis revealed that TBX3 protein expression levels were increased in the uterine tissues of ADM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These findings suggested a potential association between the expression of TBX3 and ADM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eActivation of Wnt/β-catenin and EMT pathways in the uterine tissues of ADM mice\u003c/h2\u003e \u003cp\u003eResults of previous studies demonstrated that TBX3 expression may be regulated by the Wnt signaling pathway in various cancers, which also regulates cell proliferation and migration[ 24, 26]. Therefore, changes in the Wnt3a/β-catenin pathway and the expression of EMT-related proteins were determined in the uterine tissues of ADM mice. Results of the western blot analysis demonstrated that the expression of nuclear β-catenin was significantly higher in the ADM group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, levels of Wnt3a, a member of the Wnt family, were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results demonstrated abnormal activation of the Wnt3a/β-catenin pathway in the uterine tissues of ADM mice.\u003c/p\u003e \u003cp\u003eThe expression of EMT-related proteins in mouse uterine tissues was detected, and changes in EMT were investigated following activation of the Wnt3a/β-catenin pathway. Results of the western blot analysis demonstrated an increase in Slug and N-cadherin protein expression levels in the ADM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Results of the IHC assay further demonstrated the decreased protein expression of EMT marker, E-cadherin, in the uterine tissues of ADM mice, compared with the control group. In addition, results of the present study demonstrated that Twist1 protein expression levels were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, protein expression levels of α-SMA and vimentin were also significantly increased in the uterine tissues of ADM mice. Thus, results of the present study suggested that global activation of the Wnt/β-catenin pathway and EMT occurred in ADM mice following the increased expression of TBX3. Thus, increased TBX3 expression in ADM mice may be associated with activation of the Wnt/β-catenin pathway and EMT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAbnormal proliferation and apoptosis in the uterine tissues of ADM mice\u003c/h2\u003e \u003cp\u003eActivation of Wnt/β-catenin and increased TBX3 expression impacted cell proliferation and apoptosis in the uterine tissues of ADM mice. Positive expression of PCNA was used to identify \u003cem\u003ein situ\u003c/em\u003e cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Results of the present study demonstrated that PCNA expression was more prevalent in the nuclei of endometrial glands and stromal cells of uterine tissues than in smooth muscle cells. Additionally, PCNA positive staining levels were significantly higher in the uterine tissues of ADM mice than in control mice. Results of the western blot analysis demonstrated upregulation of bcl2 protein in the uterine tissues of ADM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), while protein expression of bax was reduced. These findings further indicated that cell proliferation was significantly increased when TBX3 expression was increased, and Wnt/β-catenin was activated in the uterine tissues of ADM mice.\u003c/p\u003e \u003cp\u003eMoreover, results of the present study revealed that TUNEL-positive expression was reduced in the ADM group, compared with the control group. These results indicated that cell apoptosis was decreased in ADM mice when cell proliferation increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Collectively, these results suggested that cell proliferation increased and apoptosis decreased when the Wnt3a/β-catenin signaling pathway was activated, and TBX3 was highly expressed in ADM mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTBX3 expression is regulated by the Wnt3a/β-catenin signaling pathway in the uterine tissues of ADM mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the regulatory association between TBX3 and the Wnt3a/β-catenin signaling pathway in ADM mice, mice were injected with the Wnt signaling pathway inhibitor, XAV-939. Results of the western blot analysis demonstrated that Wnt3a and nuclear β-catenin protein expression levels were reduced in the XAV-939 group, compared with the ADM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, Wnt3a and nuclear β-catenin protein expression levels were increased in the ADM group, compared with the control group. These results suggested that XAV-939 inhibited the Wnt3a/β-catenin signaling pathway in ADM mice. In addition, TBX3 protein expression levels were decreased in the XAV-939 group, compared with the ADM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), which was consistent with results of the IHC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results indicated that expression of TBX3 in the uterine tissues of ADM mice may be regulated by the Wnt3a/β-catenin signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eWnt signaling pathway inhibitor, XAV-939, causes the reversal of EMT, and inhibition of endometrium cell invasion and proliferation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAnalyses of the effects of XAV-939 revealed an expression change in EMT-related proteins post-injection. Results of the western blot analysis revealed reduced Slug protein expression levels in the XAV-939 group, compared with the ADM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Moreover, N-cadherin protein expression levels were also decreased in the XAV-939 group, demonstrated using western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and IHC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In addition, Twist1 and α-SMA protein expression was reduced, and E-cadherin protein expression was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Results of the present study demonstrated that activation of EMT in ADM mice may be reversed following treatment with XAV-939. Moreover, regulation and inhibition of EMT by Wnt3a/β-catenin may impede further development of ADM.\u003c/p\u003e \u003cp\u003eHistopathological changes in mouse uterine tissues were detected following inhibition of the Wnt3a/β-catenin pathway. As displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, H\u0026amp;E staining revealed that intrusion of the endometrium into the muscular layer of mice was limited following treatment with XAV-939, compared with the ADM group. Moreover, the infiltration depth score of ADM mice was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), indicating that treatment with XAV-939 inhibited the invasion of endometrium cells in ADM mice. These results were comparable with those obtained following investigation of cell proliferation in the endometrium. Results of the western blot analysis revealed that protein expression levels of bcl2 were lower in the XAV-939 group, compared with the ADM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In addition, results of the IHC assay demonstrated that positive staining of PCNA was lower in the XAV-939 group than in the ADM group, which, in turn, was higher than that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These results indicated that abnormal cell proliferation was inhibited in the uterine tissues of mice with ADM, following treatment with XAV-939. By contrast, positive TUNEL expression was higher in the XAV-939 group than in the ADM group, which, in turn, was lower than that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). These results suggested that cell apoptosis was increased in ADM mice following treatment with XAV-939.\u003c/p\u003e \u003cp\u003eResults of the present study demonstrated that when Wnt3a/β-catenin signaling was inhibited, the protein expression of TBX3 was suppressed, and EMT was inhibited. Moreover, the Wnt signaling pathway inhibitor, XAV-939, reversed EMT and inhibited endometrial cell invasion and proliferation; thus, further suppressing the development of ADM. Therefore, the occurrence and development of ADM may be regulated by TBX3, EMT and the Wnt3a/β-catenin signaling pathway. Overexpression of TBX3 and the occurrence of EMT is regulated by Wnt3a/β-catenin signaling in ADM mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pathogenesis of ADM, a gynecological disease, is yet to be fully elucidated. The most widely accepted theory postulates that ADM lesions, originating from the basal layer of the endometrium, deeply invaginate into the myometrium [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although some aspects remain unclear, numerous studies have investigated the molecular mechanisms of ADM. In addition, ADM is accompanied by abnormal cell proliferation, apoptosis and migration; which are similar characteristics to those of cancer cells. Therefore, the present study aimed to investigate the molecular mechanisms of ADM, using TBX3 as a target protein.\u003c/p\u003e \u003cp\u003eAs a regulatory transcription factor, TBX3 regulates cell proliferation, differentiation, tissue integrity and the process of EMT. Moreover, TBX3 regulates organ growth and development, and is also involved in the regulation of a variety of diseases [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Results of a previous study revealed that TBX3 is regulated by Wnt/β-catenin, and TBX3 overexpression may promote human colorectal cancer [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Results of our previous study demonstrated that TBX3, along with TBX2, are typical effectors of Wnt signaling in ureteral mesenchymal cells, and are regulated by the Wnt pathway when activated in the uterus [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, results of the present study demonstrated that overexpression of TBX3 in the uterine tissues of mice was associated with ADM development. Increased protein expression of TBX3 in uterine tissues and stromal cells may be associated with endometrium invasion of the myometrium.\u003c/p\u003e \u003cp\u003eThe present study also aimed to determine whether the expression of TBX3 in ADM mice was regulated by the Wnt3a/β-catenin pathway. Results of the present study demonstrated that TBX3 protein expression was suppressed by XAV-939 in ADM mice, suggesting that the expression of TBX3 in ADM may be regulated by the Wnt3a/β-catenin pathway. Results of a previous study revealed that Wnt/β-catenin signaling may regulate the development of ADM [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]; however, results of the present study also demonstrated an increase in cell proliferation and a decrease in apoptosis, following TBX3 overexpression. Moreover, when TBX3 was inhibited by XAV-939, results of the present study demonstrated a reduction in cell proliferation and increased apoptosis. Collectively, these findings suggested that TBX3 may be situated downstream of the Wnt3a/β-catenin pathway.\u003c/p\u003e \u003cp\u003eEMT also plays an important role in the development of ADM, and results of the present study illustrated the occurrence of EMT in ADM. Specifically, a loss of E-cadherin expression and an increase in the abundance of mesenchymal markers, such as vimentin, α-SMA and N-cadherin, were observed. Numerous previous studies demonstrated that functional proteins promote the occurrence of EMT by activating the Wnt/β-catenin pathway [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Inducers of EMT, such as Snail, Slug, Zeb1 and Twist, serve as target genes of the Wnt pathway [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Notably, activation of the Wnt/β-catenin pathway may promote EMT. Moreover, treatment with XAV-939 in ADM mice subsequently increased the expression of E-cadherin in the uterus, and reduced the levels of Twist1 and α-SMA. Thus, results of the present study revealed that EMT in ADM was regulated by the Wnt3a/β-catenin pathway. In addition, the occurrence of EMT in the uterus induced uterine epithelial cells to lose their polarity in acquiring a mesenchymal phenotype and migrate into the myometrium. Thus, ADM development was promoted. Results of the present study also confirmed that XAV-939 reversed EMT in ADM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChanges in cell proliferation and apoptosis are distinct phenotypes in the development of ADM regulated by the Wnt3a/β-catenin pathway. Results of the present study demonstrated an increase in cell proliferation and inhibition of apoptosis in ADM mice, following treatment with XAV-939. Thus, we hypothesized that abnormal activation of the Wnt3a/β-catenin pathway may promote cell proliferation and inhibit apoptosis which, in turn, will accelerate the development of ADM. Notably, TBX3 may directly upregulate Slug expression to promote the occurrence of EMT in breast cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and TBX3 may have a regulatory function in organ development and cancer development [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Results of a previous study demonstrated an association between TBX3 function, and cell proliferation and apoptosis, tumor formation, metastasis, cell survival, angiogenesis, invasion, and cancer stem cell expansion [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, results of previous studies demonstrated that development of ADM involves immunization and adhesion factors, angiogenesis, inflammation, cell invasion, and migration [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Thus, further investigations into the function of TBX3 in cell survival, angiogenesis, fibrosis, inflammation, cell invasion and migration in ADM are required.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, results of the present study provide a theoretical basis for the role of TBX3 in ADM, and further investigations into the regulatory association between TBX3 and relevant pathways, such as the Wnt and Hippo pathways, are required. Thus, the present study provides a reference for the potential treatment of ADM.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTBX3 \u0026nbsp; \u0026nbsp; \u0026nbsp;T-box transcription factor 3\u003c/p\u003e\n\u003cp\u003eADM \u0026nbsp; \u0026nbsp; \u0026nbsp;Adenomyosis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eICR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Institute of Cancer research\u003c/p\u003e\n\u003cp\u003eEMT \u0026nbsp; \u0026nbsp; \u0026nbsp; Epithelial-to-mesenchymal transition\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-SMA \u0026nbsp; \u0026nbsp; \u0026alpha;-smooth muscle actin\u003c/p\u003e\n\u003cp\u003eTAM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tamoxifen citrate\u003c/p\u003e\n\u003cp\u003eDMSO \u0026nbsp; \u0026nbsp; \u0026nbsp; Dimethyl sulfoxide\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eIHC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eGAPDH \u0026nbsp; \u0026nbsp; \u0026nbsp;Glyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\n\u003cp\u003ePCNA \u0026nbsp; \u0026nbsp; \u0026nbsp; Anti-proliferation cell nuclear antigen\u003c/p\u003e\n\u003cp\u003eECL \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Enhanced chemiluminescence\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Phosphate buffered saline\u003c/p\u003e\n\u003cp\u003ePMSF \u0026nbsp; \u0026nbsp; \u0026nbsp; Phenylmethanesulfonyl fluoride\u003c/p\u003e\n\u003cp\u003eBCA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bicinchoninic Acid\u003c/p\u003e\n\u003cp\u003eRIPA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Radio immunoprecipitation assay\u003c/p\u003e\n\u003cp\u003ePVDF \u0026nbsp; \u0026nbsp; \u0026nbsp; Polyvinylidene fluoride\u003c/p\u003e\n\u003cp\u003eIOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Integrated optical density\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mean optical density\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Laboratory Animal Center of Jinan University for caring for the animals in the present study. The authors would also like to thank the Central Laboratory of the School of Medicine for providing the experimental platform.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was supported by the Guangdong Provincial Hospital of Chinese Medicine - Weixian Li famous doctor studio (grant no. E43719)and National Famous Traditional Chinese Medicine Expert inheritance Studio - Jianling Huang(project no.0102016205)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWQC and QZR conceived and designed the study and revised the manuscript. MQL and TL performed the experiments and wrote the manuscript. TTJ ,YC,and SMY analyzed the data. LC and QHL provided clinical guidance. TTL provided guidance in HE experiments. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Laboratory Animal Review Committee of Jinan University\u0026nbsp;(ethics approval number, IACUC-20200905-01).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003ePatient consent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDevlieger R, D\u0026apos;Hooghe T and Timmerman D (2003) Uterine adenomyosis in the infertility clinic. 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Reprod Biomed Online 35(5):592-601. https://https://doi.org/10.1016/j.rbmo.2017.06.016\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Adenomyosis (ADM), Epithelial-to-mesenchymal transition (EMT), T-box transcription factor3.Wnt3a/β-catenin signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-2803345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2803345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTBX3 is a transcription factor that can regulate cell proliferation, apoptosis, invasion, and migration in different tumor cells; however, its role in adenomyosis (ADM) has not been previously studied. Some of ADM\u0026rsquo;s pathophysiological characteristics are similar to those of malignant tumors (e.g., abnormal proliferation, migration, and invasion).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods and results\u003c/b\u003e\u003c/p\u003e \u003cp\u003ewe hypothesized that TBX3 might have a role in ADM. We used tamoxifen-induced ICR mice to establish ADM disease model. The study procedure included western blotting and immunohistochemistry to analyze protein levels; additionally, we used intraperitoneal injection of Wnt/β-catenin pathway inhibitor XAV-939 to study the relationship between TBX3 and Wnt/β-catenin pathway as well as PCNA and TUNEL to detect cell proliferation and apoptosis, respectively. TBX3 overexpression and epithelial-to-mesenchymal transition (EMT) in ADM mice was found to be associated with activation of the Wnt3a/β-catenin pathway. Treatment with XAV-939 in ADM mice led to the inhibition of both TBX3 and EMT; moreover, abnormal cell proliferation was suppressed, the depth of invasion of endometrium cells was limited and the expression of ERα was suppressed. Thus, the use of XAV-939 effectively inhibited further invasion of endometrial cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThese findings suggest that TBX3 may play an important role in the development of ADM. The expression of TBX3 in ADM was regulated by the Wnt3a/β-catenin pathway. The activation of the Wnt3a/β-catenin pathway in ADM promoted TBX3 and ERα expression and induced the occurrence of EMT, thus promoting cell proliferation and inhibiting apoptosis, ultimately accelerating the development of ADM. The study provides a reference for the diagnosis of ADM\u003c/p\u003e","manuscriptTitle":"Abnormal activation of the Wnt3a/β-catenin signaling pathway promotes the expression of TBX3 and the EMT pathway to mediate the occurrence of adenomyosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-05 21:16:57","doi":"10.21203/rs.3.rs-2803345/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-05-03T11:19:16+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-05-02T10:17:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-04-14T14:10:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2023-04-12T22:59:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1f770833-3601-481e-9c01-89a63fcdd022","owner":[],"postedDate":"May 5th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-30T15:05:14+00:00","versionOfRecord":{"articleIdentity":"rs-2803345","link":"https://doi.org/10.1007/s11033-023-08870-y","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2023-10-25 15:02:31","publishedOnDateReadable":"October 25th, 2023"},"versionCreatedAt":"2023-05-05 21:16:57","video":"","vorDoi":"10.1007/s11033-023-08870-y","vorDoiUrl":"https://doi.org/10.1007/s11033-023-08870-y","workflowStages":[]},"version":"v1","identity":"rs-2803345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2803345","identity":"rs-2803345","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}