Invasion Of Endometrial Cells Research Articles

Sphingosine 1-phosphate signaling axis mediates neuropeptide S-induced invasive phenotype of endometriotic cells.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1 , S1P3 and S1P5 , three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3 , given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

Features of cytokine production at the systemic and local level in patients of reproductive age with external genital endometriosis

Objective: to establish the features of cytokine production in patients of reproductive age with external genital endometriosis at various stages of the disease.Materials and methods: the study included 52 patients with external genital endometriosis (EGE). Group I consisted of 31 patients with stages I–II of EGE, group II — 21 patients with stages III–IV of the disease, control group — 20 women without endometriosis. Serum samples were obtained before anesthesia, and peritoneal fluid (PF) samples were obtained during endoscopic examination of patients. Determination of the content of cytokines in blood serum and peritoneal fluid was carried out by enzyme immunoassay using a multifunctional counter for enzyme immunoassays with Victor software (Finland). TNF-α, IL-10 — using reagents from BenderMed Systems (Austria). IL-13, IL-18 using reagents from Cloud-CloneCorp (USA) according to the manufacturer’s recommendations. Application software packages (Megastat and Statistica 10.0) were used to create a database and conduct statistical research. To determine statistical differences between groups, the Mann-Whitney test was used for independent groups and the Wilcoxon test for dependent groups. P values less than 0.05 were considered statistically significant. Data are presented as median and quartiles.Results: at stages I–II of EGE, an increase in the level of TNF-α, IL-18 was detected in the blood serum and peritoneal fluid, which can stimulate proliferation, invasion of endometrial cells and neovascularization; stage III–IV of the disease was characterized by an increase in the level of TNF-α, IL-10, IL-13 in the peritoneal fluid, which promotes disease progression by evading ectopic endometrial cells from immune control.Conclusion: the identified features of the production of pro- and anti-inflammatory cytokines at the systemic and local level at various stages of EGE confirm their importance in the development of the disease. Further research is needed to understand the mechanisms underlying the progression of NGE and to develop methods for early diagnosis.

Abnormal activation of the Wnt3a/β-catenin signaling pathway promotes the expression of T-box transcription factor 3(TBX3) and the epithelial-mesenchymal transition pathway to mediate the occurrence of adenomyosis.

T-box transcription factor 3(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). We hypothesized that TBX3 might have a role in ADM. We used tamoxifen-induced Institute of Cancer research (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 Anti-proliferation cell nuclear antigen( 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. Thus, the use of XAV-939 effectively inhibited further invasion of endometrial cells. 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 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.

P-316 Involvement of collective cell migration in endometrial invasiveness in uterine adenomyosis and the role of M2 macrophages

Abstract Study question How do endometrial cells manage to invade surrounding myometrium to establish adenomyotic lesions and what is the role of macrophages in this process? Summary answer M2 macrophage-mediated invasiveness and resistance of endometrial cells to physiological cell death appear to contribute to the pathogenesis of adenomyosis. What is known already Despite the high prevalence and debilitating symptoms of uterine adenomyosis, its pathogenesis has not yet been elucidated. To date, the most acceptable theory to explain disease development suggests invasion of the myometrium by eutopic endometrial cells. Recent data suggest aberrant infiltration of immune cells but not platelets in adenomyosis, eventually leading to enhanced cell motility via collective cell migration (CCM) and resistance to physiological cell death during disease pathogenesis. Study design, size, duration A retrospective immunohistochemistry-based study was conducted on formalin-fixed paraffin-embedded uterine tissue from 17 women (8 adenomyosis patients and 9 healthy subjects). Fresh endometrial biopsies were retrieved from 16 women (6 adenomyosis patients and 10 healthy subjects) for in vitro culture of epithelial and stromal cells, and co-culture with THP-1 monocyte-derived M2 macrophages. Participants/materials, setting, methods Double immunofluorescence was performed to detect macrophages of M1 and M2 phenotypes. Invasiveness of endometrial cells upon interaction with macrophages was assessed by in vitro invasion assays and quantitative PCR for genes involved in cell motility and epithelial-mesenchymal transition (EMT). Mechanisms of disease progression were investigated by immunohistochemistry against E-cadherin, N-cadherin and matrix metalloproteinase 9 (MMP9). Caspase 3 and microtubule-associated protein light chain 3 beta (LC3B) were studied as markers of apoptosis and autophagy respectively. Main results and the role of chance Only M2 macrophages were found to accumulate in adenomyosis, in higher numbers in both endometrium and ectopic lesions from adenomyosis patients than healthy controls. Co-culture with M2 macrophages significantly increased invasion capacity in endometrial epithelial and stromal cells from both adenomyosis patients and healthy controls. Epithelial cells from adenomyosis patients were nonetheless more invasive than their healthy counterparts in the presence of M2 macrophages. No gene expression differences pointing to EMT were noted, either between co-cultured and control cells, or between adenomyotic and healthy cells, refuting the implication of this mechanism in adenomyosis invasiveness. E- and N-cadherin protein expression did not differ significantly between eutopic endometrium from adenomyosis subjects and healthy tissue, but N-cadherin expression was stronger in ectopic lesions. In adenomyosis, both E- and N-cadherin were more extensively expressed in basal (also known as invasive) glands than functional glands, suggesting maintenance of intercellular junctions and subsequent CCM. Extracellular matrix-degrading enzyme MMP9 was more abundantly expressed in eutopic stroma from adenomyosis patients compared to healthy tissue. Lower levels of caspase 3 were detected in both endometrium and lesions from adenomyosis patients, while LC3B was found significantly decreased in adenomyotic stroma compared to that of healthy subjects. Limitations, reasons for caution A limitation of the study is the 2D nature of in vitro cultures, which cannot fully reconstitute in vivo cell arrangement and cell-cell interactions, nor allow to observe CCM in real time. Wider implications of the findings M2 macrophages accumulating in endometrium and ectopic lesions from adenomyosis patients boost invasion capacity of endometrial epithelial and stromal cells, as shown by in vitro co-culture experiments. These findings indicate that M2 macrophages play a key role in disease pathogenesis and could be targeted to develop novel therapeutic options. Trial registration number not applicable

M2 macrophages enhance endometrial cell invasiveness by promoting collective cell migration in uterine adenomyosis

Research questionAre M2 macrophages implicated in endometrial invasiveness in adenomyosis? DesignSeventeen formalin-fixed paraffin-embedded uterine samples and 16 fresh endometrial biopsies were collected from women with or without adenomyosis. Double immunofluorescence was performed to determine the predominant macrophage population in adenomyosis between M1 and M2 phenotypes. The invasion capacity of endometrial cells was assessed by invasion assays and quantitative polymerase chain reaction for genes involved in cell motility and epithelial–mesenchymal transition (EMT). Specific mechanisms of invasion were investigated by immunohistochemistry for E-cadherin, N-cadherin and matrix metalloproteinase 9 (MMP9). ResultsOnly M2 macrophages were found to accumulate in adenomyosis, in higher numbers in both eutopic endometrium (P = 0.0109) and lesions (P = 0.0267) than healthy tissue. Co-culture with M2 macrophages significantly boosted invasion capacity in endometrial epithelial (P = 0.0002; P = 0.002) and stromal cells (P = 0.0469; P = 0.0047) from both adenomyosis patients and healthy controls. No gene expression differences indicating EMT were noted, either between co-cultured and control cells, or between healthy and adenomyotic cells. E- and N-cadherin protein expression did not differ significantly between endometrium from adenomyosis subjects and healthy tissue but MMP9 expression was increased in eutopic stroma from adenomyosis patients (P = 0.0492). In adenomyosis, both E-cadherin (P = 0.0379) and N-cadherin (P = 0.0196) were more extensively expressed in basal glands than functional glands. ConclusionsM2 macrophages accumulate in adenomyosis and enhance invasion capacity of adenomyotic and even healthy endometrial cells, implying that macrophage infiltration alone may be sufficient to promote the disease. This study failed to detect any changes pointing to EMT, suggesting an alternative mode of invasion. Strong E- and N-cadherin-positive intercellular junctions in basal (invasive) glands suggest the involvement of collective cell migration in the invasion process of endometrium.

Effect and mechanisms of kaempferol against endometriosis based on network pharmacology and in vitro experiments

Endometriosis is a common gynecological disease, and its underlying mechanisms remain elusive. Patients are at a higher risk of recurrence after surgery or drug withdrawal. In this study, to identify a potentially effective and safe therapy for endometriosis, we screened potential target genes of kaempferol on endometriosis using network pharmacology and further validation. Network pharmacology showed kaempferol may suppress migratory and invasive properties by modulating the phosphoinositide 3-kinase (PI3K) pathway and its downstream target matrix metalloproteinase (MMP)9. Furthermore, in vitro experiments showed that kaempferol repressed the migration and invasion of endometrial cells, and this effect may be involved in mediating the PI3K-related genes, phosphatase and tensin homolog (PTEN) and MMP9. Network pharmacology and in vitro experiments showed that kaempferol, repressed the implantation of endometrial cells and formation of ectopic lesions by inhibiting migration and invasion and regulating PTEN and MMP9, which may be associated with the PI3K pathway.

665 The state of local immune interrelations in patients with adenomyosis

ObjectiveThe frequency of adenomyosis reaches 70%. The intrusion of the basal layer of the endometrium into the myometrium is possible in case of a violation of local immunity, which is formed against the background of causative agents of genital infection. The study of immune interrelations in the uterus in patients with adenomyosis will expand the understanding of the pathogenesis. Study design: 159 women with adenomyosis were examined. In the cervical secretion, a quantitative assessment of the levels of cytokines associated with the activity of innate immune defense IL-4,8,10,18, TNF, INF, the concentration of sIgA, IgA, M, G, the concentration of C3, C4 components of complement and lysozyme. In the endometrium, Chlamydia trachomatis, Ureaplasma spp., Mycomlasma genitalium, HSV1,2 / CMV, HPV were determined by PCR. Results: In the presence of genital pathogens in patients with adenomyosis, disorders of innate immunity were revealed, manifested in an increased content of sIgA, IgA, G (p <0.05), which may be a consequence of impaired permeability of the mucous membranes. An increase in the level of pro-inflammatory (IL-8, IL-18, TNF) and a decrease in the level of anti-inflammatory cytokines (IL-4, IL-10), INF (p <0.05) were revealed. The complement system is activated, as evidenced by an increase in the level of C3, C4 (p <0.05). An increase in proinflammatory cytokines at the local level aims to limit inflammatory processes in the endometrium and promoting the development of a T-cell immune response. Conclusions: In patients with adenomyosis, signs of immunopathology are observed, leading to a weakening of immune control, which creates conditions for the development of endometrial heterotopies. Formed against the background of bacterial and viral pathogens, immune disorders can contribute to the invasion of endometrial cells into the adjacent myometrium.

MiodesinTM Positively Modulates the Immune Response in Endometrial and Vaginal Cells.

Endometriosis presents high prevalence and its physiopathology involves hyperactivation of endometrial and vaginal cells, especially by bacteria. The disease has no cure and therapies aiming to inhibit its development are highly desirable. Therefore, this study investigated whether MiodesinTM (10 µg/mL = IC80; 200 µg/mL = IC50), a natural compound constituted by Uncaria tomentosa, Endopleura uchi, and astaxanthin, could exert anti-inflammatory and anti-proliferative effects against Lipopolysaccharides (LPS) stimulation in endometrial and Candida albicans vaginal cell lines. VK2 E6/E7 (vaginal) and KLE (epithelial) cell lines were stimulated with Candida albicans (1 × 107 to 5 × 107/mL) and LPS (1 μg/mL), respectively. MiodesinTM inhibited mRNA expression for Nuclear factor kappa B (NF-κB), ciclo-oxigenase 1 (COX-1), and phospholipase A2 (PLA2), beyond the C–C motif chemokine ligand 2 (CCL2), CCL3, and CCL5 in VK2 E6/E7 cells (p < 0.05). In addition, the inhibitory effects of both doses of MiodesinTM (10 µg/mL and 200 µg/mL) resulted in reduced secretion of interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor α (TNF-α) (24 h, 48 h, and 72 h) and CCL2, CCL3, and CLL5 (p < 0.05) by VK2 E6/E7 cells. In the same way, COX-1 MiodesinTM inhibited LPS-induced hyperactivation of KLE cells, as demonstrated by reduced secretion of IL-1β, IL-6, IL-8, TNF-α (24 h, 48 h, and 72 h) and CCL2, CCL3, and CLL5 (p < 0.05). Furthermore, MiodesinTM also inhibited mRNA expression and secretion of matrix metalloproteinase-2 (MMP-2), MMP-9, and vascular endothelial growth factor (VEGF), which are key regulators of invasion of endometrial cells. Thus, the study concludes that MiodesinTM presents beneficial effects in the context of endometriosis, positively affecting the inflammatory and proliferative response.

Associations between Exposure to Organochlorine Chemicals and Endometriosis: A Systematic Review of Experimental Studies and Integration of Epidemiological Evidence.

Background:Growing epidemiological evidence suggests that organochlorine chemicals (OCCs), including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may play a role in the pathogenesis of endometriosis.Objectives:We aimed to systematically review the experimental evidence (in vivo and in vitro) on the associations between exposure to OCCs and endometriosis-related end points.Methods:A systematic review protocol was developed following the National Toxicology Program /Office of Health Assessment and Translation (NTP/OHAT) framework and managed within a web-based interface. In vivo studies designed to evaluate the impact of OCCs on the onset or progression of endometriosis and proliferation of induced endometriotic lesions were eligible. Eligible in vitro studies included single-cell and co-culture models to evaluate the proliferation, migration, and/or invasion of endometrial cells. We applied the search strings to PubMed, Web of Science, and Scopus®. A final search was performed on 24 June 2020. Assessment of risk of bias and the level of evidence and integration of preevaluated epidemiological evidence was conducted using NTP/OHAT frameworkResults:Out of 812 total studies, 39 met the predetermined eligibility criteria (15 in vivo, 23 in vitro, and 1 both). Most studies () tested TCDD and other dioxin-like chemicals. In vivo evidence supported TCDD’s promotion of endometriosis onset and lesion growth. In vitro evidence supported TCDD’s promotion of cell migration and invasion, but there was insufficient evidence for cell proliferation. In vitro evidence further supported the roles of the aryl hydrocarbon receptor and matrix metalloproteinases in mediating steroidogenic disruption and inflammatory responses. Estrogen interactions were found across studies and end points.Conclusion:Based on the integration of a high level of animal evidence with a moderate level of epidemiological evidence, we concluded that TCDD was a known hazard for endometriosis in humans and the conclusion is supported by mechanistic in vitro evidence. Nonetheless, there is need for further research to fill in our gaps in understanding of the relationship between OCCs and their mixtures and endometriosis, beyond the prototypical TCDD. https://doi.org/10.1289/EHP8421

Increased Expression of Retinol-Binding Protein 4 in Ovarian Endometrioma and Its Possible Role in the Pathogenesis of Endometriosis.

Although endometriosis is a benign disease characterized by the presence of endometrial tissues outside the uterus, ectopic endometrial cells can exhibit malignant biological behaviors. Retinol-binding protein4 (RBP4) is a novel adipocyte-derived cytokine, which has important roles in regulating insulin sensitivity and energy metabolism. RBP4 is a potent modulator of gene transcription, and acts by directly controlling cell growth, invasiveness, proliferation and differentiation. Here, we evaluated the possible role of RBP4 in the pathogenesis of endometriosis. We compared the levels of RBP4 in the tissues and peritoneal fluid (PF) of women with and without endometriosis and evaluated the in vitro effects of RBP4 on the viability, invasiveness, and proliferation of endometrial stromal cells (ESCs). RBP4 levels were significantly higher in the PF of the women in the endometriosis group than in the controls. RBP4 immunoreactivity was significantly higher in the ovarian endometriomas of women with advanced stage endometriosis than those of controls. In vitro treatment with human recombinant-RBP4 significantly increased the viability, bromodeoxyuridine expression, and invasiveness of ESCs. Transfection with RBP4 siRNA significantly reduced ESC viability and invasiveness. These findings suggest that RBP4 partakes in the pathogenesis of endometriosis by increasing the viability, proliferation and invasion of endometrial cells.

Nucleolar small molecule RNA SNORA75 promotes endometrial receptivity by regulating the function of miR-146a-3p and ZNF23.

Endometrial receptivity enables the embryo to attach, invade and develop, forming a new individual and species continuity. Small nucleolar RNAs (SnoRNAs) are a class of non-coding RNAs comprising two classes: C/D box snoRNAs and H/ACA box snoRNAs. Aberrant expression of SNORNAs has been reported in tumorigenesis. However, the role of SNORNAs in maintaining endometrial receptivity has not been reported. First, we detected SNORNA expression in endometrial tissues during proliferative and secretory endometrial periods using RNA sequencing. SNORA75 expression was higher in the secretory endometrium, and its overexpression significantly promoted the proliferation, migration and invasion of endometrial cells. The results of analysis with bioinformatics software and RNA pulldown experiments showed that miR-146a-3p interacted with SNORA75. Western blotting showed that miR-146a-3p regulated the expression of ZNF23, whose overexpression significantly promoted the proliferation, migration and invasion of endometrial cells. SNORA75 modulates endometrial receptivity through the miR-146a/ZNF23 signaling pathway.

MiR-543 Inhibits the Occurrence and Development of Intrauterine Adhesion by Inhibiting the Proliferation, Migration, and Invasion of Endometrial Cells.

Objective To explore the function of miR-543 in endometrial cells and the possible mechanism of regulating the occurrence and development of intrauterine adhesion. Method Endometrial epithelial cells and endometrial adenocarcinoma cells were transfected with miR-543 mimics and miR-543 inhibitor as the experimental group and were tested with the control group, using the CCK-8 method, scratch test, and Transwell assay, and flow cytometry was used to detect the proliferation, migration, invasion, and apoptosis of cells. RT-qPCR and Western blot were used to detect the expression of corresponding mRNA and protein. Results After the overexpression of miR-543, endometrial epithelial cells and endometrial adenocarcinoma cells have reduced migratory, proliferative, and invasive capabilities, while the apoptosis rate has increased significantly. The mRNA expression of CDH2, COL16A1, vimentin, α-SMA and fibronectin decreased, and the protein expression of CDH2, vimentin, and α-SMA also decreased, while the mRNA and protein expression of CDH1 increased. The result after interfering with miR-543 is opposite, and luciferase reporter gene confirms that CDH2 is the target gene of miR-543. Conclusion During the formation of intrauterine adhesions, the expression of CDH2, COL16A1, vimentin, and α-SMA may be inhibited by the high expression of miR-543, which may affect the degree of fibrosis and collagen content in the intrauterine adhesions, thereby inhibiting the occurrence and development of intrauterine adhesions.

Kallistatin Inhibits Anoikis Resistance and Metastasis of Ectopic Endometrium Cells by Modulating MnSOD and Caspase 3 Signaling.

Endometriosis (EM) is a disease that involves active endometrial cell invasion and migration which is an important reason for infertility. Anoikis resistance is the most important prerequisite for EM, but the molecular mechanism is not yet clear. Kallistatin (KS) is one kind of serine protease inhibitors which had extensive biological function including anti-inflammatory, antioxidant stress, anti-angiogenesis, and anti-tumor. Our preliminary data showed that the level of KS in EM patients' endometrial tissue and blood were much lower than control (non-EM) patients without endometriosis. Interestingly, the decrease of KS is correlated with the severity of endometriosis. Moreover, kallistatin recombinant protein could increase the anoikis rate of ectopic endometrium cells (EESCs), and then inhibits its metastasis and invasion. Mechanically, our data show that the EESCs have lower intracellular reactive oxygen species (ROS) production and KS can elevate the ROS levels significantly. Further, KS modulate expression of MnSOD and caspase 3 signaling in EESCs grown in suspended conditions. These findings reveal novel mechanisms of KS in inducing anoikis and metastasis in EESCs, thus inhibiting EM progression by regulation of MnSOD and caspase 3 signaling. Our findings suggest that KS is a significant protein with prospects for application in EM.

CD44 variant 6 is involved in the attachment and invasion of endometrial cells to peritoneum

To study the effects of CD44 standard (CD44s), CD44v3, and CD44v6 overexpression (OE) on immortalized human endometrial epithelial (iEECs) and stroma cells (human endometrial stromal cells [hESCs]) using in vitro assays and a nude mouse xenograft model. Menstrual endometrial cells from women with endometriosis have increased adhesion and also express higher levels of CD44 variant 6 (v6), but not v3, compared to menstrual endometrial cells from women without endometriosis.In vitro studies and in vivo xenograft model.Academic center.Deidentified immortalized endometrial epithelial tissue samples of a reproductive-age woman.Overexpression of CD44s, CD44v3, and CD44v6 was carried out using lipofectamine, and their expression was verified with mRNA and protein in iEEC and hESCs. The OE cells were used in in vitro studies and an in vivo xenograft model compared to plasmid control.The effect of CD44s, CD44v3, and CD44v6 OE on attachment and invasion assays and a xenograft model with immortalized human stromal and epithelial cells.Expression of mRNA and protein confirmed appropriate OE of CD44s, CD44v3, and CD44v6 in the different cell types. CD44v6 OE increased attachment of hESCs compared with controls. CD44v6 OE did not change the attachment of iEECs. There was no difference in attachment in iEECs or hESCs with OE of CD44s or CD44v3.Overexpression of CD44v6 increases attachment of hESCs to peritoneal mesothelial cells in an in vitro assay and an in vivo xenograft model. Menstrual endometrial cell type and CD44 variants play a complex role in the development of the early endometriotic lesion.

M6A RNA Methylation Regulators Contribute to Eutopic Endometrium and Myometrium Dysfunction in Adenomyosis.

Adenomyosis is a prevalent, estrogen-dependent uterine disorder wherein endometrial cells are abnormally present in the myometrium and are surrounded by hyperplastic/hypertrophic smooth muscle. Its etiology is unclear, although endometrial cell invasion into the myometrium has been postulated. RNA methylation, particularly N6-methyladenosine (m6A), plays an important role in regulating various physiological processes and invasive disorders. The goal of this in silico and lab-based experimental study was to explore a possible role for m6A in adenomyosis. Gene expression profiles of both the endometrium and myometrium of women with adenomyosis (cases) and without disease (controls) were obtained from the publicly available Gene Expression Omnibus (GEO) database. In the endometrium, STRING database analysis revealed that METTL3 functions as a “hub” gene of m6A RNA methylation regulators, and the genes involved in m6A regulation, including METTL3, FTO, ZC3H13, and YTHDC1 expression, were significantly decreased in cases versus controls. Functional, co-expression, and correlational analyses of endometrium from cases versus controls revealed decreased total m6A levels, induced by METTL3, and the downstream elevated insulin−like growth factor−1(IGF1) and D-Dopachrome Tautomerase (DDT), with the latter two having known functions in epithelial proliferation and cell migration, which are important processes in the pathogenesis of adenomyosis in endometrium. m6A RNA methylation regulators, including RBM15/15B, ALKBH5, FTO, YTHDF1/2, KIAA1429, HNRNPC, METTL3, ZC3H13, and YTHDC2, were also differentially expressed in the myometrium from cases versus controls. We validated decreased total m6A levels and differential expression of m6A RNA methylation regulators in the myometrium of patients with adenomyosis using qRT-PCR, immunohistochemistry and tissues available from our biorepository. Possible target genes, including cadherin 3(CDH3), sodium channelβ-subunit 4 (SCN4B), and placenta-specific protein 8 (PLAC8), which are involved in cell adhesion, muscle contraction and immune response in the myometrium of adenomyosis patients were also validated. Thus, through extensive public database mining and validation of select genes, this study, for the first time, implicates m6A and its methylation regulators in the pathogenesis of adenomyosis. Follow on functional studies are anticipated to elucidate mechanisms involving m6A and its regulators and down-stream effectors in the pathogenesis of this enigmatic reproductive disorder and potentially identify druggable targets to control its associated symptoms.

TIPE2 inhibits the migration and invasion of endometrial cells by targeting β-catenin to reverse epithelial-mesenchymal transition.

Do changes in tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TIPE2) levels in endometrium of patients with adenomyosis alter the proliferation, migration and invasion ability of endometrial cells? TIPE2 expression levels were low in eutopic and ectopic endometrium of adenomyosis patients, and TIPE2 inhibited the migration and invasion of endometrial cells, mainly by targeting β-catenin, to reverse the epithelial-mesenchymal transition (EMT). Adenomyosis is a benign disease, but it has some pathophysiological characteristics similar to the malignant tumor. TIPE2 is a novel negative immune regulatory molecule, and it also participates in the development of malignant tumors. Control endometrium (n = 48 women with non-endometrial diseases) and eutopic/ectopic endometrium from patients with adenomyosis (n = 50), human endometrial cancer cell lines, and primary endometrial cells from the eutopic endometrium of adenomyosis patients were used in the study. The expression level of TIPE2 mRNA and protein in the eutopic/ectopic endometrial tissues of adenomyosis patients and control endometrium was determined by quantitative RT-PCR (qRT-PCR), western blot and immunohistochemistry. The effects of TIPE2 overexpression and knockdown on the proliferation, migration and invasion of endometrial cell lines and primary adenomyotic endometrial cells were determined using a cell counting kit-8, 5-ethynyl-2'-deoxyuridine assay, colony-forming assay, transwell migration assay and matrigel invasion assay. The expression of EMT-related markers and signal molecules was detected by western blot. The interaction between TIPE2 and β-catenin was detected by co-immunoprecipitation and laser confocal microscopy. The mRNA and protein expression levels of TIPE2 in the eutopic and ectopic endometrial tissues of adenomyosis patients were significantly downregulated compared with the control endometrium (P˂0.01). TIPE2 could bind to β-catenin and inhibit the nuclear translocation of β-catenin, downregulate the expression of stromal cell markers, upregulate the expression of glandular epithelial cell markers, decrease the occurrence of epithelial-mesenchymal transition (EMT) and suppress the migration and invasion of endometrial cells (P˂0.01). N/A. In this study, the experiments were performed only in eutopic and ectopic endometrial tissues, endometrial cancer cell lines and primary adenomyotic endometrial cells. A mouse model of adenomyosis will be constructed to detect the effects of TIPE2 in vivo. These results suggest that TIPE2 is involved in the development of adenomyosis, which provides a potential new diagnostic and therapeutic strategy for the treatment of adenomyosis. This present study was supported by grants from the National Natural Science Foundation of China (81471437, 81771554), Natural Science Foundation of Shandong (ZR2018MH013), Science and technology development plan provided by Health and Family Planning Committee in Shandong (2014-25). The authors declare that they have no conflicts of interest.

Aberrant Overexpression of RNA-Editing Enzyme ADAR1 Promotes the Progression of Endometriosis

Considerable efforts have been invested to elucidate the potential mechanisms involved in the physiopathology of endometriosis. However, to date, prior research has not been conclusive. This research has examined one particular mechanism, i.e., the effect of ADAR1 on endometriosis lesions. Eutopic endometrium was collected from women with (n = 25) and without endometriosis (n = 25), respectively. The expression of ADAR1 mRNA was measured based on quantitative real-time polymerase chain reactions (RT-qPCR). Both Western blot and immunohistochemistry were performed to establish ADAR1 protein expression levels. The results indicated that ADAR1 mRNA and proteins were significantly greater in the eutopic endometrium of the women with endometriosis, compared to the women without (P < 0.05). The Cell Counting Kit-8 (CCK-8) and EdU method were conducted to examine the effect of ADAR1 on cell viability and proliferation in eutopic endometriosis cells. A transwell assay was also used to detect the role of ADAR1 in the invasion of endometrial cells. The results obtained showed that ADAR1 promoted endometrial cell viability, proliferation, and invasion (P < 0.05). This informed our conclusion that the ADAR1 gene is upregulated in endometriosis, potentially paying a pivotal role in the physiopathology of endometriosis.

Knockdown of vascular cell adhesion molecule 1 impedes transforming growth factor beta 1-mediated proliferation, migration, and invasion of endometriotic cyst stromal cells

PurposeEndometriosis is one of the most common, difficult, and complicated gynecological disorders. Vascular cell adhesion molecule 1 (VCAM-1) has been reported to be aberrantly expressed in patients with endometriosis. However, the exact role and mechanism of VCAM-1 in endometriosis remains unclear.MethodsThe expression of transforming growth factor beta 1 (TGF-β1) and VCAM-1 was determined by quantitative real-time polymerase chain reaction and western blotting. Human endometriotic cells were cultured and their responsiveness to TGF-β1 was evaluated by Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, and transwell migration and invasion assays.ResultsThe levels of TGF-β1 and VCAM-1 mRNA were upregulated in the endometriotic tissues. Knockdown of TGF-β1 in endometriotic cyst stromal cells caused a marked inhibition of cell proliferation, migration, and invasion. Treatment of endometriotic cyst stromal cells with TGF-β1 resulted in an obvious promotion of cell proliferation, migration, and invasion, and strikingly increased the protein expression of VCAM-1. Silencing of Smad3 abated TGF-β1-stimulated VCAM-1 expression. Furthermore, the promoting effects of TGF-β1 on the proliferation, migration, and invasion of endometriotic cyst stromal cells were blocked by silencing of VCAM-1.ConclusionKnockdown of VCAM-1 impedes TGF-β1-mediated proliferation, migration, and invasion of endometrial cells, thereby indicating that VCAM-1 may serve as a therapeutic target for endometriosis.

MicroRNA-126-5p downregulates BCAR3 expression to promote cell migration and invasion in endometriosis

PurposeEndometriosis (EMs) is an estrogen-dependent multifactorial disease. Inhibition of estrogen in endometrial cells contributes to their failure to form lesions in ectopic sites. However, whether reducing or suppressing the inhibitory effect of estrogen results in the establishment of ectopic lesions remains unclear. The BCAR3 gene induces estrogen resistance in estrogen-dependent breast cancer cells and promotes cell migration, invasion, and epithelial-mesenchymal transition (EMT). However, the expression of BCAR3 in endometriosis and its effect on endometrial cell function and the anti-estrogen effect of endometriosis have not been reported. These issues are addressed in the present study. MethodsThe study included 32 cases of ectopic endometrium and eutopic endometrium in patients with endometriosis and 31 cases of normal endometrium as controls. The expression of BCAR3 and microRNA (miR)-126-5p was detected by real-time PCR, immunohistochemistry, and western blotting. The effects of BCAR3 and miR-126-5p on the morphology and biological behavior of eutopic endometrial cells were verified using lentivirus overexpression and a vector knockdown model, the CCK-8 assay, Transwell experiments, and estrogen intervention experiments using primary cultures of epithelial and stromal cells. ResultsThe BCAR3 gene was highly expressed in ectopic endometrium and the eutopic endometrium of patients with endometriosis, and the expression level was higher in stage III-IV patients than in stage I-II patients. In vitro cell experiments showed that miR-126-5p negatively regulated the expression of BCAR3 and its effect on the migration and invasion of stromal cells. Low expression of miR-126-5p and high expression of BCAR3 promoted endometriosis stromal cell migration and invasion. Assessment of EMT in endometriosis compared with eutopic endometrium showed that the expression of vimentin was significantly increased and the expression of E-cadherin was significantly decreased in ectopic endometrium. Estrogen promoted EMT in eutopic endometrial epithelial cells and this effect was reversed by estrogen inhibitors. BCAR3 had no direct effect on EMT and did not act synergistically with estrogen on promoting EMT. ConclusionmiR-126-5p negatively regulated BCAR3 expression in eutopic endometriosis, enhanced the migration and invasion of endometrial cells, and promoted the occurrence of endometriosis. BCAR3 did not induce EMT and had no synergistic effect with estrogen, but its inhibition of anti-estrogen function may provide new insight into the mechanism of local estrogen action in endometriosis.

Naringenin ameliorates progression of endometriosis by modulating Nrf2/Keap1/HO1 axis and inducing apoptosis in rats

Endometriosis is mainly characterized by the presence of endometrial tissue exterior to the uterus, however, the exact pathophysiology of this disease still remains uncertain. Moreover, the incidence significantly contributes to infertility among women and hence, a novel treatment for endometriosis is widely investigated. Naringenin is a plant-derived flavonoid having anti-proliferative, anti-inflammatory, and anti-angiogenic properties in chronic and metabolic diseases. The current study was planned with an objective to demonstrate the anti-endometriotic therapeutic potential of naringenin in rats and to examine its impact on various cellular aspects with a view to define the mechanism involved. The endometrial lesion volumes, weight, serum TNF-α level and the histopathologic scores were significantly reduced in the naringenin- treated group as compared to the endometriotic control group. Naringenin ameliorated the expression of prognostic markers (TAK1, PAK1, VEGF and PCNA) involved in development and progression of endometriotic cells. Naringenin caused dose-dependent loss of mitochondrial membrane potential, induced apoptosis and inhibited proliferation in these cells. Further, a significant increase in level of Nrf2 and its downstream molecules (NQO1, HO-1) was found in endometriotic lesion, with a subsequent decrease in its repressor molecule Keap-1. Naringenin significantly modulated the expression of Nrf2 and its effector molecules downstream. It also inhibited the invasion of endometrial cells by reducing the expression of MMP-2 and MMP-9 in in-vitro primary culture. We conclude that naringenin may have a therapeutic potential in the treatment of endometriosis via induction of ROS-mediated apoptosis and its anti-invasive effects.