Endometrial Stromal Cells In Endometriosis Research Articles

CircFOXO3 mediates hypoxia-induced autophagy of endometrial stromal cells in endometriosis.

Endometriosis is a benign gynecological disease that shares some common features of malignancy. Autophagy plays vital roles in endometriosis and influences endometrial cell metastasis, and hypoxia was identified as the initiator of this pathological process through hypoxia inducible factor 1 alpha (HIF-1α). A newly discovered circular RNA FOXO3 (circFOXO3) is critical in cell autophagy, migration, and invasion of various diseases and is reported to be related to hypoxia, although its role in endometriosis remains to be elucidated up to now. In this study, a lower circFOXO3 expression in ectopic endometrium was investigated. Furthermore, we verified that circFOXO3 could regulate autophagy by downregulating the level of p53 protein to mediate the migration and invasion of human endometrial stromal cells (T HESCs). Additionally, the effects of HIF-1α on circFOXO3 and autophagy were examined in T HESCs. Notably, overexpression of HIF-1α could induce autophagy and inhibit circFOXO3 expression, whereas overexpressing of circFOXO3 under hypoxia significantly inhibited hypoxia-induced autophagy. Mechanistically, the direct combination between HIF-1α and HIF-1α-binding site on adenosine deaminase 1 acting on RNA (ADAR1) promoter increased the level of ADAR1 protein, which bind directly with circFOXO3 pre-mRNA to block the cyclization of circFOXO3. All these results support that hypoxia-mediated ADAR1 elevation inhibited the expression of circFOXO3, and then autophagy was induced upon loss of circFOXO3 via inhibition of p53 degradation, participating in the development of endometriosis.

Regulatory mechanism of GPER in the invasion and migration of ectopic endometrial stromal cells in endometriosis

ABSTRACT Endometriosis (EMS) is a chronic inflammatory disorder of high incidence that causes serious reproductive consequences. High estrogen production is a consistently observed endocrine feature of EMS. The present study aims to probe the molecular mechanism of G protein-coupled estrogen receptor 1 (GPER) in the invasion and migration of ectopic endometrial stromal cells (Ect-ESCs) and provides a new rationale for EMS treatment. Eutopic and ectopic endometrial tissues were collected from 41 EMS patients, and primary ESCs were separated. GPER, miR-16-5p, and miR-103a-3p levels in cells and tissues were determined by qRT-PCR or Western blot assay. Cell viability, proliferation, invasion, and migration were evaluated by CCK-8, colony formation, and Transwell assays. The upstream miRNAs of GPER were predicted by databases, and dual-luciferase assay was performed to validate the binding of miR-16-5p and miR-103a-3p to GPER 3’UTR. GPER was highly expressed in EMS tissues and Ect-ESCs. Inhibition of GPER mitigated the proliferation, invasion, and migration of Ect-ESCs. GPER was regulated by miR-16-5p and miR-103a-3p. Overexpression of miR-16-5p and miR-103a-3p negatively regulated GPER expression and inhibited the invasion and migration of Ect-ESC. In conclusion, GPER promoted the invasion and migration of Ect-ESCs, which can be reversed by upstream miR-16-5p and miR-103a-3p.

Retracted: miR-206 Targets MALAT1 to Suppress Cell Progression of Ectopic Endometrial Stromal Cells in Endometriosis.

[This retracts the article DOI: 10.1155/2022/8094385.].

METTL3 is aberrantly expressed in endometriosis and suppresses proliferation, invasion, and migration of endometrial stromal cells.

Endometriosis (EM) is one of the leading gynecological disorders, and associated with excessive functioning of endometrial stromal cells (ESCs). The current study was conducted to determine the expression and role of methyltransferase-like 3 (METTL3) in the proliferation, invasion, and migration of ESCs in EM. The documented expression levels of METTL3, microRNA (miR)-21-5p, and WNT inhibitory factor 1 (WIF1) in eutopic (Eut) and ectopic (Ect) endometrial tissues and ESCs were determined by a combination of real-time quantitative polymerase chain reaction and Western blot assay. After transfection with pcDNA3.1-METTL3, miR-21-5p mimic, and WIF1 small interfering RNA, cell counting kit-8, colony formation, and Transwell assays were performed in the Ect ESCs (Ect-ESCs). Subsequently, the binding of miR-21-5p to METTL3 was analyzed, along with quantification of the N6-methyladenosine (m6A) level, the enrichments of METTL3 and m6A on WIF1, and the mRNA stability of WIF1. In our findings, METTL3 was downregulated in the EM tissues and cells. METTL3 overexpression intrinsically reduced the proliferation, invasion, and migration of Ect-ESCs. miR-21-5p inhibited the METTL3 expression while METTL3 enhanced the mRNA stability and expression of WIF1 via m6A modification. Additionally, a negative correlation of METTL3 was identified with miR-21-5p along with a positive correlation with the WIF1 mRNA in EM tissues. The miR-21-5p overexpression or WIF1 downregulation enhanced the proliferation, invasion, and migration of Ect-ESCs. Collectively, miR-21-5p inhibited the METTL3-mediated m6A modification and mRNA stability of WIF1, thereby facilitating the proliferation, invasion, and migration of Ect-ESCs.

MicroRNA-100 shuttled by human umbilical cord MSC-secreted extracellular vesicles induces endometriosis by inhibiting HS3ST2

In recent years, the function of human umbilical cord mesenchymal stem cell-originated extracellular vesicles (hUC-MSC-EVs) on endometriosis has been reported, while its specific mechanisms remain largely unknown. This study aimed at investigating the mechanisms underlying the modulation of EVs harboring miR-100 derived from hUC-MSCs in the growth dynamics of endometrial stromal cells in endometriosis. Endometriosis mouse models were established. miR-100 was upregulated and HS3ST2 was downregulated in endometriosis. Ectopic endometrial tissues and umbilical cord tissues were obtained to extract endometrial stromal cells and hUC-MSCs, from which EVs were isolated. Next, the endometrial stromal cells were co-cultured with hUC-MSC-EVs, during which gain- or loss-of-function approaches were employed for gene overexpression or silencing. The binding affinity among miR-100 and HS3ST2 was identified using multiple assays. It was unveiled that miR-100 could target and inhibit HS3ST2. miR-100 from hUC-MSCs could be transferred into the endometrial stromal cells via EVs. Moreover, miR-100 shuttled by hUC-MSC-EVs facilitated endometrial stromal cell proliferation, invasion, and migration, as well as EMT by inhibiting HS3ST2. In vivo experiments also confirmed that hUC-MSC-derived EVs carrying miR-100 induced the occurrence and development of endometriosis. Collectively, hUC-MSC-EV-loaded miR-100 downregulated HS3ST2 to facilitate the development of endometriosis, which highlights a promising therapeutic target for treating endometriosis.

Long non‑coding RNA LINC01960‑201 hinders decidualization of endometrial stromal cell in endometriosis: Relevance to endometrial receptivity.

Emerging data have indicated that long non‑coding RNAs (lncRNA) are associated with the pathogenesis of endometriosis. However, few are associated with endometriosis‑associated infertility. In addition, to the best of our knowledge, the role of lncRNAs in decidual formation during the window of implantation with endometriosis has not been reported to date. Based on our previous results, the aim of the present study was to explore the role of lncRNA long intergenic non‑protein coding RNA (LINC)01960‑201 in invitro decidualization of endometrial stromal cells in endometriosis during the window of implantation, as well as to explore the biological function of LINC01960‑201, and the regulation of a disintegrin and metalloproteinase with thrombospondin motifs7 (ADAMTS7), hsa‑microRNA (miR)‑760 and hsa‑miR‑608 in the decidualization of endometrial stromal cells with endometriosis. Using miRanda, PITA and RNAhybrid, the present study predicted which miRs share the common target gene ADAMTS7 with LINC01960‑201 and the existence of regulatory targets. Dual luciferase vectors were constructed to extract the plasmids and measure the relative fluorescence values in order to estimate target regulatory association between LINC01960‑201, ADAMTS7 and miRs. Mid‑secretory endometrial tissues were collected from women with endometriosis‑associated infertility. From these tissues, endometrial stromal cells were extracted and cultured as primary cultures. Medroxyprogesterone acetate (MPA) and 8‑Bromoadenosine 3',5'‑cyclic monophosphate (8‑Br‑cAMP) were added to induce invitro decidualization, and to knockdown LINC01960‑201 and transfect a hsa‑miR‑608 mimic at the same time. Reverse transcription‑quantitative PCR and western blotting were conducted to compare the difference in gene expression between the experimental and negative control groups. No regulatory sites between LINC01960‑201 and hsa‑miR‑608 were identified; however, potential regulatory sites were detected between hsa‑miR‑608 and the 3'‑untranslated region (UTR) of ADAMTS7, whereas neither the 3'‑UTR of LINC01960‑201 or the 3'‑UTR of ADAMTS7 had any regulatory targets with hsa‑miR‑760. During the process of decidualization of endometrial stromal cells by invitro induction, the expression of hsa‑miR‑608 in the knockdown group was significantly higher compared with that of the negative control group after LINC01960‑201‑knockdown, and the expression of ADAMTS7 in the transfection group was significantly lower compared with that of the negative control group after hsa‑miR‑608 mimic transfection. In conclusion, it was hypothesized that LINC01960‑201 played a notable regulatory role in the decidualization of endometrial stromal cells in women with endometriosis during the window of implantation, and its abnormal expression may lead to the decline of endometrial receptivity and recurrent abortions.

Silencing of circ_0007299 suppresses proliferation, migration, and invasiveness and promotes apoptosis of ectopic endometrial stromal cells in endometriosis via miR-424-5p-dependent modulation of CREB1.

The abnormality of endometrial stromal cells (ESCs) can contribute to endometriosis pathogenesis. Circular RNAs (circRNAs) possess critical roles in endometriosis pathogenesis. Here, we defined the activity and mechanism of human circ_0007299 in the regulation of ectopic ESCs in vitro. Circ_0007299, miR-424-5p and cAMP response element-binding protein 1 (CREB1) were quantified by qRT-PCR or immunoblotting. Cell viability, proliferation, apoptosis, invasion and motility were gauged by CCK-8, 5-Ethynyl-2'-Deoxyuridine (EdU), flow cytometry, transwell, and wound-healing assays, respectively. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to verify the direct relationship between miR-424-5p and circ_0007299 or CREB1. Our data showed that circ_0007299 was upregulated in human ectopic endometrium tissues and ectopic ESCs. Silencing endogenous circ_0007299 impeded the proliferation, invasiveness, and motility and enhanced apoptosis of ectopic ESCs. Mechanistically, circ_0007299 regulated miR-424-5p expression. Moreover, circ_0007299 silencing impeded the proliferation, invasiveness, and motility and enhanced apoptosis of ectopic ESCs via its regulation on miR-424-5p. CREB1 was identified as a direct miR-424-5p target, and miR-424-5p overexpression suppressed ectopic ESC proliferation, migration, and invasiveness and promoted apoptosis by downregulating CREB1. Furthermore, circ_0007299 positively modulated CREB1 expression through miR-424-5p competition. Our findings establish that circ_0007299 silencing impedes the proliferation, invasiveness, and motility and promotes apoptosis of ectopic ESCs at least in part via miR-424-5p-dependent modulation of CREB1.

Exosomal AFAP1-AS1 binds to microRNA-15a-5p to promote the proliferation, migration, and invasion of ectopic endometrial stromal cells in endometriosis.

BackgroundEndometriosis (EMS) remains a major challenge to reproductive health due to multifactorial etiology, disease heterogeneity, and the lack of appropriate diagnostic markers and treatment. Eexosome (Exo) has become a major factor in progression of a variety of diseases. However, the mechanisms directing their role in the pathophysiology of EMS are ill-defined. Here, we aimed to investigate the clinical implications of actin filament associated protein 1-Antisense RNA 1 (AFAP1-AS1) in EMS.MethodsBioinformatics analysis was used to predict the expression and interaction of AFAP1-AS1, miR-15a-5p and BCL9 in EMS, and dual luciferase reporter assay was used to verify the targeted relationship of AFAP1-AS1, miR-15a-5p, and BCL9. The Exo from endometrial stromal cells (ESCs) was isolated and characterized by transmission electron microscopy (TEM) and Nanoparticle tracking analysis (NTA). Exosome uptake studies were performed. For in vitro assay, ectopic ESCs (EcESCs) proliferation, migration, and invasion were assessed by CCK-8 and Transwell assays. In vivo assay was performed by establishment of EMS mice to validate the result derived from in vitro assay.ResultsThe Exo was successfully isolated from ESCs and we observed high expression of AFAP1-AS1 and BCL9 but low expression of miR-15a-5p in EMS. Moreover, Exo derived from EcESCs could deliver AFAP1-AS1 to EcESCs and thus promoting proliferation, migration, and invasion of ESCs. AFAP1-AS1 bound to BCL9, which was targeted by miR-15a-5p in EMS. In vivo experiments in nude mice revealed that inhibition of Exosomal AFAP1-AS1 suppressed migration and invasion of EcESCs through miR-15a-5p/BCL9.ConclusionsCollectively, these findings suggested that ESCs-derived Exo carrying AFAP1-AS1 contributed to EMS pathogenesis. This study might help us realize the etiology of EMS and improve the treatment of the related complications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12958-022-00942-1.

Effects of miR-363 on the Biological Activities of Eutopic Endometrial Stromal Cells in Endometriosis.

EMs is a kind of benign disease with certain malignant behaviors. The adhesion, invasive growth, and angiogenesis of ectopic endometrial cells are the pathological basis of EMs occurrence, but its etiology and pathogenesis have not been completely illustrated yet. In our research, we aim to investigate the role of miR-363 in the pathogenesis of endometriosis. Real-time quantitative PCR was used to detect the expression of miR-363 before and after ESC/NSC transfection. CCK-8, flow cytometry, and transwell assay were used to detect the effect of the miR-363 expression on cell proliferation, apoptosis, and invasion. The effects of the miR-363 expression on the contents of Fas/APO-1 and ICAM-1 in cell culture supernatant were detected by ELISA. qRT-PCR and WB assay were used to detect the effects of the miR-363 expression on the mRNA and protein expression levels of ICAM-1, MMP-7, and VEGF in ESC. The increased expression of miR-363 could inhibit the proliferation and invasion of ESC, promote apoptosis, and inhibit the secretion of FAS/APO-1 and ICAM-1. The knockdown expression of miR-363 promoted proliferation and invasion of NSC, inhibited apoptosis, and promoted secretion of FAS/APO-1 and ICAM-1. VCAM-1, VEGF, and MMP-7 were detected in ESCs before transfection. The protein expression level was higher than that of NSCs. Compared with pretransfection, the protein levels of VCAM-1, VEGF, and MMP-7 in the M-363 group were significantly downregulated. The downregulated expression of miR-363 was associated with a stronger cell proliferation ability, a lower cell apoptosis rate, and a stronger ESC. Migration is associated with invasiveness, proliferation, angiogenesis, and immune escape. The low expression of miR-363 promotes endogenesis through posttranscriptional regulation of target genes VCAM-1, MMP-7, and VEGF. The differential expression of miR-363 between ESC and NSC may be an important factor in the many biological differences between ESC and NSC.

MiR-206 Targets MALAT1 to Suppress Cell Progression of Ectopic Endometrial Stromal Cells in Endometriosis.

Background miR-206 was reported to be a tumor suppressor in bladder cancer. In this study, we explore the expression and function of miR-206 in endometriosis (EM). Methods 40 EM patients undergoing total hysterectomy were selected as the experimental group. RT-qPCR assay was adopted to detect the expression of MALAT1 and miR-206 in EM. Cell proliferation was detected by EdU incorporation and colony formation assay. Cell migration and invasion viability of ESCs were examined by transwell assay and wound healing assay. Flow cytometry was carried out to assess cell apoptosis of ESCs. The protein expressions of Bcl-2 and Bax were examined by western blot assay. The relationship between miR-206 and MALAT1 was verified by the dual-luciferase reporter assay and RNA pull-down assay. Results In this work, miR-206 was found to be downregulated in EM. Functional experiments displayed that miR-206 mimic repressed cell proliferation, migration, and invasion of ESCs and promoted cell apoptosis of ESCs. Furthermore, miR-206 mimic reduced the expression of Bcl-2 but enhanced the expression of Bax. MALAT1 was found to be upregulated in EM. Furthermore, MALAT1 was indicated to be a target of miR-206. Additionally, MALAT1 was found to alleviate the influence of miR-206 on cell progression of ESCs. Furthermore, miR-206 inhibited tumor growth in vivo. Conclusion This study indicated that miR-206 inhibited cell progression by regulating MALAT1 in EM. Hence, miR-206 was suggested to be a possible target for EM treatment.

Inhibition of METTL3/m6A/miR126 promotes the migration and invasion of endometrial stromal cells in endometriosis†.

N 6-methyladenosine (m6A), one of the most abundant RNA modifications, is involved in the progression of many diseases, but its role and related molecular mechanisms in endometriosis remain unknown. To address these issues, we detected m6A levels in normal, eutopic, and ectopic endometrium and found the m6A levels decreased in eutopic and ectopic endometrium compared with normal endometrium. In addition, we proved that methyltransferase-like 3 (METTL3) downregulation accounted for m6A reduction in endometriosis. Furthermore, we observed that METTL3 knockdown facilitated the migration and invasion of human endometrial stromal cells (HESCs), whereas METTL3 overexpression exerted opposite effects, suggesting that METTL3 downregulation might contribute to endometriosis development by enhancing cellular migration and invasion. Mechanistically, METTL3-dependent m6A was involved in the DGCR8-mediated maturation of primary microRNA126 (miR126 and pri-miR126). Moreover, miR126 inhibitor significantly enhanced the migration and invasion of METTL3-overexpressing HESCs, whereas miR126 mimics attenuated the migration and invasion of METTL3-silenced HESCs. Our study revealed the METTL3/m6A/miR126 pathway, whose inhibition might contribute to endometriosis development by enhancing cellular migration and invasion. It also showed that METTL3 might be a novel diagnostic biomarker and therapeutic target for endometriosis.

SMURF1-mediated ubiquitylation of SHP-1 promotes cell proliferation and invasion of endometrial stromal cells in endometriosis.

BackgroundEndometriosis is a widespread benign gynecological disorder. The signal transducer and activator of transcription 3 (STAT3) signaling pathway plays an important role in the pathogenesis of endometriosis through regulating proliferation and invasion of endometrial stromal cells. Furthermore, the protein tyrosine phosphatase (PTP), SH2 domain-containing phosphatase 1 (SHP-1), negatively regulates STAT3 activation. However, regulation of the SHP-1-STAT3 pathway in the pathogenesis of endometriosis remains unclear.MethodsCell proliferation and invasion were assessed by Cell Counting Kit-8 (CCK-8) assay and Transwell analysis, respectively, to investigate the role and regulation of the SHP-1-STAT3 pathway in the proliferation and invasion of endometrial stromal cells. Expression of Smad ubiquitin regulatory factor 1 (SMURF1), SHP-1, matrix metalloproteinase 2 (MMP2), MMP9, STAT3, and phospho-STAT3 (p-STAT3) level in patients with endometriosis were measured by Western blotting and/or immunohistochemical staining. The interaction between SMURF1 and SHP-1 was investigated by co-immunoprecipitation and ubiquitylation analysis.ResultsThe present study demonstrated that downregulation of SHP-1 expression in patients with endometriosis was negatively correlated with SMURF1 expression. SMURF1, an E3 ubiquitin ligase, activated the STAT3 pathway via ubiquitylation and degradation of SHP-1. Furthermore, SMURF1 promoted cell proliferation and invasion of endometrial stromal cells by activating STAT3 signaling and expression of its downstream targets, MMP2 and MMP9, whereas SHP-1 demonstrated an inverse effect. Additionally, SHP-1 inhibited SMURF1-mediated cell invasion and proliferation of endometrial stromal cells.ConclusionsOur findings indicate that SMURF1-mediated ubiquitylation of SHP-1 regulates endometrial stromal cell proliferation and invasion during endometriosis.

The AMPK-mTOR axis requires increased MALAT1 expression for promoting granulosa cell proliferation in endometriosis.

Endometriosis is a common reproductive disorder in women, with a global prevalence of 10-15%. Long noncoding RNAs (lncRNAs) are critical to gene transcription, cell cycle modulation and immune response. The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) reportedly mediates autophagy of endometrial stromal cells in endometriosis. The present study aimed to evaluate the role and mechanism of MALAT1 in granulosa cells (GCs) in endometriosis. Consequently, MALAT1 expression was upregulated in GCs obtained from patients with endometriosis and in the steroidogenic human granulosa-like tumor cell line KGN. However, MALAT1 knockdown consequently decreased the proliferation and viability of these cells, as determined by MTT and 5-ethynyl-2'-deoxyuridine staining assays. Both Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometry and western blotting performed to detect proapoptotic factors indicated that MALAT1 depletion might promote KGN cell apoptosis. Furthermore, MALAT1 knockdown increased GC autophagy, as evidenced by microtubule-associated protein 1A/1B-light chain 3 (LC3) cleavage upregulation and p62 degradation. In addition, although 5'-AMP-activated protein kinase (AMPK) mRNA expression and protein levels decreased in GCs obtained from patients with endometriosis and KGN cells, MALAT1 knockdown restored AMPK levels. However, addition of BML-275 (MALAT1 inhibitor) to MALAT1-knockdown KGN cells recovered their viability and proliferative capacity and simultaneously reduced their apoptotic and autophagic capacity. Therefore, MALAT1 may regulate GC proliferation via AMPK-mTOR-mediated cell apoptosis and autophagy.

Baicalein inhibits FURIN-MT1-MMP-mediated invasion of ectopic endometrial stromal cells in endometriosis possibly by reducing the secretion of TGFB1.

Endometriosis (EMs) is characterized by the presence of endometrial stroma and glands outside the uterus. Our previous study showed that baicalein inhibited proliferation and induced apoptosis in EMs. However, the effects of baicalein on the invasiveness of ectopic endometrial stromal cells (EcESCs) remain unclear. The aim of this study was to assess the potential anti-invasive effect of baicalein and determine the underlying mechanism. The invasive and migratory properties of EcESCs were assessed in vitro using Transwell and wound healing assays. The expression of functional markers of EcESCs, including matrix metalloproteases (MMPs), FURIN, and TGFB1, was analyzed using WB and ELISA. Additionally, a mouse model of EMs was treated with baicalein (10mg/kg/d and 35mg/kg/d) for 4weeks. The weight and number of ectopic lesions were determined, and the expression of markers was assessed using immunohistochemistry. Baicalein inhibited the invasion of EcESCs and the expression of certain invasion-related proteins, including MMP9, MMP2, and MT1-MMP. Exposure to baicalein reduced the extracellular levels of TGFB1 in EcESCs and the reduced expression of TGFB1, resulting in decreased expression of FURIN in EcESCs, which serves a pivotal role in the transformation of pro-MT1-MMP to activated MT1-MMP. In the mouse model of EMs, intraperitoneal injection of baicalein inhibited the growth of ectopic lesions and reduced MT1-MMP, FURIN, and TGFB1 expression. Baicalein reduced the invasion of EMs, potentially by restricting the FURIN-MT1-MMP-mediated cell invasion of EcESCs maybe through reduction of the autocrine of TGFB1.

Fibrinogen alpha chain promotes the migration and invasion of human endometrial stromal cells in endometriosis through focal adhesion kinase/protein kinase B/matrix metallopeptidase 2 pathway†.

Fibrinogen alpha chain (FGA), a cell adhesion molecule, contains two arginyl-glycyl-aspartic acid (RGD) cell adhesion sequences. Our previous study demonstrated that FGA, as an up-regulated protein in endometriosis (EM), was closely related to disease severity and involved in the development of EM. However, the biological functions and underlying mechanism of FGA in EM have not been fully understood. To explore the roles of FGA in EM, we analyzed the effects of FGA on the biological behaviors of human primary eutopic endometrial stromal cells (EuESC). The results indicated FGA knockdown suppressed the migration and invasion ability of EuESC, which also altered the distribution of cytoskeletal filamentous and cell morphology. Western blot analysis demonstrated that knockdown of FGA attenuated the migration-related protein levels of vimentin and matrix metallopeptidase 2 (MMP-2), but not integrin subunit alpha V (ITGAV) and integrin subunit beta 3 (ITGB3). Meanwhile, integrin-linked transduction pathways were detected. We found FGA knockdown significantly suppressed the expression of focal adhesion kinase (FAK) level and protein kinase B (AKT) phosphorylation, without extracellular-signal-regulated kinase (ERK) dependent pathways. Treatment with the AKT inhibitor MK2206 or RGD antagonist highly decreased the effects of FGA on the migration and invasion of EuESC. RGD antagonist treatment strongly inhibited FAK- and AKT-dependent pathways, but not ERK pathways. Our data indicated that FGA may enhance the migration and invasion of EuESC through RGD sequences binding integrin and activating the FAK/AKT/MMP-2 signaling pathway. This novel finding suggests that FGA may provide a novel potential approach to the treatment of EM, which provides a new way to understand the pathogenesis of EM.

Suppression of PRDX4 inhibits cell proliferation and invasion of ectopic endometrial stromal cells in endometriosis

Oxidative stress (OS) has been proposed to play a role in the development of EMs. Peroxiredoxins are a family of antioxidant proteins that exhibit peroxidase activity in a thioredoxin-dependent manner, protecting cells against OS. The Western blotting results showed that the relative expression of PRDX4 was significantly increased in ectopic endometria compared with the normal endometria of EMs-free (p < .05). The H2O2 concentration was also significantly higher in the ectopic endometrium. PRDX4 siRNA was transfected into primary ectopic endometrial stromal cells (EESCs). The viability of the transfected EESCs was measured by CCK-8 assay, and the results showed significantly decreased cell viability. Furthermore, the apoptosis rate and ROS generation in flow cytometry assays were significantly increased after the knockdown of PRDX4 expression (p < .05). Scratch assays and transwell assays revealed that decreased expression of PRDX4 mediated by siRNA inhibited EESC migration and invasion. In conclusion, these findings indicate the potential role of PRDX4 in the development of EMs and PRDX4 as a possible therapeutic target for EMs treatment.

TRIM59 inhibits PPM1A through ubiquitination and activates TGF-β/Smad signaling to promote the invasion of ectopic endometrial stromal cells in endometriosis.

This study was conducted to define the underlying molecular mechanism of tripartite motif (TRIM) 59-induced invasion of ectopic endometrial stromal cells in endometriosis. Primary endometriosis ectopic endometrial stromal cells and normal endometrial cells were isolated and purified. Western blot was used to detect the expression of TRIM59, protein phosphatase Mg2+/Mn2+-dependent 1A (PPM1A), smad2/3, and phosphorylated (p)-smad2/3. Lentiviral vector-mediated TRIM59 interference and overexpression were established. Cell Counting Kit-8 assay was used to detect cell proliferation, and the Transwell migration assay was used to detect cell invasion. Matrix metalloproteinase (MMP-2), MMP9, smad2/3, and p-smad2/3 expressions were also detected using Western blot analysis; degradation of PPM1A was verified to be through ubiquitination. We found that TRIM59 expression levels in the endometriosis group was significantly higher compared with the normal group (P < 0.05), whereas the expression levels of PPM1A in the endometriosis group were significantly lower (P < 0.05). Endometriosis did not alter smad2/3 (P > 0.05) expression. However, after activating smad2/3 by phosphorylation, the expression of p-smad2/3 in the endometriosis group was significantly higher compared with the normal group (P < 0.05). The content of PPM1A in the TRIM59 overexpression group was significantly lower than that in the control group (P < 0.001), whereas the content of PPM1A in the siTRIM59 group was significantly higher than that in the control group (P < 0.001). In addition, there were no significant differences in the mRNA levels of PPM1A among the five groups, indicating that TRIM59 affects the expression of PPM1A at the posttranslational level (P < 0.05). Overexpression of TRIM59 significantly promoted the ubiquitination of PPM1A. We conclude that TRIM59 inhibits PPM1A through ubiquitination and activates the transforming growth factor-β/Smad pathway to promote the invasion of ectopic endometrial stromal cells in endometriosis.

Dysregulation of miR-202-3p Affects Migration and Invasion of Endometrial Stromal Cells in Endometriosis via Targeting ROCK1.

Dysregulation of microRNAs in endometrial cells plays a pivotal role in the pathogenesis of endometriosis (EM). This study aims to investigate the implication of aberrant miR-202-3p expression in EM and the underlying mechanisms. We demonstrated that miR-202-3p was significantly downregulated in eutopic endometrium of EM in comparison to normal endometrial samples (P< 0.05). Primary endometrial stromal cells (ESCs) isolated from eutopic or ectopic endometrium also showed a significant decrease in miR-202-3p level compared to ESCs from normal endometrium (P< 0.01). Functional studies using MTT, wound healing assay and transwell assay indicated that overexpression of miR-202-3p greatly impaired cell viability, migration, and invasion, whereas suppression of miR-202-3p exhibited the opposite effects (P< 0.05 or P< 0.01). miR-202-3p mimics or inhibitors transfection significantly decreased or increased expression of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1), respectively, in ESCs (P< 0.01). Using dual luciferase reporter assay, we validated ROCK1 as a direct target of miR-202-3p. Moreover, negative correlations between miR-202-3p and ROCK1 mRNA/protein levels were determined in both eutopic and normal control endometrium (P< 0.01). In conclusion, these findings suggest that suppression of miR-202-3p in ESCs results in enhanced cell viability, invasion, and migration at least partially via upregulation of its target ROCK1, which eventually contributes to the development of endometriosis.

T-cadherin inhibits invasion and migration of endometrial stromal cells in endometriosis.

What is the expression level of T-cadherin in endometriosis, and does T-cadherin play a role in regulating invasion and migration of endometrial stromal cells? T-cadherin expression was reduced in ectopic endometriotic lesions compared to eutopic endometrium, and T-cadherin overexpression inhibited the invasion and migration of endometrial stromal cells. Endometriosis is a disease that involves active cell invasion and migration. T-cadherin can inhibit cell invasion, migration and proliferation in various cancer cells, but its role in endometriosis has not been investigated. We explored the expression status of T-cadherin in 40 patients with and 24 without endometriosis. We also isolated endometrial stromal cells to study the invasion, migration and signaling pathway regulation of T-cadherin overexpression. Patients were recruited at the Guangzhou Women and Children's Medical Center to study the expression levels of T-cadherin. The expression of T-cadherin was detected by immunohistochemistry staining and western blot. H-score was used to evaluate the staining intensity of T-cadherin. The correlation between T-cadherin expression levels (H-score) and endometriosis patients' age, stage, lesion size and adhesion was analyzed. Endometrial stromal cells from patients with and without endometriosis were isolated, and cell invasion and migration were detected by transwell assays after T-cadherin overexpression. The expression of vimentin in T-cadherin-overexpressed cells was detected by western blot. After T-cadherin overexpression, the phosphorylation profile of signaling pathway proteins was detected with the Proteome Profiler Human Phospho-Kinase Array Kit. There was no difference in the expression of T-cadherin in the normal endometrium of control patients and the eutopic endometrium of endometriotic patients, but it was significantly decreased in the ectopic endometrium of endometriotic patients, compared with control endometrium and eutopic endometrium of endometriosis patients (P< 0.0001, for both). Western blot analysis also showed that the expression of T-cadherin was decreased in ectopic endometriotic lesions, but not the normal control endometrium or the endometriotic eutopic endometrium. The results of transwell assays indicated that T-cadherin overexpression inhibited the invasion and migration of endometrial stromal cells. In addition, T-cadherin overexpression promoted the phosphorylation of HSP27 (S78/S82) and JNK 1/2/3 (T183/Y185, T221/Y223) and decreased the expression of vimentin, MMP2 and MMP9 in eutopic endometriosis stromal cells. N/A. The control group were patients with benign gynecological conditions (e.g. uterus myoma, endometrial or cervical polyp), which may have genetic or epigenetic variations associated with T-cadherin expression and signaling pathways. The case numbers of involved endometriosis and control patients were limited. This study only used endometrial stromal cells from patients with or without endometriosis. Ideally, ectopic endometrial stromal cells of the ovarian endometriotic lesions should also be utilized to explore the function of T-cadherin. Further investigation of the role of T-cadherin in endometriosis may generate new potential therapeutic targets for this complex disorder. This study was supported by the Natural Science Foundation of Guangdong Province (2016A030313495), National Natural Science Foundation of China (81702567, 81671406, 31871412), the Science and Technology Programs of Guangdong (2017A050501021), Medical Science Technology Research Fund of Guangdong Province (A2018075), the Science and Technology Programs of Guangzhou City (201704030103), Internal Project of Family Planning Research Institute of Guangdong Province (S2018004), Post-doc initiation fund of Guangzhou (3302) and Post-doc science research initiation fund of Guangzhou Women and Children's Medical Center (20160322). There are no conflicts of interest.

GLI1 is increased in ovarian endometriosis and regulates migration, invasion and proliferation of human endometrial stromal cells in endometriosis.

Endometriosis is a benign gynecological disorder which shares certain characteristics with malignant tumor like migration, invasion and proliferation. Glioma-associated oncogene homolog 1 (GLI1) has been implicated in some cancers including endometrial cancer, however, its role in endometriosis remains unknown. The aim of this study was to explore the expression pattern of GLI1 in endometriosis, and further investigate the effect of GLI1 regulation on human endometrial stromal cells. The expression of GLI1 in normal endometrium and ectopic tissues was analyzed by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. The Short hairpin RNA (ShRNA) intervention technique and GLI1 inhibitor GANT-61 were used to silence GLI1. The expression levels of GLI1, MMP2 and MMP9 was detected by qRT-PCR and western blot. The migration and invasion ability of human endometrial stromal cells was determined by wound healing assay and transwell migration/invasion assay. The viability and proliferation potentiality of cells was detected by MTT assays and colony formation assay, respectively. We found that the expression of GLI1 mRNA and protein were significantly higher in ectopic endometrium from patients with endometriosis. Our analyses also show that GLI1 downregulation attenuated cells migration, invasion and proliferation abilities. What's more, reduced expression of GLI1 inhibited the expression of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9). Our findings suggest that high levels of GLI1 may contribute to the development of endometriosis by promoting cell migration, invasion and proliferation involving regulation of MMP2 and MMP9 expression. Therefore, inhibition of GLI1 might be a novel potential therapeutic approach to the treatment of endometriosis, which sheds new light on our understanding of the pathogenesis of endometriosis.