Green walnut husk extracts restrain proliferation and invasion of endometrial stromal cells by inhibiting the PI3K/AKT signaling pathway

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 level of CCL19 increased in the peritoneal fluid of women with endometriosis, but the precise mechanism of CCL19/CCR7 in the pathogenesis of endometriosis remains unknown. ELISA and immunohistochemistry were performed to analyze CCL19/CCR7 expressions in peritoneal fluid and endometrium from women with endometriosis (n=38) and controls (n=32). Cell proliferation and transwell invasion assays were applied to detect proliferation and invasion of human endometrial stromal cells (ESCs). Expressions of Bcl2, MMP2, MMP9, and p-AKT/AKT were analyzed by Western blot. Peritoneal fluid concentration of CCL19 in patients with endometriosis was higher than that in controls. Those patients with moderate/severe endometriosis had significantly higher peritoneal fluid concentrations of CCL19 compared to those with minimal/mild endometriosis. Higher CCL19 and CCR7 were found in the endometrium with endometriosis compared to control. CCL19 significantly enhanced ESC proliferation and invasion through CCR7 via activating PI3K/Akt signal pathways. CCL19/CCR7 interaction significantly enhanced phosphorylation of Akt, Bcl2, MMP2, and MMP9 in ESCs. These data indicate CCL19/CCR7 contributes to proliferation and invasion of ESCs, which are conducive to the pathogenesis of endometriosis through activating PI3K/Akt pathway.

SKP2 regulates ZEB1 expression and stimulates eutopic endometrial stromal cell invasion and proliferation of adenomyosis

Although immune dysregulation is implicated in the pathogenesis of endometriosis(EMs), the specific role of prostate transmembrane protein androgen induced 1 (PMEPA1) in modulating the function of regulatory T cells (Tregs) remains inadequately understood. This study aimed to elucidate the regulatory mechanisms by which PMEPA1 influences the activity of Tregs, thereby facilitating the invasion of endometrial stromal cells (ESCs). Single-cell RNA sequencing (scRNA-seq) was performed on matched ectopic ovarian lesions and eutopic endometria from 3 patients. Clinical specimens from patients with EMs and control subjects were examined for PMEPA1 expression. Primary human Tregs isolated from peripheral blood mononuclear cells were subjected to PMEPA1 overexpression (via plasmid) or knockdown (via siRNA). Modulation of the PI3K pathway was conducted via the activator 740Y-P or the inhibitor LY294002. The secretion of IL-10 and TGF-β by Tregs was quantified using an enzyme-linked immunosorbent assay. Ectopic ESCs cocultured with modified Tregs were assessed for their proliferation, migration, and invasion capabilities. scRNA-seq data revealed significant upregulation of PMEPA1 in Tregs from ectopic ovarian lesions compared with paired eutopic endometria. PMEPA1 expression was increased in the ectopic lesions and peritoneal fluid mononuclear cells of patients with EMs. Tregs overexpressing PMEPA1 demonstrated reduced secretion of IL-10 and TGF-β but exhibited hyperactivation of the PI3K/AKT signaling pathway. Treatment with LY294002 ameliorated the impairment in cytokine secretion. Coculture experiments with Tregs expressing high levels of PMEPA1 resulted in increased invasion, migration, and proliferation of ESCs. PMEPA1 impairs Treg-mediated immunosuppression by hyperactivating the PI3K/AKT pathway, thereby facilitating the invasiveness of ESCs in EMs.

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.

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.

To explore the expression of CCN5 in endometriotic tissues and its impact on proliferation, migration and invasion of human endometrial stromal cells (HESCs). We collected ovarian endometriosis samples from 20 women receiving laparoscopic surgery and eutopic endometrium samples from 15 women undergoing IVF-ET for comparison of CCN5 expression. Cultured HESCs were transfected with a recombinant adenovirus Ad-CCN5 for CCN5 overexpression or with a CCN5-specific siRNA for knocking down CCN5 expression, and the changes of cell proliferation, migration and invasion were evaluated using CCK-8 assay, wound healing assay and Transwell chamber assay. RT-qPCR and Western blotting were used to examine the expression levels of epithelial-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin, Snail-1 and vimentin in HESCs with CCN5 overexpression or knockdown. CCN5 expression was significantly decreased in ovarian endometriosis tissues as compared with eutopic endometrium samples (P < 0.01). CCN5 overexpression obviously inhibited the proliferation, migration and invasion of HESCs, significantly increased the expression of E-cadherin and decreased the expressions of N-cadherin, Snail-1 and vimentin (P < 0.01). CCN5 knockdown significantly enhanced the proliferation, migration and invasion of HESCs and produced opposite effects on the expressions of E-cadherin, N-cadherin, Snail-1 and vimentin (P < 0.01). CCN5 can regulate the proliferation, migration and invasion of HESCs and thus plays an important role in EMT of HESCs, suggesting the potential of CCN5 as a therapeutic target for endometriosis.

Chemokines have been reported to play a sovereign role in the establishment and progression of endometriosis. Fractalkine is a chemokine that is upregulated in many inflammatory diseases including endometriosis. Fractalkine functions as a chemotactic role for lymphocytes and monocytes. In this study, we investigated the role of fractalkine/CX3CR1 in the pathogenesis of endometriosis. In this study, immunohistochemistry was performed on normal endometrium (taken from controls), eutopic endometrium (taken from patients with endometriosis), and ectopic tissues to analyze fractalkine/CX3CR1 expression. The levels of fractalkine in peritoneal fluid and the cell culture supernatant were examined by enzyme-linked immunosorbent assay (ELISA). Bromodeoxyuridine (BrdU) cell proliferation assay was applied to detect the proliferation of endometrial stromal cells (ESCs). The invasion of ESCs was measured by transwell invasion assay. The protein levels of Bcl2, MMP2, MMP9, p-AKT/AKT, p-p38/p38, p-JNK/JNK, and p-ERK/ERK were analyzed by Western blot. We found that the eutopic endometrium had significantly higher expression of fractalkine and CX3CR1 compared to normal endometrium, and the ectopic tissues had the highest expression. The concentrations of fractalkine in peritoneal fluid of endometriosis patients were obviously higher than that of the control and correlate very well with the severity of endometriosis. Fractalkine enhanced ESCs proliferation and invasion via activating AKT and p38 signal pathways. Moreover, high concentration of estradiol (10(-7) , 10(-6) molL(-1) ) induced fractalkine expression while high concentration of progesterone (10(-6) , 10(-5) molL(-1) ) inhibited fractalkine expression in ESCs. The results revealed that the high levels of fractalkine in ectopic milieu promoted proliferation and invasion of ESCs through activating AKT and p38 signal pathways. Estradiol has a stimulating effect on the expression of fractalkine. The present results increase our understanding of the significance of fractalkine in the progression of endometriosis and shed some lights on the targeted fractalkine/CX3CR1 therapies.

Endometriosis (EM) is a benign gynecological disease that shares some characteristics with malignancy, such as proliferation and invasion. So far, the pathogenesis of EM is still unclear. In this study, we investigated whether TRIM65 can play a role in the development of EM. TRIM65 expression levels in eutopic, ectopic, and normal endometrium were detected by quantitative real-time PCR and Western blot. Cell proliferation and invasion of primary endometrial stromal (EMS) cells were detected by CCK-8 and Transwell analysis. The interaction between TRIM65 and DUSP6 or C-myc was measured by coimmunoprecipitation, ubiquitylation, dual luciferase, and chromatin immunoprecipitation analysis. We found that TRIM65 was identified as an up-regulated gene in ectopic endometrial tissues and EMS cells compared with control groups without EM. TRIM65 expression was positively correlated with the levels of p-ERK1/2, C-myc, matrix metalloproteinase-2, and integrin β1 in ectopic endometrial tissues in patients and mice. TRIM65 promoted the cell proliferation and invasion of EMS cells via the ERK1/2/C-myc pathway through ubiquitination of DUSP6. C-myc promoted TRIM65 expression through inducing TRIM65 promoter activity. Additionally, the increased expression of TRIM65, C-myc, matrix metalloproteinase-2, integrin β1, and p-ERK1/2 and the decreased expression of DUSP6 in ectopic endometrial tissues were significantly suppressed by inhibition of ERK1/2 signaling pathway in ectopic endometrial tissues in experimental mice model. In conclusion, TRIM65 promotes invasion of ectopic EMS cells by activating a feedback loop with the ERK1/2/C-myc signaling pathway and may be a potential therapeutic target for EM.

Background: Dysregulated migration and invasion of endometrial stromal cells is implicated in the pathogenesis of endometriosis. Hypoxia functions as critical microenvironmental factor that results in promotion of endometrial stromal cells migration and invasion through up-regulation of autophagy. Paeonol functioned as a tumor suppressor in human ovarian cancer and promoted cytoprotective autophagy. However, the role of paeonol in hypoxia-induced autophagy in endometriosis remains unknown. Methods: Stromal cells were isolated from endometriotic patients by enzymatic digestion of ectopic endometrial tissues, and then characterized by immunohistochemical analysis of cytoskeleton 19 (CK19) and vimentin. Cellular morphology was evaluated under microscope. Cell viability, proliferation and apoptosis of stromal cells were assessed by Cell Counting Kit-8, EdU labeling and flow cytometry, respectively. Wound healing and transwell assays were performed to detect metastasis of the stromal cells. Hypoxia-induced autophagy was evaluated through immunohistochemistry and western blot. Results: Paeonol treatment dosage dependently decreased cell proliferation and metastasis of the ectopic endometrial stromal cells (ecESCs), while promoted the cell apoptosis. Hypoxia-induced autophagy in the ecESCs was repressed by paeonol through down-regulation of LC3-II/LC3-I and Beclin-1, while up-regulation of p62. Hypoxia-inducible factor-1α (HIF-1α) was reduced post paeonol treatment, and paeonol-induced increase of p62 and decrease of LC3-II/LC3-I and Beclin-1 were reversed by over-expression of HIF-1α. Over-expression of HIF-1α also attenuated the suppressive effect of paeonol on cell growth of ecESCs. Conclusions: Paeonol attenuated HIF-1α-induced promotion of ecESCs migration and invasion through reducing autophagy, and reduced HIF-1α-induced endometriotic lesion in rats, providing potential therapeutic strategy for the treatment of endometriosis.

Polychlorinated biphenyl 104 promotes migration of endometrial stromal cells in endometriosis

Menstrual cycle–specific inhibition of the proliferation of endometrial stromal cells by interleukin 6 and its soluble receptor

O-5: Activin A increases invasion of endometrial stromal cells and endometrial epithelial cells into modeled human peritoneum

Endometriosis is a benign gynecologic disease that causes chronic pelvic pain, dysmenorrhea and infertility and shares several characteristics with malignant tumors, afflicting women of reproductive age. Hexokinase 2 plays an essential role as the first rate-limiting enzyme in the metabolic glycolysis pathway, and its abnormal elevation in tumors is associated with tumor genesis and metastasis. However, the expression and role of hexokinase 2 in endometriosis remain unclear. We sequenced the primary endometrial stromal cells from patients with endometrioma and utilized immunohistochemistry, quantitative real-time PCR, and western blot to determine the expression of hexokinase 2. Then wound healing assays, cell invasion assays, and cell proliferation assays were performed to explore the functions of hexokinase 2 in endometrial stromal cells. Furthermore, mice models of endometriosis were used to observe the effects of hexokinase 2 inhibitors in vivo. Lastly, glycolysis metabolism detection and transcriptome sequencing were carried out in hexokinase 2-knockdown endometrial stromal cells to analyze the mechanism of hexokinase 2 affecting cell function. Endometrial stromal cells of endometrioma displayed active glycolysis metabolism and elevated expression of hexokinase 2. Downregulating hexokinase 2 reduced the migration, invasion, and proliferation capacity of endometrial stromal cells. Knockdown of hexokinase 2 induced upregulation of signal transducer and activator of transcription 1 and their phosphorylation to attenuate the proliferation of endometrial stromal cells. Hexokinase 2 is associated with the migration, invasion, and proliferation of endometrial stromal cells, which might provide new insights into the pathogenesis and treatment of endometriosis. HK2 is upregulated in ovarian endometrioma and knockdown of HK2 induced upregulation of signal transducer and activator of transcription 1 (STAT1) and their phosphorylation to attenuate the proliferation of endometrial stromal cells.

Endometriosis (EMS) is associated with an abnormal immune response to endometrial cells, which can facilitate the implantation and proliferation of ectopic endometrial tissues. It has been reported that human endometrial stromal cells (ESCs) express interleukin (IL)15. The aim of our study was to elucidate whether or not IL15 regulates the cross talk between ESCs and natural killer (NK) cells in the endometriotic milieu and, if so, how this regulation occurs. The ESC behaviors in vitro were verified by Cell Counting Kit-8 (CCK-8), Annexin/PI, and Matrigel invasion assays, respectively. To imitate the local immune microenvironment, the co-culture system between ESCs and NK cells was constructed. The effect of IL15 on NK cells in the co-culture unit was investigated by flow cytometry (FCM). In this study, we found that ectopic endometrium from patients with EMS highly expressed IL15. Rapamycin, an autophagy inducer, decreased the level of IL15 receptors (i.e. IL15Rα and IL2Rβ). IL15 inhibits apoptosis and promotes the invasiveness, viability, and proliferation of ESCs. Meanwhile, a co-culture with ESCs led to a decrease in CD16 on NK cells. In the co-culture system, IL15 treatment downregulated the levels of Granzyme B and IFN-γ in CD16(+)NK cells, NKG2D in CD56(dim)CD16(-)NK cells, and NKP44 in CD56(bright)CD16(-)NK cells. On the one hand, these results indicated that IL15 derived from ESCs directly stimulates the growth and invasion of ESCs. On the other hand, IL15 may help the immune escape of ESCs by suppressing the cytotoxic activity of NK cells in the ectopic milieu, thereby facilitating the progression of EMS.