Elevated GPX3 expression in endometriosis: implications for pathogenesis and immune cell infiltration.

Endometriosis (EMs) is a common and complex gynecological disease with an unclear pathogenesis. Splicing factors play critical roles in the progression of various diseases. However, the expression pattern and biological function of serine/arginine-rich splicing factor 3 (SRSF3) in EMs remain incompletely understood. This study aimed to investigate whether the splicing factor SRSF3 participates in the pathogenesis of endometriosis by regulating the alternative splicing of the Adducin 3 (ADD3) gene. Ectopic and eutopic endometrial tissues were collected from patients with EMs, with normal endometrial tissues from non-EMs women serving as controls. The expression levels of SRSF3 and ADD3 were detected by qRT-PCR and Western blot. Overexpression vectors for SRSF3 and two splice isoforms of ADD3 (ADD3(+ 14) and ADD3(− 14)) were constructed and transfected into ectopic endometrial stromal cells (ESCs). Subsequently, the effects on cell proliferation, apoptosis, migration, and invasion were assessed using CCK-8 assay, flow cytometry, wound healing assay, and Transwell assay. The binding of SRSF3 to the precursor mRNA of ADD3 was validated by RNA immunoprecipitation (RIP) assay. The results showed that SRSF3 expression was downregulated while ADD3 expression was upregulated in ectopic endometrial tissues. Overexpression of SRSF3 inhibited the splicing of ADD3 exon 14, leading to reduced expression of both isoforms. Functionally, overexpression of ADD3(− 14) enhanced the proliferation, migration, and invasion abilities of ESCs while inhibiting apoptosis, whereas overexpression of SRSF3 exerted the opposite effects. RIP-qPCR confirmed that SRSF3 could bind to the exon 14 region of the ADD3 precursor mRNA. In conclusion, the downregulation of SRSF3 induces the skipping of ADD3 exon 14, generating the ADD3(− 14) isoform, thereby promoting the progression of endometriosis. These findings provide new insights into the pathogenesis of this disease and suggest a potential therapeutic target.

To observe the levels of leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in blood, peritoneal fluid, ectopic endometrial tissue, and ectopic endometrial stromal cells of patients with endometriosis, and to compare expression of IL-6, LIF and VEGF expression between endometriotic and non-endometriotic patients underwent laparoscopic surgery. Thirty-one patients who underwent laparoscopic surgery for endometriosis in our hospital from January 2018 to January 2020 were included in the observation group, and 32 patients who underwent laparoscopic surgery for uterine fibroids, ovarian serous cystadenoma, and ovarian teratomas, were included in the control group. The levels of LIF, IL-6 and VEGF in the blood and peritoneal fluid of the two groups of patients were detected. The levels of LIF, IL-6 and VEGF in ectopic endometrial tissue and self-paired eutopic endometrial tissue, ectopic endometrial stromal cells and self-paired eutopic endometrial stromal cells of patients in the observation group were detected. After treating the primary cultured ectopic endometrial stromal cells with LIF and IL-6 alone or in combination, the changes of VEGF mRNA of ectopic endometrial stromal cells and the VEGF levels in the supernatant were observed. The levels of LIF, IL-6 and VEGF in the blood and peritoneal fluid of the observation group were all higher than those of the control group (P < 0.05), and the levels of LIF, IL-6 and VEGF in the peritoneal fluid of the observation group were significantly higher than those in the blood (P < 0.05). In the observation group, the expression levels of LIF-mRNA and IL-6 mRNA in the ectopic endometrial tissue were higher than those in the self-paired eutopic endometrial tissues (P < 0.05), while the expression level of VEGF mRNA in the ectopic endometrial tissues was lower than that in the self-paired eutopic endometrial tissues (P < 0.05). Besides, the mRNA expression levels of LIF, IL-6 and VEGF in ectopic endometrial stromal cells of the observation group were all higher than those in the self-paired eutopic endometrial stromal cells (P < 0.05). After stimulating ectopic endometrial stromal cells with LIF, IL-6 and LIF + IL-6, respectively, the VEGF levels in the supernatant were all significantly higher than that in the blank control group (P < 0.05). The LIF, IL-6 and VEGF levels in blood and peritoneal fluid were increased in patients with endometriosis, and increased LIF and IL-6 in ectopic endometriosis stromal cells can play a synergistic role in increasing the VEGF levels, which may be involved in the occurrence and development of endometriosis.

Endometriosis is a chronic gynecological disorder defined as the presence of endometrial tissue within extra‐uterine sites. The proliferation of topic and ectopic endometrial tissue was demonstrated in several trials. In endometriotic tissues and cultured endometriotic cells the expression profile of cell cycle and inflammatory proteins are modified, particularly p27 protein – reduced expression – and IL‐1β – increased expression –, both leading to an increased cell proliferation. The therapeutic use of an adenoviral p27 super‐expression vector (Adp27EGFP) in endometriotic cell cultures induces the cells back to normal proliferative levels. In order to improve the therapeutic potential of this gene therapy we combined it with cell therapy using human umbilical cord mesenchymal stem cells (hUCMSC). The levels of IL‐1β were evaluated in the culture medium of normal and endometriotic endometrial cells transfected or not with Adp27EGFP and Adnull, and treated with hUCMSC. Treatment with hUCMSC leads to increased IL‐1β expression reaching endometriotic levels after 14 days of treatment even in the normal endometrial cells. The therapeutic effects of Adp27EGFP were totally inhibited in the endometriotic cells treated with hUCMSC. These results suggest that the mesenchymal stem cells can have a role in the progression of the endometriosis and cannot be used to treat the disease.

Growing evidence indicates that aberrant methylation is pivotal in the development and progression of endometriosis (EMs). This study explores the relationship between abnormal methylation of the ENPP3 promoter and the pathogenesis of ovarian EMs, focusing on its regulatory effect on ENPP3 expression. We analyzed the methylation levels of ENPP3 in ectopic endometrial tissues from ovarian EMs patients and in normal endometrial tissues from women without EMs. The expression and distribution of ENPP3 were evaluated using RT-qPCR and immunohistochemistry. Transwell assays were conducted to examine the impact of ENPP3 overexpression on the migratory and invasive capabilities of endometrial stromal cells. Our results demonstrated significantly reduced methylation levels at the CpG sites of the ENPP3 promoter region in ectopic endometrial tissues compared to normal endometrial tissues. RT-qPCR findings revealed a marked increase in ENPP3 expression in ovarian EMs tissues relative to endometrial tissues from patients without EMs, and this upregulation was negatively correlated with the methylation levels of the ENPP3 promoter region. Immunohistochemical analyses confirmed elevated ENPP3 expression in the glandular epithelial cells and stroma of ovarian EMs tissues. Furthermore, in vitro experiments showed that overexpressed ENPP3 notably intensified the invasion and migration of endometrial stromal cells. Transcriptome sequencing and functional analyses indicated that the increased ENPP3 expression activated the AKT/mTOR/4EBP1 signaling pathway. In summary, the study suggests that hypomethylation in the ENPP3 promoter region may contribute to the initiation and advancement of ovarian EMs by activating the AKT/mTOR/4EBP1 pathway, supporting the theory that EMs might be an epigenetically regulated disorder.

Adenomyosis, defined as ectopic endometrial tissue within the myometrium, can often be misdiagnosed as multiple uterine leiomyomata or endometrial thickening. We therefore performed a combined mRNA and long noncoding (lnc)RNA microarray and bioinformatic analysis of eutopic and ectopic endometria in women with adenomyosis to better understand its pathogenesis and help in the development of a semi-invasive diagnostic test. A total of 586 mRNAs were increased and 305 mRNAs decreased in the ectopic endometrium of adenomyosis compared with the eutopic endometrium, while 388 lncRNA transcripts were up-regulated and 188 down-regulated in ectopic compared with paired eutopic endometrial tissue. Bioinformatic analysis suggested a series of metabolic and molecular abnormalities in adenomyosis, which have many similarities with endometriosis. Furthermore, our study constitutes the first known report of lncRNA expression patterns in human adenomyosis ectopic and eutopic endometrial tissue.

To investigate the expression of human β-defensin-2 (hBD-2) in the endometrium of patients with endometriosis (EMS) and explore the potential role of hBD-2 in the pathogenesis of EMS. Prospective controlled study. 50 women including EMS patients undergoing laparoscopic ovarian cystectomy and non-EMS patients undergoing hysterectomy for uterine fibroids. Large university teaching hospital. Patients were divided into EMS and non-EMS groups. The gene expressions of hBD-2, interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the endometrial tissues of each group were detected with real-time quantitative polymerase chain reaction (PCR), and hBD-2 protein expression with immunohistochemical method. The gene expression levels of hBD-2, TNF-α, and IL-1β as well as the positive expression rate of hBD-2 protein in the ectopic endometrium of EMS patients were significantly higher than those in the eutopic endometrium of EMS and non-EMS patients (all P < 0.05). Correlation analysis showed that the gene expression levels of hBD-2 in the ectopic and eutopic endometrium of EMS patients were positively correlated with the gene expression levels of IL-1β and TNF-α (P < 0.01). High levels of hBD-2 gene and protein expressions in the ectopic endometrium of EMS patients may be an important contributor in the pathogenesis of EMS. TNF-α and IL-1β may promote the upregulation of hBD-2 expression.

Endometriosis (EM) is known as a common estrogen-dependent chronic inflammatory disease. Elevated levels of Forkhead box L2 (FOXL2) have been observed in uterine diseases, including EM. However, the molecular mechanism of FOXL2 in EM needs to be further illustrated. This study aimed to investigate the regulatory role of FOXL2 in EM rats and isolated ectopic endometrial stromal cells (EC-ESCs). FOXL2 knockdown were designed to evaluate the effects of FOXL2 in EM model rats and EC-ESCs. Hematoxylin-eosin (HE) staining was used to evaluate the pathological morphology of ectopic endometrium. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis and immunohistochemistry (IHC) were applied to detect the expression of FOXL2, EM-related genes, and epithelial to mesenchymal transition-related proteins. The proliferation, migration, invasion, and apoptosis of EC-ESCs were determined by 5-ethynyl-2'-deoxyuridine (EDU) assay, Transwell assay, and flow cytometry. The FOXL2 level was remarkably higher in the ectopic endometrial tissue than that in the normal endometrial tissue. Knockdown of FOXL2 notably improved the pathological morphology of EM in rats, and decreased expression levels of ER-α, ER-ß, and Cyp19a. Additionally, down-regulation of FOXL2 suppressed cells proliferation, migration and invasion, and stimulated more apoptotic cells in EC-ESCs. Besides, FOXL2-small interfering RNA (FOXL2-siRNA) treatment resulted in enhanced cleaved-Caspase3 protein expression and cleaved-Caspase3/Caspase3 ratio in EC-ESCs. FOXL2 participates in the occurrence and development of EM through promoting epithelial-mesenchymal transition procession and enhancing the migration and invasion of EC-ESCs, suggesting that FOXL2 may be a new therapeutic target for the EM therapy.

HOXA10 expression in ectopic endometrial tissue

Expression and possible implication of growth hormone–releasing hormone receptor splice variant 1 in endometriosis

How does hypoxia promote growth of lesions in the development of endometriosis? Hypoxia induces the epithelial-mesenchymal transition (EMT) of endometrial cells, resulting in changes in cellular characteristics, which may be a prerequisite for the establishment of endometriotic lesions. Up-regulated hypoxia-inducible factor 1α (HIF-1α) has recently been found in ectopic endometrial lesions. There is increasing evidence that EMT, in which epithelial cells acquire mesenchymal and migratory properties, may also play a role in the establishment of the disease. EMT induced by HIF-1α has been reported to play a role in the development of many tumor types. We investigated expression changes of N-cadherin, E-cadherin, β-catenin, HIF-1α and vimentin using immunohistochemistry in normal, eutopic and ectopic endometria. Endometrial tissues from 96 additional females without related pathology were collected, and these tissues were subjected to subsequent primary cell culture for further experiments. The expression of N-cadherin, E-cadherin, β-catenin, HIF-1α and vimentin in 20 normal, 21 eutopic and 21 ectopic endometrial samples was assessed by immunohistochemistry. Human primary endometrial epithelial cells were isolated from 96 normal endometrial tissues. Times for hypoxia treatment for western blot analysis were 1, 2, 4 and 8 h, for transwell experiments was 48 h. Ishikawa cells were used instead of primary endometrial epithelial cells for transfection and part of the transwell experiments. The impact of hypoxia on invasion was evaluated by transwell assays. Overexpression and inhibition of HIF-1α were achieved by transfection of pG/CMV/HIF-1α/IRES/EGFP and MCS-shHIF-1α-EGFP-IRES plasmids separately. The effect of hypoxia, overexpression and knockdown of HIF-1α on hypoxia-induced changes of EMT markers and β-catenin were analyzed by western blot and reverse transcriptase polymerase chain reaction (RT-PCR). Overexpression of HIF-1α and changes associated with EMT were observed in normal, eutopic and ectopic endometrial tissues. Primary cultured human endometrial epithelial cells responded to hypoxia with classic EMT changes (fibroblastoid phenotype, increased expression of snail family zinc finger 1, β-catenin and mesenchymal markers, and decreased expression of E-cadherin) and increased invasiveness. The decreased invasiveness of Ishikawa cells by knockdown of HIF-1α was observed under hypoxic conditions. While up-regulation of HIF-1α induced changes characteristic of EMT, down-regulation of HIF-1α had the opposite effect. Statistical significance was defined as P < 0.05. A weakness of this study is the relatively small sample size for immunohistochemistry. Ishikawa cells were used instead of primary endometrial epithelial cells for transfection and part of the transwell experiments. Hypoxia-stabilized HIF-1α may play an important role in the invasion of endometrial cells in ectopic endometrial lesions, and it may induce EMT in the development of endometriosis. HIF-1α may be a new and important target for the endometriosis treatment. This study was funded by the National Nature Science Foundation of China (grant number 81170545 and 81471439). The authors declare no competing interests.

Expression of dioxin-related transactivating factors and target genes in human eutopic endometrial and endometriotic tissues

Inflammation and immune dysregulation play critical roles in Kawasaki disease (KD) pathogenesis, yet specific biomarkers and immune signatures remain elusive. This study aims to identify key biomarkers and characterize immune cell infiltration scores in KD using bioinformatic approaches. The GSE73461 dataset, downloaded from the Gene Expression Omnibus (GEO) database, includes 78 KD patients and 55 normal controls collected by Imperial College London from 2015 to 2023, and was analyzed to identify differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed significant involvement of these DEGs in acute inflammatory responses, plasma membrane components, PI3K-Akt signaling, and cytokine interactions. Protein-protein interaction (PPI) network was constructed, and five candidate hub genes (AURKB, BUB1, CCL2, IL-4, and TOP2A) were identified. Immune cell infiltration analysis gusing the XCell algorithm showed increased levels of Monocytes, neutrophils, and other immune cells in KD, while B cells and T cells were decreased. Correlation analysis indicated that these candidate hub genes are associated with immune dysregulation and inflammation in KD. These findings provide potential diagnostic biomarkers and therapeutic targets for KD, warranting further validation in larger studies.

Endometriosis is a debilitating disease characterized by chronic inflammation. The transporter multidrug resistance-associated protein 4 (MRP4/ABCC4) is expressed in human endometrial tissue; it is overexpressed in ectopic endometrial tissue, and is modulated by the anti-inflammatory lipid Lipoxin A4 (LXA4). Recently, it was demonstrated that aspirin induces platelet MRP4 over-expression, through genomic modulation in megakaryocytes. Since patients with endometriosis frequently use aspirin or other non-aspirin Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), the aim of this study was to verify whether aspirin and other NSAIDs enhance MRP4 expression in 12Z human endometriotic epithelial cells and whether this was peroxisome proliferator-activated receptor alpha (PPARa) dependent. MRP4 and PPARa expression was analyzed by Q-RT-PCR using TaqMan® Master Mix and TaqMan® Assay Reagents (Life Technologies, Monza, Italy) and Western blot. In 12Z cells, aspirin and other NSAIDs enhanced MRP4 mRNA and protein expression; these treatments also induced PPARa expression. Aspirin and diclofenac-induced increases in MRP4 expression were not observed in cells where PPARa was knocked down using siRNA. NSAIDs-induced MRP4 expression was correlated with augmented PGE2 secretion, indicating functional relevance. MRP4 expression was increased in cells treated with NSAIDs and the nuclear receptor PPARa is involved. Elevated PGE2 levels in cell supernatants correlate with its increased transport by MRP4 after NSAID treatment. More importantly, we provide evidence that in endometriotic epithelial cells aspirin and non-aspirin NSAIDs treatments alter gene expression.

Glutathione peroxidase 3 (GPX3), an important part of the anti-peroxidase system, plays a crucial role in the removal of Reactive oxygen species (ROS) and maintenance of the homeostasis of the human body. Current research indicates that the abnormal expression of GPX3 is significantly associated with the occurrence and development of malignant tumors. In malignant tumors, the high methylation of GPX3 promoter was reported to suppress the expression of GPX3, thus affecting the occurrence and development of tumors. However, the regulation mechanism of GPX3 expression is unclear and under-research, that will elucidate the mechanism of tumor occurrence and development, provide a theoretical basis and new strategies for individual precision treatment to patients with malignant tumor. Key words: Glutathione peroxidase 3; Malignant tumor; Reactive oxygen species; Methylation

BackgroundImmune system dysregulation plays a critical role in aortic valve calcification (AVC) and metabolic syndrome (MS) pathogenesis. The study aimed to identify pivotal diagnostic candidate genes for AVC patients with MS.MethodsWe obtained three AVC and one MS dataset from the gene expression omnibus (GEO) database. Identification of differentially expressed genes (DEGs) and module gene via Limma and weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, protein–protein interaction (PPI) network construction, and machine learning algorithms (least absolute shrinkage and selection operator (LASSO) regression and random forest) were used to identify candidate immune-associated hub genes for diagnosing AVC with MS. To assess the diagnostic value, the nomogram and receiver operating characteristic (ROC) curve were developed. Finally, immune cell infiltration was created to investigate immune cell dysregulation in AVC.ResultsThe merged AVC dataset included 587 DEGs, and 1,438 module genes were screened out in MS. MS DEGs were primarily enriched in immune regulation. The intersection of DEGs for AVC and module genes for MS was 50, which were mainly enriched in the immune system as well. Following the development of the PPI network, 26 node genes were filtered, and five candidate hub genes were chosen for nomogram building and diagnostic value evaluation after machine learning. The nomogram and all five candidate hub genes had high diagnostic values (area under the curve from 0.732 to 0.982). Various dysregulated immune cells were observed as well.ConclusionFive immune-associated candidate hub genes (BEX2, SPRY2, CXCL16, ITGAL, and MORF4L2) were identified, and the nomogram was constructed for AVC with MS diagnosis. Our study could provide potential peripheral blood diagnostic candidate genes for AVC in MS patients.