Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

Background Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women. Methods Ishikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting. Results Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers. Conclusions EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

Dysregulation of X-Ray Repair Cross Complementing 4 Expression in the Eutopic Endometrium of Women with Endometriosis

Recent data suggest that the DNA damage response (DDR) is altered in the eutopic endometrium (EE) of women with endometriosis and this probably ensues in response to higher DNA damage encountered by the EE in endometriosis. DDR operates in a tissue-specific manner and involves different pathways depending on the type of DNA lesions. Among these pathways, the non-homologous end joining (NHEJ) pathway plays a critical role in the repair of double-stranded DNA breaks. The present study was undertaken to explore whether NHEJ is affected in the EE of women with endometriosis. Towards this, we focused on the X-Ray Repair Cross-Complementing 4 (XRCC4) protein, one of the core components of the NHEJ pathway. Endometrial XRCC4 protein levels in the mid-proliferative phase were found significantly (p<0.05) downregulated in women with endometriosis, compared to control women. Investigation of a microarray-based largest dataset in the GEO database (GSE51981) revealed a similar trend at the transcript level in the EE of women with endometriosis, compared to control women. Further in-vitro studies were undertaken to explore the effects of H2O2-induced oxidative stress on DNA damage, as assessed by γ-H2AFX and 8-hydroxy-2’-deoxyguanosine (8-OHdG) immunolocalization, and XRCC4 protein levels in endometrial stromal (ThESCs) and epithelial (Ishikawa) cells. A significant decrease in XRCC4 protein levels and significantly higher localization of γ-H2AFX and 8-OHdG were evident in ThESCs and Ishikawa cells experiencing oxidative stress. Overall, the study demonstrates that the endometrial XRCC4 expression is dysregulated in women with endometriosis and this could be due to higher oxidative stress in endometriosis.

Insulin exposed endometrial epithelial cells cultured in a microfluidic device alters transcripts involved in translation that may contribute to reduced implantation capacity of the endometrium.

Obesity is a rapidly growing public health issue among women of reproductive age. It is also associated with decreased reproductive function including implantation failure. Implantation failure can result from a myriad of factors including impaired gametes and endometrial dysfunction. The mechanisms of how obesity-related hyperinsulinaemia disrupts endometrial function and implantation are poorly understood. Our study aims to investigate potential mechanisms by which insulin alters endometrial transcript expression, which may affect endometrial receptivity. Ishikawa cells mimicking human endometrial epithelium were seeded into a microfluidics organ-on-chip device to produce an in vitro endometrium. Syringe pump was attached to the microfluidics device to deliver three varying treatments into Ishikawa cells: 1) media control 2) vehicle control (PBS acidified to pH3 with acetic acid) 3) Insulin (2mg/mL) at a constant flow rate of 1uL/min for 24 hours to mimic secretion in vivo. Three biological replicates were obtained. Insulin-induced transcriptomic response of the in vitro endometrium was quantified via RNA sequencing, and subsequently analysed using DAVID and Webgestalt to identify Gene Ontology (GO) terms and signalling pathways. A Total of 29 transcripts showed differential expression levels across two comparison groups (control v vehicle control; vehicle control v insulin). There were nine transcripts significantly differentially expressed in vehicle control v insulin group (p<0.05). Functional annotation analysis of transcripts altered by insulin (n=9) identified three significantly enriched GO terms: SRP-dependent cotranslational protein targeting to membrane, poly(A) binding, and RNA binding (p<0.05). Over-representation analysis found three significantly enriched signalling pathways relating to insulin-induced transcriptomic response: protein export, glutathione metabolism, and ribosome pathways (p<0.05). Insulin-induced dysregulation of biological functions and pathways highlight potential mechanisms by which high insulin concentrations within maternal circulation may perturb endometrial receptivity.

TET3- and OGT-Dependent Expression of Genes Involved in Epithelial-Mesenchymal Transition in Endometrial Cancer

TET3 is a member of the TET (ten-eleven translocation) proteins family that catalyzes the conversion of the 5-methylcytosine into 5-hydroxymethylcytosine. TET proteins can also affect chromatin modifications and gene expression independently of their enzymatic activity via interactions with other proteins. O-GlcNAc transferase (OGT), the enzyme responsible for modification of proteins via binding of N-acetylglucosamine residues, is one of the proteins whose action may be dependent on TET3. Here, we demonstrated that in endometrial cancer cells both TET3 and OGT affected the expression of genes involved in epithelial to mesenchymal transition (EMT), i.e., FOXC1, TWIST1, and ZEB1. OGT overexpression was caused by an increase in TWIST1 and ZEB1 levels in HEC-1A and Ishikawa cells, which was associated with increased O-GlcNAcylation of histone H2B and trimethylation of H3K4. The TET3 had the opposite effect on gene expressions and histone modifications. OGT and TET3 differently affected FOXC1 expression and the migratory potential of HEC-1A and Ishikawa cells. Analysis of gene expressions in cancer tissue samples from endometrial cancer patients confirmed the association between OGT or TET3 and EMT genes. Our results contribute to the knowledge of the role of the TET3/OGT relationship in the complex mechanism supporting endometrial cancer progression.

Increased METTL3-mediated m6A methylation inhibits embryo implantation by repressing HOXA10 expression in recurrent implantation failure

Background Recurrent implantation failure (RIF) is a major limitation of assisted reproductive technology, which is associated with impaired endometrial receptivity. Although N6-methyladenosine (m6A) has been demonstrated to be involved in various biological processes, its potential role in the endometrium of women with RIF has been poorly studied. Methods Global m6A levels and major m6A methyltransferases/demethylases mRNA levels in mid-secretory endometrium from normal and RIF women were examined by colorimetric m6A quantification strategy and quantitative real-time PCR, respectively. The effects of METTL3-mediated m6A modification on embryo attachment were evaluated by an vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids, and the expression levels of homeo box A10 (HOXA10, a well-characterized marker of endometrial receptivity) and its downstream targets were evaluated by quantitative real-time PCR and Western blotting in METTL3-overexpressing Ishikawa cells. The molecular mechanism for METTL3 regulating HOXA10 expression was determined by methylated RNA immunoprecipitation assay and transcription inhibition assay. Results Global m6A methylation and METTL3 expression were significantly increased in the endometrial tissues from women with RIF compared with the controls. Overexpression of METTL3 in Ishikawa cells significantly decreased the ration of BeWo spheroid attachment, and inhibited HOXA10 expression with downstream decreased β3-integrin and increased empty spiracles homeobox 2 expression. METTL3 catalyzed the m6A methylation of HOXA10 mRNA and contributed to its decay with shortened half-life. Enforced expression of HOXA10 in Ishikawa cells effectively rescued the impairment of METTL3 on the embryo attachment in vitro. Conclusion Increased METTL3-mediated m6A modification represents an adverse impact on embryo implantation by inhibiting HOXA10 expression, contributing to the pathogenesis of RIF.

Hsa_Circ_0001860 Promotes Smad7 to Enhance MPA Resistance in Endometrial Cancer via miR-520h

Background: Medroxyprogesterone acetate (MPA) is one of the most commonly prescribed progestin for the treatment of endometrial cancer (EC). Despite initial benefits, many patients ultimately develop progesterone resistance. Circular RNA (circRNA) is a kind of noncoding RNA, contributing greatly to the development of human tumor. However, the role of circular RNA in MPA resistance is unknown.Methods: We explored the expression profile of circRNAs in Ishikawa cells treated with (ISK/MPA) or without MPA (ISK) by RNA sequencing, and identified a key circRNA, hsa_circ_0001860. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify its expression in MPA-resistant cell lines and tissues. CCK8, Transwell, and flow cytometry were used to evaluate the functional roles of hsa_circ_0001860 in MPA resistance. The interaction between hsa_circ_0001860 and miR-520 h was confirmed by bioinformatics analysis, luciferase reporter assay, and RNA pull-down assay.Results: The expression of hsa_circ_0001860 was significantly downregulated in MPA-resistant cell lines and tissues, and negatively correlated with lymph node metastasis and histological grade of EC. Functional analysis showed that hsa_circ_0001860 knockdown by short hairpin RNA (shRNA) promoted the proliferation, inhibited the apoptosis of Ishikawa cells, and promoted the migration and invasion of Ishikawa cells treated with MPA. Mechanistically, hsa_circ_0001860 promoted Smad7 expression by sponging miR-520 h.Conclusion: Hsa_circ_0001860 plays an important role in the development of MPA resistance in EC through miR-520h/Smad7 axis, and it could be targeted to reverse the MPA resistance in endometrial cancer.

Knockdown of LMTK3 in the Endometrioid Adenocarcinoma Cell Line Ishikawa: Inhibition of Growth and Estrogen Receptor α

ObjectiveThe curative effect of high-efficiency progesterone and other therapeutic drugs for endometrioid adenocarcinoma patients with preservation of reproductive capacity has not been satisfactory so far. Novel therapeutic drugs need to be explored.MethodsWe investigated the cytoplastic and nuclear expression levels of LMTK3 between endometrioid adenocarcinoma tissues and adjacent endometrial tissues by immunohistochemistry. We detected the effects of LMTK3 on cell viability of Ishikawa cells by CCK-8. We detected the effects of LMTK3 on cell cycle and apoptosis of Ishikawa cells by flow cytometry. We also detected the effects of LMTK3 knockdown on mRNA and protein levels of ERα by qRT-PCR and western blotting, respectively. We also used the cBioPortal online database to analyze the coexpression of LMTK3 and ESR1 in 1647 UCEC samples.ResultsWe used TMAs to identify that LMTK3 was mainly detected in the cytoplasm of endometrioid tissues, and cytoplasmic LMTK3 expression in endometrioid tissues was higher than that in adjacent endometrial tissues (P &lt; 0.05). LMTK3 knockdown decreased the proliferation of Ishikawa cells through decreasing cell viability (P &lt; 0.01), increasing G1 (P &lt; 0.001) arrest, and promoting apoptosis (P &lt; 0.01). There was a positive correlation between the mRNA expression levels of LMTK3 and ESR1 (Spearman: P=2.011e-5, R=0.13; Pearson: P=7.18e-8, R=0.17). Knockdown of LMTK3 also reduced the mRNA (P &lt; 0.001) and protein (P &lt; 0.001) levels of ERα.ConclusionsInhibitors of LMTK3 may be a possible future treatment for ERα and LMTK3 highly expressed endometrioid adenocarcinoma following appropriate studies.

The Effects of the Trifolium Pratense L. Extract on the Leukemia Inhibitory Factor and its Receptor in The Endometrial Epithelial Cell Line

Isoflavones have attracted much notice due to their health advantages; however, a comprehensive understanding of the effects of isoflavones on endometrium biology remains undiscovered. The expression and deficiency of leukemia inhibitory factor (LIF) and LIF receptor (LIFR) has been shown to be involved in multiple implantations failures in female infertility. Mechanisms implicated in the failure of implantations require further researches, thus our aim is to investigate the effect of the Trifolium pratense L. isoflavone extract with abundant formononetin content on implantation through assessing LIF and LIFR expressions. The Ishikawa cells were cultured with 20, 30, and 40 µg/mL concentrations of Trifolium pratense L. isoflavone extracts for 24 h and detected staining intensity of LIF and LIFR by immunocytochemistry and immunofluorescence staining using image analysis software. As compared with the control and 20 µg/mL Trifolium pratense L. groups, the staining intensity of LIF and LIFR in 30 and 40 µg/mL Trifolium pratense L. groups were significantly increased ( P < .0001). Our findings suggest that Trifolium pratense L. isoflavone extract may alter the endometrium expression of LIF and LIFR in the human endometrial adenocarcinoma cell line.

GRIM19 is involved in WT1 expression and epithelial-to-mesenchymal transition in adenomyotic lesions

The epithelial-to-mesenchymal transition may play a role in adenomyosis. GRIM19 expression is downregulated in adenomyotic lesions, and the effects of this downregulation in adenomyosis remain relatively unclear. We aimed to explore whether aberrant GRIM19 expression is associated with the epithelial-to-mesenchymal transition in adenomyosis. In this study, expression of both GRIM19 and WT1 was low, and epithelial-to-mesenchymal transition, which included significant changes in CDH1, CDH2 and KRT8 expression, occurred in adenomyotic lesions, as confirmed by western blotting and quantitative real-time polymerase chain reaction. We provided novel insights into WT1 expression in adenomyosis, revealing that WT1 expression was increased in the endometrial glands of adenomyotic lesions by immunohistochemistry. In vitro, knockdown of GRIM19 expression by small-interfering RNA promoted the proliferation, migration and invasion of Ishikawa cells, as measured by Cell Counting Kit-8, wound healing assay and Transwell assays. Western blotting and quantitative real-time polymerase chain reaction confirmed that WT1 expression increased and epithelial-to-mesenchymal transition was induced, including upregulation of CDH2 and downregulation of CDH1 and KRT8 after transfecting the GRIM19 small interfering RNA to Ishikawa cells. Furthermore, WT1 expression was upregulated and epithelial-to-mesenchymal transition was observed, including downregulation of CDH1 and KRT8 in GRIM19 gene-knockdown mice. Upregulation of WT1 expression in the endometrial glands of GRIM19 knockdown mice was also verified by immunohistochemistry. Taken together, these results reveal that low expression of GRIM19 in adenomyosis may upregulate WT1 expression and induce epithelial-to-mesenchymal transition in the endometria, providing new insights into the pathogenesis of adenomyosis.