Normal Endometrial Epithelial Cells Research Articles

MiR-200a-3p promotes the malignancy of endometrial carcinoma through negative regulation of epithelial-mesenchymal transition

BackgroundmiR-200a-3p is involved in the progression of malignant behavior in various tumors, and its mechanism of action in endometrial cancer is speculated to be related to epithelial-mesenchymal transition (EMT). Therefore, this study explored the metastatic mechanism of miR-200a-3p and EMT in endometrial cancer, with the aim of identifying potential therapeutic targets.MethodsqRT-PCR was used to analyze miR-200a-3p expression in HEC-1B and Ishikawa cell lines. The cell proliferation assay, transwell assay, and cell scratch test were used to assess changes in the malignant phenotypes of cells after regulating miR-200a-3p expression. Changes in EMT-related protein zinc finger E-box binding homeobox 1 (ZEB1) were detected after regulating miR-200a-3p expression. An endometrial carcinoma transplantation mouse tumor model was constructed, and multiple EMT-related proteins were examined.ResultsThe expression of miR-200a-3p and ZEB1 in the endometrial cancer cell lines was higher than in normal endometrial epithelial cell lines (P < 0.05). After silencing miR-200a-3p, the expression of EMT-related protein ZEB1 increased, indicating a negative correlation. Simultaneously, the proliferation, invasion, and metastasis of endometrial cancer cells were significantly enhanced. After miR-200a-3p overexpression, the corresponding malignant phenotype was reversed (P < 0.05). In in vivo experiments, the degree of tumor malignancy and the expression level of EMT-related proteins were significantly reduced in the miR-200a-3p mimic group (P < 0.05).ConclusionThis study found that miR-200a-3p is a promising target, regulating the EMT process and promoting endometrial cancer progression.

Surface-Enhanced Raman Spectroscopy-Based Detection of EMT-Related Targets in Endometrial Cancer: Potential for Diagnosis and Prognostic Prediction.

Epithelial-mesenchymal transformation (EMT) is one of the important mechanisms of malignancy in endometrial cancer, and detection of EMT targets is a key challenge to explore the mechanism of endometrial carcinoma (EC) malignancy and discover novel therapeutic targets. This study attempts to use surface-enhanced Raman spectroscopy (SERS), a highly sensitive, ultrafast, and highly specific analytical technology, to rapidly detect microRNA-200a-3p and ZEB1 in endometrial cancer cell lines. The silver nanoparticles were decorated with iodine and calcium ions, can capture the SERS fingerprints of microRNA-200a-3p and ZEB1 protein, and effectively avoid the interference of impurity signals. At the same time, the method has high sensitivity for the detection of the above EMT targets, and the lowest detection limits for microRNA-200a-3p and ZEB1 are 4.5 pmol/mL and 10 ng/mL, respectively. At the lowest detection concentration, the method still has high stability. In addition, principal component analysis can not only identify microRNA-200a-3p and ZEB1 protein from a variety of EMT-associated microRNA and proteins but also identify them in the total RNA and total protein of endometrial cancer cell lines and normal endometrial epithelial cell lines. This study modified silver nanoparticles with iodine and calcium ions and for the first time captured the fingerprints of EMT-related targets microRNA-200a-3p and ZEB1 at the same time without label, and the method has high sensitivity and stability. This SERS-based method has immense potential for elucidating the molecular mechanisms of EMT-related EC, as well as identifying biomarkers for malignant degree and prognosis prediction.

Fanconi anemia complementation group D2 promotes sensitivity of endometrial cancer cells to chemotherapeutic agents by inhibiting the ferroptosis pathway

BackgroundResistance can develop during treatment of advanced endometrial cancer (EC), leading to unsatisfactory results. Fanconi anemia complementation group D2 (Fancd2) has been shown to be closely related to drug resistance in cancer cells. Therefore, this study was designed to explore the correlation of Fancd2 with EC resistance and the mechanism of Fancd2.MethodsReal-time quantitative PCR (RT-qPCR) was used to detect the expression of Fancd2 in EC tissues and cells. EC cells (Ishikawa) and paclitaxel-resistant EC cells (Ishikawa/TAX) were transfected to knock down Fancd2. In addition, the ferroptosis inhibitor Ferrostatin-1 was adopted to treat Ishikawa/TAX cells. The sensitivity of cancer cells to chemotherapeutic agents was observed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and inhibitory concentration (IC)50 was calculated. Reactive oxygen species (ROS) levels were measured by flow cytometry, the activity of malondialdehyde (MDA) and the levels of glutathione (GSH) and Fe2+ in cells were detected by corresponding kits, and protein expression of solute farrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) was obtained through western blot.ResultsCompared with the normal tissues and endometrial epithelial cells, Fancd2 expression was significantly increased in EC tissues and Ishikawa cells, respectively. After knock-down of Fancd2, Ishikawa cells showed significantly increased sensitivity to chemotherapeutic agents. Besides, compared with Ishikawa cells, the levels of ROS, the activity of MDA, and the levels of GSH and Fe2+ were significantly decreased in Ishikawa/TAX cells, while the expression levels of SLC7A11 and GPX4 were significantly increased. Knock-down of Fancd2 significantly increased the ferroptosis levels in Ishikawa/TAX cells, but this effect could be reversed by Ferrostatin-1.ConclusionFancd2 increases drug resistance in EC cells by inhibiting the cellular ferroptosis pathway.

Abstract 1214: Establishment and characterization of reversibly immortalized endometrial and ovarian epithelial cell lines using Sendai virus

Abstract Objective: We aimed to establish reversibly immortalized cell lines from human uterine and ovary cells using the Sendai virus (SeV) vector. The immortalized cells derive from normal and benign ovarian epithelial cells and endometrial epithelial cells. Furthermore, we sought to elucidate the mechanisms of carcinogenesis using the immortalized cell lines. Methods: Cells were collected at the time of surgery after obtaining patient consent. The cells used in this study were as follows: ovarian epithelial cells (normal epithelium, Ov n; normal epithelium with germline BRCA1 or BRCA2 mutation, Ov BRCA1 2; ovarian endometrioma, Ov endo; mucinous cystadenoma; Ov m), normal fallopian tube (FT) cells, and endometrial epithelium (normal epithelium, Em n). These cells were infected with temperature-sensitive SeV vectors carrying three immortalization genes, Bmi-1, hTERT, and SV40T.The presence of infection was confirmed through Green Fluorescent Protein (GFP) and Orange Fluorescent Protein (OFP). Immunoreactivity to the anti-human EpCAM antibody (a marker derived from epithelial carcinoma) in each SeV-infected cell was confirmed through flow cytometry. QH and multicolor FISH staining were performed for karyotyping of metaphase chromosomes in each cell line to determine chromosome number and structural abnormalities. Human transcriptome sequencing analysis was performed with NovaSeq 6000 (Illumina) using total RNA from each cell line. Some genes that showed significant expression in each cell line were subjected to real-time PCR (RT-PCR). Results: We established the immortalized cell lines from human uterine and ovarian tissues. SeV-infected cells exhibited GFP and OFP fluorescence, while non-infected cells did not. SeV infection allowed all primary cell lines to grow for 25 or more passages, while non-infected SeV cells lacked the proliferative capacity and showed senescence-like morphology. SeV-infected cells senesced in a temperature-dependent manner. Ov n SeV- infected cells ion causedshowed a small increase in chromosome structural abnormalities. But, Ov BRCA1 and 2 SeV-infected cells showed larger than thatand . Llong-term passaged cells did not show immune response to anti-human EpCAM antibodies in normal cells. Eleven Three genes were predominantly expressed in Ov BRCA1 and Ov BRCA2 cells and were not expressed in Ov n cells. SomeTwo out of ththe three genesem were found to be predominantly expressed in Ov endo cells compared to the expression in the Ovn cell line. Furthermore, RT-PCR results also indicated substantially higher expression of two genes in Ov BRCA1/2 cells and in Ov endo cells compared to the expression in Ov n cells. Conclusion: We succeeded in the reversible immortalization of endometrial and ovarian epithelial cells by using SeV infection. We identified several candidate genes that may be involved in the oncogenic mechanism of ovarian cancer associated with endometriosis or germline BRCA1 and BRCA2. Citation Format: Masayo Okawa, Hiroaki Komatsu, Kohei Hikino, Yuki Iida, Masayo Hosokawa, Mayumi Sawada, Akiko Kudoh, Jun Chikumi, Shinya Sato, Genki Hichiwa, Yasuhiro Kazuki, Kanako Kazuki, Fuminori Taniguchi, Mitsuo Oshimura, Tasuku Harada. Establishment and characterization of reversibly immortalized endometrial and ovarian epithelial cell lines using Sendai virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1214.

FHL1 mediates HOXA10 deacetylation via SIRT2 to enhance blastocyst-epithelial adhesion

Recurrent implantation failure (RIF) is a rather thorny problem in the clinical practice of assisted reproductive technology. Due to the complex aetiology of RIF, its pathogenesis is far from fully understood, and there is no effective treatment available. Here, We explored the regulatory mechanism of the four half-domains of LIM domain 1 (FHL1), which is significantly downregulated in the endometrium of RIF patients, in blastocyst-epithelial adhesion. Indeed, FHL1 expression was dramatically increased in normal female mid-secretory endometrial epithelial cells and was abnormally reduced in RIF patients. Furthermore, FHL1 overexpression promoted blastocyst-epithelial adhesion, and interfering with FHL1 expression in the mouse uterus significantly inhibited embryo implantation. Mechanistically, FHL1 did not regulate HOXA10 mRNA expression but increased HOXA10 protein stability and activated HOXA10, thereby promoting its regulation of downstream gene expression and the β3 integrin/FAK pathway. Meanwhile, FHL1 regulates HOXA10 function by increasing HOXA10 deacetylation through enhanced binding of HOXA10 and SIRT2. SIRT2-specific inhibitors can significantly inhibit this effect. In the endometrial epithelial cells of RIF patients, the correlation between FHL1 and HOXA10 and its downstream target genes has also been verified. Finally, our data indicated FHL1 is a regulatory molecule that promotes blastocyst-epithelial adhesion. Altogether, downstream dysfunction due to aberrant FHL1 expression is an important molecular basis for embryo implantation failure in patients with RIF and to provide new potential therapeutic targets.

CircWDR26 regulates endometrial carcinoma progression via miR-212-3p-mediated typing genes MSH2

BackgroundCircular RNAs (circRNA) are important in mediating tumor progression, but their roles in endometrial carcinoma (EC) are not fully understood yet. Many circRNAs are dysregulated and may contribute to EC progression. The functions of circWDR26 in EC remain unknown.MethodsThe expression of circWDR26 in EC and adjacent normal tissues, and cell lines was determined by qPCR. The proliferation, apoptosis, migration, and invasion of EC cells was examined by CCK-8 assay, flow cytometry, wound healing assay and Transwell assay. The interaction between circWDR26, MSH2 and miR-212-3p was determined by luciferase assay. EC cells were inoculated into nude mice and tumor burden was determined by measuring tumor dimensions, size, and weight. The proliferative marker Ki67 in EC tissue was determined by immunohistochemistry.ResultsThe expression of circWDR26 in EC tissues or cell lines was higher than in the normal tissue or endometrial epithelial cells. Downregulation of circWDR26 resulted in attenuated proliferation, increased apoptosis, reduced migration and invasion of EC cells. Mechanistically, circWDR26 targeted and suppressed the expression of miR-212-3p. We further found that MSH2 was the novel target of miR-212-3p and was upregulated by circWDR26 via inhibiting miR-212-3p. In vivo experiment demonstrated that circWDR26 was essential for EC tumor growth.ConclusioncircWDR26 promoted EC progression by regulating miR-212-3p/MSH2 axis and provided novel insights into anti-cancer treatment.

P-380 Single-cell transcriptome analysis reveales that expression changes of the endometrium in repeated implantation failure are altered by HPV-mediated CXCL chemokine secretion

Abstract Study question What are the mechanisms and molecular expression patterns of reduced endometrial receptivity in repeated implantation failure (RIF) after human papillomavirus (HPV) infection? Summary answer The single-cell transcriptomic analysis identifies the expression changes of endometrium in RIF via HPV-mediated CXCL chemokines secretion in single-cell resolution. What is known already Regardless of the advance of in vitro fertilization (IVF), RIF is still a formidable challenge for couples and physicians in clinical treatment. In infertile couples, a reduction in natural and assisted cumulative pregnancy rate and an increase in miscarriage rate are related to the HPV infection. Study design, size, duration Cross-sectional clinical studies with 322 infertile couples undergoing IVF were integrated to demonstrate the associations between HPV infection and reproductive outcomes (pregnancy rate and miscarriage). Descriptive analysis of single-cell transcriptome data of uteruses, and transcriptome profiles of mid-secretory endometrium from 16 healthy fertile women and 38 repeated IVF failure women were analyzed to identify the expression patterns of endometrium in RIF. In vitro assays were used to validate the expression patterns in endometrium. Participants/materials, setting, methods 322 infertile couples, single-cell transcriptome data of uteruses (human and mouse), and transcriptome profiles of endometrium (16 normal vs. 38 RIF) were used to analyze the association between HPV infection and reduced endometrial receptivity. HPV genes (E1, E2, E4, and E5) were transfected into a human normal endometrial epithelial cell line (hEM3), and immunohistochemistry, Westerns, quantitative PCR were used to validate the changes of CXCL chemokines in the endometrium in vitro. Main results and the role of chance Integrated cross-sectional studies demonstrate that HPV+ women exhibit a decreased pregnancy rate (83.09%) as compared with HPV- women (55.17%, P &amp;lt;0.001), and a higher miscarriage rate (62.5% vs. 16.7%, P &amp;lt;0.001) and the relative risk of spontaneous abortion (odd ratio=2.84, P &amp;lt;0.0001) were observed in HPV+ women. Transcriptome profiling analysis identified the enrichment of the processes related to viral protein interaction with cytokine and cytokine receptor and cytokine-cytokine receptor interaction, especially in the CXCL chemokine family. Further analysis of single-cell transcriptome demonstrated that the changed expression patterns were associated with endometrial epithelial cells and immune cells, including macrophage dendritic cells, monocytes, and granulocytes. Moreover, in vitro assays validated the HPV-mediated CXCL chemokines secretion, which played the role in recruiting immune cells. Limitations, reasons for caution The current findings are based on the single-cell profiling analysis in normal endometrium. In addition, the in vivo response of the HPV infection may differ from the in vitro assay, which should be validated in the HPV infection couples. Wider implications of the findings Our study demonstrated the expression changes of endometrium in RIF via HPV-mediated CXCL chemokines secretion, which provided insight into the mechanisms of HPV-induced reduced endometrial receptivity in single-cell resolution. Trial registration number not applicable

Effect of Protease Activated Receptor‐2 antagonist in the transition from endometrium to endometriosis and ovarian cancer

Endogenous proteases are associated with the ovulation process and inflammation, among other human physiological processes. Still their role in the transition from normal tissue to cancer development remains uncertain. For example, the Protease activated receptor 2 (PAR‐2) is a G Protein Couple receptor expressed in mostly in epithelial cells, including the female reproductive system. Previous work from our lab showed that the PAR‐2 antagonist‐FSLLRY‐NH2 inhibited the cell‐mediated inflammation in the airways. The aim of this study is to evaluate the effect of the PAR‐2 antagonist when a normal cell progresses to a cancerous one, using trypsin as a gold standard enzyme for Par‐2 activation. In this case, the transition from normal endometrium, to endometriosis, ovarian. We hypothesized that PAR‐2 antagonist will decrease the activation of its receptor reducing the transition to endometriosis and ovarian cancer. To test this, we performed western blot to determine PAR‐2 expression in endometriosis (12Z) cells, human endometrial stromal cells (HESC) cells, ovarian cancer (A2780) cells, and breast cancer (MCF‐7) cells. In addition, we quantified the activation of PAR‐2 receptor using a Fluorogenic intracellular calcium assay in human normal endometrial (uterine) epithelial cells (HEUEC), ovarian cancer (Caov‐3), and MCF‐7 cells as internal positive control. We also performed the BrdU proliferation assay on these cells. Our results showed a higher expression (p&lt;0.05) of PAR‐2 in A2780 cells (ovarian aggressive cancer) when compared with the other cells lines. The intracellular calcium assay resulted in a significant reduction (p&lt;0.05) once exposed to the PAR‐2 antagonist in the HEUEC cells and Caov‐3 when compared to cells treated with trypsin (p&lt;0.05). In contrast, there was not significant difference in the MCF‐7 cell line upon exposure to treatments. We observed a significant decrease in MCF‐7 proliferation when treated with the PAR‐2 antagonist in comparison to trypsin treatment. Our results suggested that PAR‐2 antagonist can be used as treatment or adjunct therapy for specific female reproductive cancers. Next steps of this study will include the active full‐length PAR‐2 recombinant and test other concentrations of the antagonist to determine the LD50 in the selected cells. Proliferating markers will be measured for all the previous cell lines as well for endometrial cancer.

Long non-coding RNA NNT-AS1 positively regulates NPM1 expression to affect the proliferation of estrogen-mediated endometrial carcinoma by interacting.

Objective: This study aims to investigate the mechanism of long non-coding RNA NNT-AS1 in the proliferation of estrogen-mediated endometrial carcinoma (EC).Materials and methods: NNT-AS1, miR-30c, and Nucleophosmin 1 (NPM1) expressions were measured by quantitative real-time PCR and Western blotting. Cell Counting Kit-8 assay and 5-Ethynyl-2'-deoxyuridine (EdU) assay were used to detect the viability and proliferation of Ishikawa and HEC-1-A cells, respectively. RNA immunoprecipitation assay was used to confirm the interaction between NNT-AS1 and miR-30c. Luciferase reporter assay was performed to confirm the interaction between miR-30c and NPM1.Results: NNT-AS1 and NPM1 expressions in EC tissues and cell lines were higher than in benign endometrium and normal endometrial epithelial cells (EECs). miR-30c expression in EC tissues and cell lines was lower than in benign endometrium and normal EECs. NNT-AS1 interacted with miR-30c, and miR-30c negatively regulated NPM1 expression. Overexpression of NNT-AS1 increased NPM1 expression in EC cells, while overexpression of miR-30c reversed the effect. NNT-AS1 interference inhibited the mRNA level of NPM1, while the miR-30c inhibitor reversed the result. Estradiol (E2) promoted the proliferation of EC cells, small interfering RNA (siRNA) against NNT-AS1 inhibited EC cell proliferation, miR-30c inhibitor promoted cell proliferation, and NPM1 siRNA inhibited cell proliferation. E2 increased tumor volume, and NNT-AS1 interference reduced tumor volume in vivo.Conclusion: NNT-AS1 promoted the proliferation of estrogen-mediated EC by regulating miR-30c/NPM1.

Eosinophilic endometrial metaplasia - case report and brief literature review on its immunohistochemical characteristics

Endometrial epithelial metaplasia is described as transition of the normal endometrial epithelial cells by benign complex proliferation of cells. These metaplastic changes have been frequently reported as associated changes in endometrial hyperplasia and adenocarcinoma more than non-neoplastic samples and are also known to appear atypical occasionally, and hence can be a diagnostic challenge. Eosinophilic cell change is one of the most frequently encountered endometrial metaplasias. Eosinophilic syncytial change is a form of eosinophilic endometrial metaplasia, and is known to mimic endometrial serous carcinoma, again posing a diagnostic challenge. In this article, we have presented a case of endometrial eosinophilic metaplasia in a 47-year-old patient along with a brief discussion on immunohistochemical characteristics of eosinophilic syncytial change that could help pathologists to differentiate them from malignancies in challenging scenarios.

Circ_0109046 promotes the malignancy of endometrial carcinoma cells through the microRNA-105/SOX9/Wnt/β-catenin axis.

Emerging evidence suggests the important involvements of circular RNAs (circRNAs) in cancer progression. This study focuses on the function of Circ_0109046 on the malignancy of endometrial carcinoma (EC) cells and the molecules involved. First, high expression of Circ_0109046 was found in EC tissues compared to the adjacent tissues, and it predicted unfavorable prognosis in patients. Similarly, high expression of Circ_0109046 was confirmed in EC cells relative to that in normal endometrial epithelial cells. Silencing of Circ_0109046 in AN3-CA cells inhibited proliferation and aggressiveness but increased apoptosis of cells. Circ_0109046 was mainly sub-localized in cytoplasm, and it mediated SOX9 expression through sponging microRNA (miR)-105. The proliferation and aggressiveness of EC cells suppressed by Circ_0109046 downregulation was recovered upon SOX9 overexpression. SOX9 activated the Wnt/β-catenin pathway. Furthermore, downregulation of Circ_0109046 reduced the growth of xenograft tumors in nude mice. This study evidenced that Circ_0109046 upregulates SOX9 expression through sponging miR105, leading to activation of Wnt/β-catenin signaling and the malignant growth of EC. This study may offer novel understanding in EC treatment.

MiR-326 regulates the proliferation and apoptosis of endometrial cancer by targeting Bcl-2.

MiR-326 has been investigated to be correlated with multiple types of malignancies; however, the role of miR-326 in endometrial cancer (EC) remains rarely reported. The aim of our research is to investigate the functions of miR-326 in EC and the potential molecular mechanism. RT-qPCR was performed to compare the expression of miR-326 and Bcl-2 in normal endometrial epithelial cell line (End1/e6e7) and EC cells lines (HEC-1A, Ishikawa), respectively. Bioinformatic analysis and luciferase assay verified the relationship between miR-326 and the 3'-UTR of Bcl-2. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, soft agar colony formation assay and the flow cytometry were performed to investigate the functions of miR-326 and Bcl-2 on proliferation and apoptosis in EC. Western blotting was employed to explore the expression of Bcl-2, Bcl2-associated X (Bax) and caspase-3. The expression of miR-326 decreased in EC cell lines compared to normal endometrial epithelial cell line, while Bcl-2 expression was increased in EC cells. Results of MTT and soft agar colony formation assays showed that miR-326 suppressed proliferation in EC cells. In addition, flow cytometry revealed that miR-326 promoted apoptosis in EC cells. Western blotting showed that silencing miR-326 promoted the expression of Bcl-2. Bioinformatics analysis and luciferase assay verified the 3'-UTR of Bcl-2 was a target of miR-326. Furthermore, MTT assay, soft agar colony formation assay and the flow cytometry proved that miR-326 acts as tumor suppressor via inhibiting the expression of Bcl-2. MiR-326 acts as a cancer suppressor to inhibit proliferation and promote apoptosis via targeting Bcl-2 axis in EC.

Establishment and characterization of cell lines from human endometrial epithelial and mesenchymal cells from patients with endometriosis

To establish and characterize cell lines derived from human endometrial epithelial cells (ECs) and mesenchymal cells (MCs) from patients with and without endometriosis. Invitro experimental study. University and national cancer center research institute. Two women with endometriosis and two women without endometriosis. Sampling of endometrial ECs and MCs. Establishing immortalized endometrial ECs and MCs with quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunocytochemical analysis, and RNA sequence profiling performed to characterize the immortalized cells and a cell proliferation assay, three-dimensional culture, and assays for hormone responses performed to characterize the features of ECs. The qRT-PCR, immunocytochemical analysis, and Western blot analysis revealed that the ECs and MCs maintained their original features. Moreover, the immortalized cells were found to retain responsiveness to sex steroid hormones. The ECs formed a gland-like structure in three-dimensional culture, indicating the maintenance of normal EC phenotypes. The RNA sequence profiling, principal component analysis, and clustering analysis showed that the gene expression patterns of the immortalized cells were different from those of cancer cells. Several signaling pathways that were statistically significantly enriched in ECs and MCs with endometriosis were revealed. We successfully obtained four paired immortalized endometrial ECs and MCs from patients with and without endometriosis. Using these cells could help identify diagnostic and therapeutic targets for endometriosis. The cell lines established in this study will thus serve as powerful experimental tools in the study of endometriosis.

The NF-κB signalling pathway regulates GLUT6 expression in endometrial cancer

BackgroundGene and protein expression of the glucose transporter GLUT6 are elevated in multiple cancers, including endometrial cancer. However, the extrinsic and intrinsic mechanisms that regulate GLUT6 expression in this malignancy are unknown. Herein we investigate the potential mechanisms regulating GLUT6 expression in endometrial cancer. MethodsData mining of the GLUT6 gene (SLC2A6) in The Cancer Genome Atlas (TCGA) PanCan datasets was performed in cBioPortal. A transcriptome PCR array was used to identify regulators of GLUT6 expression. The role of RELA in regulating GLUT6 gene and protein expression was investigated by overexpressing constitutively active and dominant-negative RELA in endometrial cells. Endometrial cells were treated with the pro-inflammatory cytokine TNFα and the expression of RELA, IκBα, TNFα, and GLUT6 were examined by Western blotting and RT-qPCR. ResultsGLUT6 is altered in 1% of all cancer samples (157 of 10, 967 samples) within TCGA datasets including 4.7% of uterine (endometrial) cancers. GLUT6 expression was positively co-expressed with multiple members of the NF-κB signalling pathway including NFKB2, RELB, NFKBIE, and TNF in endometrial cancer samples. A transcriptome PCR array identified RELA as the top potential transcriptional regulator of GLUT6 expression. Overexpression of constitutively active RELA increased GLUT6 gene expression in normal endometrial epithelial cells (hUE-Ts), while overexpression of dominant-negative RELA decreased GLUT6 expression in cancerous RL95–2 endometrial cells. TNFα treatment activated canonical NF-κB signalling and increased the expression of GLUT6, but not that of other glucose transporters (GLUTs 1, 3, 4, 8, 10, or 12) in endometrial cells. ConclusionsTNFα is a cytokine that is commonly increased in obesity-related endometrial cancer and the findings herein support a potential mechanism whereby TNFα may contribute to endometrial cancer initiation or progression by increasing GLUT6 expression. Furthermore, we identified RELA, an important downstream mediator of the TNFα signalling cascade, as a regulator of GLUT6 expression in endometrial cells. Future studies are warranted to determine how GLUT6 expression affects endometrial tumourigenesis or cancer progression.

Advanced oxidation protein products change biological behaviors of rat endometrial epithelial cells by activating ERK/P38 signaling pathways.

ABSTRACTAdvanced oxidation protein products (AOPPs) are a family of oxidized protein compounds and could induce oxidative stress and inflammatory lesion in various cells. The accumulation of AOPPs was associated with female reproductive diseases such as polycystic ovary syndrome (PCOS), leiomyoma and endometriosis. However, the relationship between AOPPs and endometrial cells is unclear. To explore the effects of accumulated AOPPs on endometrial cells, we treated normal rat endometrial epithelial cells (rEECs) and endometriosis model rats with AOPPs. Primary rEECs were collected from 8-week-old female Wistar rats. Increasing the amount of AOPPs in the media of rEECs enhanced rEEC proliferation and migration, and inhibited apoptosis. Moreover, AOPPs triggered the production of reactive oxygen species and nitrite along with activated ERK and P38 signal and this, in turn, led to an upregulation of proliferation and migration. With the treatment of antioxidants or the inhibitors of ERK and P38, the above effects of AOPPs on rEECs were attenuated. Additionally, in an endometriosis rat model, a similar phenomenon was observed in that the growth of endometriotic implants were promoted by AOPPs and EECs were significantly increased. This study indicated that the accumulation of AOPPs could promote rEEC proliferation and migration through ERK and P38 signal both in vivo and in vitro.This article has an associated First Person interview with the first author of the paper.

Melatonin inhibits 17\u03b2-estradiol-induced migration, invasion and epithelial-mesenchymal transition in normal and endometriotic endometrial epithelial cells

BackgroundMelatonin is a potential therapeutic agent for endometriosis, but its molecular mechanism is unclear. Here, we investigated the effect of melatonin on the epithelial-mesenchymal transition (EMT) in endometriotic endometrial epithelial cells and explored the pathway that might be involved.MethodsThis hospital-based study included 60 women of reproductive age using the endometrium for immunohistochemistry, 6 women of reproductive age undergoing bilateral tubal ligation and 6 patients with endometriosis for isolation of endometrial epithelial cells or subsequent analysis, respectively. We examined the expression of Notch1/Numb signaling and EMT markers by immunohistochemistry analysis and western blot analysis, the invasion and migration of endometrial epithelial cells by transwell assays, and the cell proliferation by CCK8 assays.ResultsCompared with normal endometrium, the endometriotic eutopic endometrium showed increased expression of Notch1, Slug, Snail, and N-cadherin, and decreased expression of E-cadherin and Numb. Melatonin or Notch inhibition by specific inhibitor blocked 17β-estradiol-induced cell proliferation, invasion, migration and EMT-related markers in both normal and endometriotic epithelial cells.ConclusionsOur data suggest that aberrant expression of Notch1/Numb signaling and the EMT is present in endometriotic endometrium. Melatonin may block 17β-estradiol-induced migration, invasion and EMT in normal and endometriotic epithelial cells by upregulating Numb expression and decreasing the activity of the Notch signaling pathway.

Obesity, Insulin Resistance and Cancer: The PI3K connection

Retrospective studies have revealed that cancer rates in certain organ sites increase with obesity and some studies have suggested that the subset of obese individuals who develop insulin resistance are more likely to develop cancers than those who are obese but remain insulin sensitive. The risk of developing uterine and breast cancers are particularly high in women who are obese and insulin resistant and these two cancers have the highest rates of activating mutations in PIK3CA, the gene that encodes phosphoinositide 3‐kinase 1 alpha (PI3K1alpha). PI3K1alpha mediates insulin dependent cell growth and the activating mutations in PIK3CA allow this enzyme to be more readily activated by insulin in tumors that express the insulin receptor. Normal endometrial and breast epithelial cells express the insulin receptor and tumors in these tissues often have elevated expression of the insulin receptor. These observations raise the possibility that the elevated serum insulin in patients who are insulin resistant may be contributing to the development of tumors, especially tumors in tissues with PI3K pathway mutations. Many PI3K inhibitors have entered clinical trials and some have been approved. However, since the inhibitors that target PI3K1alpha cause insulin resistance in liver, muscle and fat, they cause hyperglycemia and hyperinsulinemia. Our studies indicate that the elevated insulin in the serum can ultimately re‐activate PI3K1alpha in the tumor, compromising the effect of these drugs. Strategies for making PI3K inhibitors more effective by preventing this feedback reactivation of PI3K1alpha in tumors will be discussed.Support or Funding InformationSupported by the NCI, the Breast Cancer Research Foundation, the Gray Family Foundation and the Hertog family.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biología Gestacional y Predicción del Parto en la Perra

La gestación en la perra transcurre entre la fecundación y el parto, y su duración tiene importancia clínica. La compleja biología de los gametos y embriones influye en la duración de la gestación, cuyo lapso fisiológico fluctúa entre los 57 y 70 días. Este amplio rango puede estar influenciado por los métodos referenciales para la estimación del inicio de la gestación. El realizar montas o inseminaciones en base a detección de la ovulación permite estrechar el rango a 62 a 64 días. Una condición de alto impacto obstétrico es la gestación de un solo cachorro, la cual es más prolongada y representa alto riesgo de distocia. Dada la importancia clínica de una adecuada proyección de la duración de la gestación y de la predicción de la fecha de parto, se han desarrollado fórmulas matemáticas para el cálculo de la edad fetal y tiempo al parto a través de la sistematización de la ultrasonografía gestacional.

The roles of tricellular tight junction protein lipolysis-stimulated lipoprotein receptor in malignancy of human endometrial cancer cells.

Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a novel molecular constituent of tricellular contacts that have a barrier function for the cellular sheet. LSR recruits tricellulin (TRIC), which is the first molecular component of tricellular tight junctions. Knockdown of LSR increases cell motility and invasion of certain cancer cells. However, the behavior and the roles of LSR in endometrial cancer remain unknown. In the present study, we investigated the behavior and roles of LSR in normal and endometrial cancer cells in vivo and in vitro. In endometriosis and endometrial cancer, LSR was observed not only in the subapical region but also throughout the lateral region as well as in normal endometrial epithelial cells in the secretory phase, and LSR in the cancer was reduced in correlation with the malignancy. Knockdown of LSR by the siRNA in cells of the endometrial cancer cell line Sawano, induced cell migration, invasion and proliferation, while TRIC relocalized from the tricellular region to the bicellular region at the membrane. In Sawano cells and normal HEEs, a decrease of LSR induced by leptin and an increase of LSR induced by adiponectin and the drugs for type 2 diabetes metformin and berberine were observed via distinct signaling pathways including JAK2/STAT. In Sawano cells, metformin and berberine prevented cell migration and invasion induced by downregulation of LSR by the siRNA and leptin treatment. The dissection of the mechanism in the downregulation of endometrial LSR during obesity is important in developing new diagnostic and therapy for endometrial cancer.

The involvement of osteopontin and matrix metalloproteinase- 9 in the migration of endometrial epithelial cells in patients with endometriosis.

BackgroundEndometriosis, which shares certain characteristics with cancers, may cause abnormal expression of proteins involved in cell migration. Endometrial epithelial cells (EECs) are believed to play an important role in endometriotic migration. The aim of this study was to investigate the relationship between the expression of osteopontin (OPN) and matrix metalloproteinase-9 (MMP-9) in endometriotic migration.MethodsWe performed primary culture of EECs and investigated the expression of OPN and MMP-9 in EECs regulated by 17beta-estradiol (E2). OPN-specific siRNA interference was used to down-regulate OPN and to explore the corresponding change in MMP-9 expression. Real-time RT-PCR, western blot analysis and flow cytometry were used to determine the expression levels of OPN and MMP-9. Gelatin zymography was performed to observe the enzymatic activity of MMP-9 in conditioned media. Transwell and wound scratch assays were performed to investigate the migration ability of EECs.ResultsThe expression levels of OPN and MMP-9 in normal EECs (NEECs) were inferior to those in EECs from patients with endometriosis (EEECs). The expression levels of OPN and MMP-9 from stage III/IV EEECs and secretory-phase EECs were higher than those of stage I/II EEECs or proliferative-phase EECs. The expression levels of OPN and MMP-9 in EEECs were increased by E2 treatment and remarkably decreased by siRNA interference. Active MMP-9 expression increased with E2 treatment and decreased with siRNA treatment in EEECs compared with the same treatments in NEECs. The migratory abilities of EEECs were enhanced after cells were treated with E2; in contrast, these abilities were reduced by siRNA interference. In NEECs, active MMP-9 and cellular migration abilities were only minimally influenced by E2 and siRNA treatment.ConclusionsThe present study suggests that the up-regulation of MMP-9 via activation of OPN induced by estrogen may correlate with the migration of endometrial epithelial cells in patients with endometriosis.