Related clinical factors of platinum-based chemotherapy resistance in patients with epithelial ovarian cancer.

OBJECTIVE: Ovarian cancer is the second most common malignancy in women, but it is the most fatal gynecological tumor. Although it has a standard treatment regimen, resistance to chemotherapy makes patients more prone to early recurrence, leading to poor survival rate. Therefore, this study aimed to investigate the factors related to platinum resistance through a complete analysis of clinical data. DESIGN: This study collected clinical data of patients with ovarian cancer and divided the petients into platinum-sensitive and platinum-resistant groups.By comparing the differences in clinical data between the two groups, the key factors affecting platinum resistance were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: We collected the clinical data of patients with epithelial ovarian cancer (EOC) who were admitted to the Department of Oncology of the General Hospital of Ningxia Medical University between January 1, 2019 and December 31, 2020. We conducted univariate and multivariate analyses and evaluated their overall survival and progression-free survival by using the Kaplan-Meier method. RESULTS: We enrolled 161 patients with EOC, of whom 124 demonstrated platinum sensitivity and 37 demonstrated platinum resistance after the initial platinum-based chemotherapy. Univariate analyses revealed that International Federation of Gynecology and Obstetrics (FIGO) stage, neoadjuvant chemotherapy, and Fagotti score were associated with an increased risk of platinum resistance for the first recurrence. In multivariate logistic regression analysis, only Fagotti score and neoadjuvant chemotherapy were associated with an increased risk of platinum resistance (odds ratio: 0.372 and 0.328, 95% confidence interval: 0.160-0.863 and 0.141-0.762, P = 0.021 and 0.010, respectively). LIMITATIONS: The sample size in this study is still relatively small, due to non-standard treatment of some patients,the absence of some clinial data,and the failure of follow-up,etc. CONCLUSION: Patients with EOC exhibiting platinum resistance had a very poor prognosis. Fagotti score and neoadjuvant chemotherapy appeared to increase the risk of platinum resistance at first recurrence.

A large-scale causal analysis of gut microbiota and endometriosis associated infertility: A Mendelian randomization study.

Endometriosis is a prevalent condition with notable impacts on fertility. Recent studies have implicated gut microbiota in the development of endometriosis associated infertility (EAI). This study employs Mendelian randomization (MR) to elucidate the causal relationship between specific gut microbes and EAI. Using MR, we selected single nucleotide polymorphisms associated with 211 gut microbiota taxa from large-scale genome-wide association studies summary data. We applied statistical methods including inverse variance weighting, weighted median, and MR-Egger for analysis. Outliers were identified through the leave-one-out method. MR-Egger intercept tests were conducted to address horizontal pleiotropy, while Cochran Q and P values assessed heterogeneity. The false discovery rate method was used for multiple testing correction. Sensitivity analysis and F statistics evaluated the reliability and potential biases of our results. The inverse variance weighting method indicated a significant association of the genus Actinomyces (OR = 1.657, 95% CI: 1.187-2.312, P = .00298) with an increased risk of EAI. Conversely, genera Holdemania (OR = 0.630, 95% CI: 0.444-0.894, P = .00969) and Ruminococcaceae NK4A214 group (OR = 0.689, 95% CI: 0.481-0.999, P = .0439) appeared as protective factors. MR-PRESSO global test and MR-Egger regression indicated no significant horizontal pleiotropy (P > .05). Leave-one-out analysis confirmed the robustness of these findings. Our study provides evidence of a causal relationship between specific gut microbiome taxa and EAI. These findings offer novel insights and may guide the development of new preventive and therapeutic strategies for managing EAI.

Enhancing precision medicine: a nomogram for predicting platinum resistance in epithelial ovarian cancer.

BACKGROUND: This study aimed to develop a novel nomogram that can accurately estimate platinum resistance to enhance precision medicine in epithelial ovarian cancer(EOC). METHODS: EOC patients who received primary therapy at the General Hospital of Ningxia Medical University between January 31, 2019, and June 30, 2021 were included. The LASSO analysis was utilized to screen the variables which contained clinical features and platinum-resistance gene immunohistochemistry scores. A nomogram was created after the logistic regression analysis to develop the prediction model. The consistency index (C-index), calibration curve, receiver operating characteristic (ROC) curve, and decision curve analysis (DCA) were used to assess the nomogram's performance. RESULTS: The logistic regression analysis created a prediction model based on 11 factors filtered down by LASSO regression. As predictors, the immunohistochemical scores of CXLC1, CXCL2, IL6, ABCC1, LRP, BCL2, vascular tumor thrombus, ascites cancer cells, maximum tumor diameter, neoadjuvant chemotherapy, and HE4 were employed. The C-index of the nomogram was found to be 0.975. The nomogram's specificity is 95.35% and its sensitivity, with a cut-off value of 165.6, is 92.59%, as seen by the ROC curve. After the nomogram was externally validated in the test cohort, the coincidence rate was determined to be 84%, and the ROC curve indicated that the nomogram's AUC was 0.949. CONCLUSION: A nomogram containing clinical characteristics and platinum gene IHC scores was developed and validated to predict the risk of EOC platinum resistance.

U0126 and BAY11-7082 Inhibit the Progression of Endometriosis in a Rat Model by Suppressing the MEK/ERK/NF-κB Pathway.

Endometriosis is an aggressive disease. It is the main cause of chronic pelvic pain, dysmenorrhea, and infertility, affecting the well-being of women. This study aimed to explore the role of U0126 and BAY11-7082 in endometriosis (EMs) treatment in rats by targeting the MEK/ERK/NF-κB pathway. The EMs model was generated and the rats were divided into model, dimethyl sulfoxide, U0126, BAY11-708, and control groups (Sham operation group). After 4 weeks of treatment, the rats were sacrificed. Compared with model group, U0126 and BAY11-7082 treatment significantly inhibited ectopic lesion growth, glandular hyperplasia, and interstitial inflammation. Compared to control group, PCNA and MMP9 levels were significantly increased in the eutopic and ectopic endometrial tissues of model group; the levels of MEK/ERK/NF-κB pathway proteins also increased significantly. Compared with model group, MEK, ERK, and NF-κB levels decreased significantly after U0126 treatment and NF-κB protein expression decreased significantly after BAY11-7082 treatment, with no significant difference in MEK and ERK levels. The proliferation and invasion activities of eutopic and ectopic endometrial cells also significantly decreased after U0126 and BAY11-7082 treatment. In summary, our results showed that U0126 and BAY11-7082 inhibited ectopic lesion growth, glandular hyperplasia, and interstitial inflammatory response in EMs rats by inhibiting the MEK/ERK/NF-κB signaling pathway.

Oocyte Arrested at Metaphase II Stage were Derived from Human Pluripotent Stem Cells in vitro.

Initiation of meiosis is the most difficult aspect of inducing competent oocytes differentiation from human stem cells in vitro. Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured with follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Expression of surface markers and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing. To induce the differentiation of hiPSCs into OLCs, cells were firstly cultured with a primordial germ cell medium for 10 days. The cells exhibited similar morphological features to primordial germ cells (PGCs), high expressing of germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured with the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cultured cells developed cumulus-oocyte-complexes (COCs) structures and OLCs with different sizes (50-150 µm diameter) and a zona pellucida. The in vitro matured OLCs had polar bodies and were arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet to be proved. in vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.

The role of Connexin26 regulated by MiR-2114-3p in the pathogenesis of ovarian cancer.

OBJECTIVES: The purpose of our research was to determine the expression of Cx26 and miR-2114-3p, and their effects on proliferation, migration, and invasion in ovarian cancer and their mechanisms. MATERIALS AND METHODS: Transcriptome sequencing was performed and differentially expressed Cx26 was screened. The mRNA and protein levels of Cx26 in EOC and normal ovarian tissues were verified. The relationship between Cx26 levels and prognostics was analyzed. Cx26 Lentiviral vectors were constructed to detect its effect on ovarian cancer. WB verified that PI3K/AKT pathway was the possible signal pathway regulated by Cx26. The interaction between miR-2114-3p and Cx26 was detected by double luciferase reporter assay and qrt-PCR. CCK8, clone formation, transwell, and flow cytometry assays were conducted in cells transfected miR-2114-3p plasmids. The vivo experiment investigated the effects of Cx26 on subcutaneous tumor growth, PI3K expression, proliferation proteins Ki67 and PCNA. RESULTS: Cx26 was up-regulated in EOC tissue and cell lines, and was associated with poor prognosis of ovarian cancer, while miR-2114-3p was down-regulated in EOC cell lines. Cx26 was a direct target of miR-2114-3p. Cx26 overexpression and miR-2114-3p inhibition promoted the growth, motility, invasiveness, and S phase arrest of EOC cells. Additionally, Cx26 could activated PI3K pathway whatever in vivo and in vitro. CONCLUSIONS: Dysregulation of Cx26 is critical in EOC patients. Manipulation of this mechanism may influence the survival of EOC patients. MiR-2114-3p regulates the tumor-promoting activity of Cx26 in EOC. By inhibiting the PI3K pathway or knocking down Cx26 effectively inhibits tumor growth in EOC cells and Nude mouse model.

Value of the risk of ovarian malignancy algorithm index in predicting the recurrence of epithelial ovarian cancer.

Aim: This study aimed to assess the predictive and diagnostic value of the risk of ovarian malignancy algorithm (ROMA) index for epithelial ovarian cancer (EOC) recurrence. Materials & methods: The clinical features and follow-up data of 159 EOC cases were studied. The ROMA index was calculated by serum CA125 and HE4 levels with menopausal status. Recurrence-free survival was evaluated for an end point. Results: The ROMA was strongly associated with clinical characteristics. The ROMA index above the cutoff value (34.71%) was significantly associated with recurrence-free survival. The ROMA index had a significantly higher sensitivity (90.59%) than CA125 (84.71%) and HE4 (80.80%) for recurrence diagnosis, and its optimal cutoff value was 17.07%. Conclusion: The primary ROMA index is a predictive factor in EOC recurrence and has better performance in the diagnosis of EOC recurrence.

Establish of an Initial Platinum-Resistance Predictor in High-Grade Serous Ovarian Cancer Patients Regardless of Homologous Recombination Deficiency Status.

Backgrounds: Ovarian cancer (OC) is still the leading aggressive and lethal disease of gynecological cancers, and platinum-based regimes are the standard treatments. However, nearly 20%-30% of patients with OC are initial platinum resistant (IPR), and there is a lack of valid tools to predict whether they will be primary platinum resistant or not prior to chemotherapy. Methods: Transcriptome data from The Cancer Genome Atlas (TCGA) was downloaded as the training data, and transcriptome data of GSE15622, GSE102073, GSE19829, and GSE26712 were retrieved from Gene Expression Omnibus (GEO) as the validation cohorts. Differentially expressed genes (DEGs) were selected between platinum-sensitive and platinum-resistant patients from the training cohort, and multiple machine-learning algorithms [including random forest, XGboost, and least absolute shrinkage and selection operator (LASSO) regression] were utilized to determine the candidate genes from DEGs. Then, we applied logistic regression to establish the IPR signature based on the expression. Finally, comprehensive clinical, genomic, and survival feature were analyzed to understand the application value of the established IPR signature. Results: A total of 532 DEGs were identified between platinum-resistant and platinum-sensitive samples, and 11 of them were shared by these three-machine learning algorithms and utilized to construct an IPR prediction signature. The area under receiver operating characteristic curve (AUC) was 0.841 and 0.796 in the training and validation cohorts, respectively. Notably, the prediction capacity of this signature was stable and robust regardless of the patients' homologous recombination deficiency (HRD) and mutation burden status. Meanwhile, the genomic feature was concordant between samples with high- or low-IPR signature, except a significantly higher prevalence of gain at Chr19q.12 (regions including CCNE1) in the high-IPR signature samples. The efficacy of prediction of platinum resistance of IPR signature successfully transferred to the precise survival prediction, with the AUC of 0.71, 0.72, and 0.66 to predict 1-, 3-, and 5-year survival, respectively. At last, we found a significantly different tumor-infiltrated lymphocytes feature, including lower abundance of CD4+ naive T cells in the samples with high-IPR signature. A relatively lower tumor immune dysfunction and exclusion (TIDE) value and more sensitivity to multiple therapies including Gefitinib may suggest the potency to transfer from platinum-based therapy to immunotherapy or target therapies in patients with high-IPR signature. Conclusion: Our study established an IPR signature based on the expression of 11 genes that could stably and robustly distinguish OC patients with IPR and/or poor outcomes, which may guide therapeutic regimes tailoring.

Differential Expression and Bioinformatics Analysis of tRF/tiRNA in Endometriosis Patients.

Background: Endometriosis (EMs) is a benign chronic condition that tends to recur in women of childbearing age, with an incidence of approximately 10%. It is a multifactorial disease for which the pathogenesis is currently unclear. This study is aimed at investigating the expression and clinical significance of tRNA-derived small RNA (tsRNA), a novel noncoding small RNA with potential regulatory functions, in endometriosis. Methods: The tRF/tiRNA expression profiles in endometrial tissues from three pairs of endometriosis patients and controls were detected by tRF&tiRNA PCR microarray technology and then verified by quantitative real-time polymerase chain reaction (qPCR). The target genes and target sites of TRF396, tiRNA-5030-GlnTTG-3, TRF308, and TRF320 were predicted by miRanda, and the network diagram of their interaction with miRNA was drawn. The impact of tRNA-derived fragments on the pathogenesis of endometriosis was analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results: Two upregulated and 19 downregulated tRNA-derived fragments were identified. The qRT-PCR results of 2 upregulated and 2 downregulated RNA-derived fragments were consistent with the RNA Seq data. The OR2B4 gene related to TRF396, the DGAT1 gene related to tiRNA-5030-GlnTTG-3, the KLF16 gene of TRF308, and the RNF213 gene of TRF320 had significant correlations. Gene Ontology and pathway analysis showed that the target genes of TRF396 and tiRNA-5030-GlnTTG-3 were mainly involved in the intrinsic components of the membrane and the overall composition of the membrane in cell components; molecular functions mainly involve olfactory conduction and G protein-coupled receptor activity. In the biological process, it was mainly involved in the detection of sensory stimuli. The target genes of TRF308 and TRF320 were mainly involved in the intracellular part; molecular functions are mainly related to DNA binding transcription factor activity and protein binding and mainly related to biological regulation of biological processes. Pathway analysis showed that the RAP1 signaling pathway and the AXON GUIDANCE signaling pathway may participate in the progression of endometriosis. Conclusion: The differential expression of tRF/tiRNA in endometriosis may be related to the pathogenesis of endometriosis. Furthermore, tRF/tiRNA may be a biomarker for the diagnosis and treatment of EMs in the future.

Cost-effectiveness Analysis of Dienogest Compared With Gonadotropin-Releasing Hormone Agonist After Conservative Surgery for Endometriosis in China.

PURPOSE: Although the clinical effect of dienogest for endometriosis after conservative surgery has been proven, the cost-effectiveness of this new pharmacotherapy remains to be determined. We aimed to assess the health economic implications of dienogest versus a gonadotropin-releasing hormone agonist (GnRH-a; goserelin in the Chinese setting. METHODS: A decision tree model was developed to evaluate the cost-effectiveness of dienogest compared with a GnRH-a (goserelin) after conservative surgery for endometriosis during a 2-year time horizon from the perspective of a health care system in China. The cost of drugs, use of outpatient care facilities, administration of medications, routine laboratory work and imaging studies, and treatment of drug-related adverse events were considered. We obtained clinical efficacy data from the peer-reviewed literature. Base case findings were further tested with 1-way and probabilistic sensitivity analyses. FINDINGS: The model projects that treatment with dienogest would result in a modest incremental 0.02 quality-adjusted life-year gains compared with a GnRH-a (goserelin) (1.48 vs 1.46) at a cost saving of ¥7274 (¥22,809 vs ¥30,164). Probabilistic sensitivity analysis found that dienogest has a 100% probability of % being considered cost-effective compared with a GnRH-a (goserelin) at the willingness-to-pay threshold of 3 times the gross domestic product per capita (¥64,644 × 3) of China in 2018 (¥1 = US$0.1454 and €0.1248). IMPLICATIONS: Dienogest is more effective and cost-saving compared with a GnRH-a (goserelin) in the treatment of patients with endometriosis after conservative surgery in China.

Real-world Experience of Olaparib Treatment in Patients with Ovarian Cancer: A Chinese Multicenter Study.

The objective of this study was to evaluate the real-world application, efficacy, and safety data of olaparib for maintenance therapy and active treatment in patients with ovarian cancer in China. Patients with ovarian cancer from 17 institutions in China treated with olaparib as maintenance or active therapy from January 2018 to March 2020 were included in this study. The medical records were reviewed, and follow-up information was collected for analysis of the patients' clinicopathologic characteristics as well as the effectiveness and safety of olaparib. A total of 251 patients receiving olaparib were included, with 84 as maintenance therapy after first-line chemotherapy (FL-M), 97 as maintenance therapy after platinum-sensitive recurrence (PSR-M), and 70 as active treatment (AT). The probability of progression-free survival (PFS) at 12 months was 87.6% in the FL-M group and 63.8% in the PSR-M group. According to the multivariate analysis, complete response (CR) to chemotherapy for the PSR-M patients was the only factor affecting the PFS (HR = 0.414, P = 0.014), and platinum sensitivity was the only factor affecting PFS improvement in the AT group (HR = 0.317, P = 0.009). In the AT group, the objective response rate was 37.1%, the CR rate was 7.1%, and 30% of the patients had stable disease. Eight (3.2%) patients discontinued olaparib due to toxicity. Anemia was the most common adverse event. In conclusion, olaparib is effective and well tolerated in the real-world setting of ovarian cancer treatment. Platinum sensitivity is positively correlated to the effectiveness of olaparib in both maintenance and active treatment.

Integrated histologic and molecular analysis of uterine leiomyosarcoma and 2 benign variants with nuclear atypia.

Uterine leiomyosarcoma (LMS) is a rare but deadly disease. Due to poor understanding of the molecular and genetic causes of the disease, the diagnosis of LMS has been based primarily on histology. Nuclear atypia is one of hallmarks in LMS, however, it also occurs in 2 clinically benign variants, including smooth muscle tumors with fumarate hydratase alteration (SMT-FH) and leiomyoma with bizarre nuclei (LM-BN). In addition to nuclear atypia, many well recognized biomarkers used for LMS are also frequently overexpressed in LM-BN, and the histogenesis and molecular natures for LM-BN and LMS remain largely unknown. To characterize the molecular profiling of LMS, SMT-FH, and LM-BN, we performed integrated comprehensive genomic profiling including whole-genome sequencing (WGS) and RNA sequencing and genomic microarray analyses to assess genome-wide copy number alterations (CNAs) and immunohistochemistry (IHC) in all 3 tumor types. We found that both LM-BN and LMS showed genomic instability and harbored extensive CNAs throughout the whole genome. By contrast, the SMT-FH presented its characteristic 1q43-44 deletions in all cases tested, with minimal CNAs in the rest of genomic regions. Further analyses revealed that LMS and LM-BN groups showed similar patterns of CNAs that are tended to cluster together and separated from the SMT-FH group. The integrated molecular profiling enabled the detection of novel and traditional biomarkers and showed excellent discrimination between LM-BN and LMS. Our study suggests that LM-BN, despite having similar nuclear atypia to SMT-FH, showed similar genomic instability but distinct genomic alterations with its malignant counterpart of LMS. The integrated molecular profiling is of clinical importance in characterizing these rare uterine smooth muscle tumors.

Development of a Genomic Signatures-Based Predictor of Initial Platinum-Resistance in Advanced High-Grade Serous Ovarian Cancer Patients.

BACKGROUND: High grade serous ovarian cancer (HGSOC) is the most common subtype of ovarian cancer. Although platinum-based chemotherapy has been the cornerstone for HGSOC treatment, nearly 25% of patients would have less than 6 months of interval since the last platinum chemotherapy, referred to as platinum-resistance. Currently, no precise tools to predict platinum resistance have been developed yet. METHODS: Ninety-nine HGSOC patients, who have finished cytoreductive surgery and platinum-based chemotherapy in Peking University Third Hospital from 2018 to 2019, were enrolled. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) were performed on the collected tumor tissue samples to establish a platinum-resistance predictor in a discovery cohort of 57 patients, and further validated in another 42 HGSOC patients. RESULTS: A high prevalence of alterations in DNA damage repair (DDR) pathway, including BRCA1/2, was identified both in the platinum-sensitive and resistant HGSOC patients. Compared with the resistant subgroup, there was a trend of higher prevalence of homologous recombination deficiency (HRD) in the platinum-sensitive subgroup (78.95% vs. 47.37%, p=0.0646). Based on the HRD score, microhomology insertions and deletions (MHID), copy number changes load, duplication load of 1-100 kb, single nucleotide variants load, and eight other mutational signatures, a combined predictor of platinum-resistance, named as DRDscore, was established. DRDscore outperformed in predicting the platinum-sensitivity than the previously reported biomarkers with a predictive accuracy of 0.860 at a threshold of 0.7584. The predictive performance of DRDscore was validated in an independent cohort of 42 HGSOC patients with a sensitivity of 90.9%. CONCLUSIONS: A multi-genomic signature-based analysis enabled the prediction of initial platinum resistance in advanced HGSOC patients, which may serve as a novel assessment of platinum resistance, provide therapeutic guidance, and merit further validation.

Cancer-associated fibroblast-derived exosomal microRNA-98-5p promotes cisplatin resistance in ovarian cancer by targeting CDKN1A.

BACKGROUND: Ovarian cancer (OC) is a gynecological malignancy with a high mortality. Cisplatin-based treatment is the typical treatment regimen for OC patients; however, it may cause unfavorable resistance. The current study intends to explore the function of cancer-associated fibroblast (CAF)-derived exosomal microRNA-98-5p (miR-98-5p) in cisplatin resistance in OC, and the participation of CDKN1A. METHODS: Bioinformatics analysis was employed in order to obtain cisplatin resistance-related differential genes in OC as well as possible upstream regulatory miRs. After gain- and loss-of-function assays in OC cells, RT-qPCR and western blot analysis were employed to measure CDKN1A and miR-98-5p expression. Dual luciferase reporter assay was applied to verify the targeting relationship between miR-98-5p and CDKN1A. CAFs were treated with miR-98-5p inhibitor, and then exosomes were isolated and co-cultured with OC cells. CCK-8, colony formation and flow cytometry assays were conducted to assess cell proliferation, cell colony formation, cell cycle distribution and cell apoptosis, respectively. At last, xenograft tumor in nude mice was carried out to test whether exosomal miR-98-5p could affect cisplatin resistance in OC in vivo. RESULTS: CDKN1A was highly expressed in cisplatin-sensitive OC cell lines, and silencing CDKN1A significantly promoted proliferation and cell cycle entry but decreased apoptosis in cisplatin-sensitive OC cells. miR-98-5p targeted CDKN1A to inhibit CDKN1A expression. CAF-derived exosomal miR-98-5p increased OC cell proliferation and cell cycle entry, but suppressed cell apoptosis. Furthermore, exosomal miR-98-5p promoted cisplatin resistance and downregulated CDKN1A in nude mice. CONCLUSION: Collectively, CAF-derived exosomes carrying overexpressed miR-98-5p promote cisplatin resistance in OC by downregulating CDKN1A.

Development and characterization of a polarized human endometrial cell epithelia in an air-liquid interface state.

Human endometrial epithelia undergo injury repair and regeneration with the menstrual cycle; however, mechanisms underpinning the roles of endometrial epithelial cells in endometrial lesions and regeneration remain incompletely understood, mainly owing to the difficulty in the isolation and expansion of primary endometrial epithelial cells and the lack of reliable models for in vitro and in vivo studies. In this report, we sought to improve methods for the isolation and expansion of human endometrial epithelial cells with a Rho-associated protein kinase (ROCK) inhibitor-modified medium and subsequently characterize endometrial epithelium generated with primary cells cultured in an air-liquid interface (ALI) state. Immunocytochemistry staining revealed the expression of epithelial cellular adhesion molecule (EpCam) and stage-specific embryonic antigen-1 (SSEA-1) but a lack of CD13 in endometrial epithelial cells. Meanwhile, a large number of proliferative Ki67+ cells were observed in isolated epithelial cells. Importantly, the EpCam+/CD13- cells were capable of forming spheroids, a characteristic of epithelial stem/progenitor cells. Interestingly, these cells also exhibited a capacity to reconstitute epithelial layers in an ALI state. Morphological analysis revealed mucosal secretion of differentiated epithelial cells with cilia and microvilli in ALI epithelial cells as determined by electronic microscopy. Immunoblotting assay further demonstrated the expression of endometrial epithelial cell markers keratin 17/19 and EpCam and stem cell marker OCT3/4 but not stromal cell marker Vimentin protein and CD13 in cell expansions. Furthermore, molecular analysis also showed that the exposure of cells to estrogen elevated the expression of estrogen receptor and progesterone receptors in ALI cultures. Our results shed light on the possibility of expanding sufficient numbers of endometrial epithelial cells for stem cell biology studies, and they provide a feasible and alternative model that can recapitulate the characteristics and physiology of endometrial epithelium in vivo.

Overexpression of microRNA-205 predicts lymph node metastasis and indicates an unfavorable prognosis in endometrial cancer.

As an integral component of the surgical staging system, lymphadenectomy for patients with endometrial cancer (EC) remains controversial, particularly in clinical stage I disease that includes not only low-risk, but also high-risk subgroups. In order to maximize the therapeutic effect of lymph node excision for high-risk patients who can potentially obtain survival benefits from it while minimizing its reverse effects in low-risk patients, pre-operative risk stratification of lymph node metastasis is necessary. The upregulation of microRNA-205 (miR-205) in carcinoma of the endometrium has been consistently reported recently and has been found to correlate with poor survival. The current study aimed to investigate whether the overexpression of miR-205 in curettage samples of EC could identify patients who are at a high risk for lymph node metastasis prior to surgery and validate the role of miR-205 as a prognostic marker in EC. Relative quantification detection of miR-205 in curettage and hysterectomy specimens of patients with EC was performed. Prediction of lymph node metastasis based on miR-205 expression, as well as tumor type and grade in curettage samples, was performed for all EC patients and patients with clinical stage I disease. Moreover, survival analysis was conducted. It was observed that miR-205 was significantly and consistently elevated in the curettage and hysterectomy samples of EC relative to normal controls. Furthermore, the overexpression of miR-205 could predict lymph node metastasis with a high accuracy and was revealed again to be associated with a poor prognosis in EC. Prospective and multicentric studies are required to further clarify the value of miR-205 as a promising predictor to stratify risk for lymph node metastasis in EC.

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

BACKGROUND: Endometriosis, 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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: The 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.

Alisertib, an Aurora kinase A inhibitor, induces apoptosis and autophagy but inhibits epithelial to mesenchymal transition in human epithelial ovarian cancer cells.

Ovarian cancer is a leading killer of women, and no cure for advanced ovarian cancer is available. Alisertib (ALS), a selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects, and is under clinical investigation for the treatment of advanced solid tumor and hematologic malignancies. However, the role of ALS in the treatment of ovarian cancer remains unclear. This study investigated the effects of ALS on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT), and the underlying mechanisms in human epithelial ovarian cancer SKOV3 and OVCAR4 cells. Our docking study showed that ALS, MLN8054, and VX-680 preferentially bound to AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS had potent growth-inhibitory, proapoptotic, proautophagic, and EMT-inhibitory effects on SKOV3 and OVCAR4 cells. ALS arrested SKOV3 and OVCAR4 cells in G2/M phase and induced mitochondria-mediated apoptosis and autophagy in both SKOV3 and OVCAR4 cell lines in a concentration-dependent manner. ALS suppressed phosphatidylinositol 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways but activated 5'-AMP-dependent kinase, as indicated by their altered phosphorylation, contributing to the proautophagic activity of ALS. Modulation of autophagy altered basal and ALS-induced apoptosis in SKOV3 and OVCAR4 cells. Further, ALS suppressed the EMT-like phenotype in both cell lines by restoring the balance between E-cadherin and N-cadherin. ALS downregulated sirtuin 1 and pre-B cell colony enhancing factor (PBEF/visfatin) expression levels and inhibited phosphorylation of AURKA in both cell lines. These findings indicate that ALS blocks the cell cycle by G2/M phase arrest and promotes cellular apoptosis and autophagy, but inhibits EMT via phosphatidylinositol 3-kinase/Akt/mTOR-mediated and sirtuin 1-mediated pathways in human epithelial ovarian cancer cells. Further studies are warranted to validate the efficacy and safety of ALS in the treatment of ovarian cancer.

IgG expression in trophoblasts derived from placenta and gestational trophoblastic disease and its role in regulating invasion.

Immunoglobulin G (IgG) is an important humoral immune factor, which plays a role in innate immunity of the fetus. IgG immunoreactivity was often seen in trophoblasts of placenta. Traditionally, IgG in trophoblasts was believed to be transported from the maternal blood through neonatal Fc receptor (FcRn). Here, we explored the phenomenon of IgG expression and its role in regulating invasion in trophoblasts derived from normal placenta and gestational trophoblastic disease (GTD). IgG expression was detected with an emphasis on mRNA transcripts by using reverse transcription-polymerase chain reaction and hybridization in situ, besides evaluated at the protein level with immunohistochemistry and immunofluorescence. The migration and attachment of normal trophoblast cell line (TEV-1) and choriocarcinoma cell line (JAR) were inhibited with down-regulation of IgG expression. Methotrexate promoted the differentiation of JAR cell line; however, it had little effect on the differentiation of TEV-1 cell line. IgG expression, migration, and attachment of JAR and TEV-1 cell lines were decreased in the presence of methotrexate. Furthermore, statistical analysis showed that the differences in migration and attachment were significant (P < 0.05) for JAR cell line, while no significant difference was found for TEV-1 cell line. Collectively, these results confirmed that with the progression from normal placenta to GTD, the expression of IgG was increased in trophoblasts, which might actively promote the migration and attachment of trophoblasts as an important regulating factor.

Molecular implication of ADAM-15 and -17 in intrauterine adhesions.

OBJECTIVES: To investigate the regulation of the proteins ADAM-15 and ADAM-17 in intrauterine adhesions (IUA). STUDY DESIGN: 68 patients were found to have IUA in a study performed at our Department of Gynecology, and 18 control volunteer participants were recruited in the study. The patients with IUA were assigned to three groups according to the classification of March et al.: IUA-I (n=28), IUA-II (n=22), and IUA-III (n=18). All the volunteers were assigned to the control group (Con, n=18). The expression of ADAM-15 and ADAM-17 in the adhesive band tissue in patients and the endometrium in volunteers was detected by western blot, real-time PCR, and immunohistochemistry. RESULTS: The expression of ADAM-15 and ADAM-17 was significantly upregulated in both protein level and transcript level in IUA patients compared to that in controls. ADAM-15 expression was significantly higher in IUA-III (4.59±0.15) compared to IUA-II (3.18±0.12) and IUA-I (2.11±0.17; P<0.01). ADAM-17 expression was also significantly higher in IUA-III (3.25±0.11) compared to IUA-II (2.21±0.15) and IUA-I (1.78±0.21; P<0.01). The transcript levels of ADAM-15 and ADAM-17 showed similar patterns, and were markedly higher in grade III IUA patients compared to grade II and grade I. The severity of IUA was positively correlated to the protein and transcript expression level of ADAM-15 and ADAM-17 in uterine tissue. CONCLUSIONS: The development of IUA is associated with regulation of ADAM15 and ADAM-17, which may be potential biological markers for evaluating the severity of intrauterine adhesions.