Tumor-associated macrophages promote cisplatin resistance in ovarian cancer cells by enhancing WTAP-mediated N6-methyladenosine RNA methylation via the CXCL16/CXCR6 axis.

PURPOSE: Tumor-promotive tumor-associated macrophages (TAMs) and the CXCL16/CXCR6 axis have been reported to be correlated with the limited efficacy of chemotherapy in ovarian cancer (OC). However, the role of TAM-secreted CXCL16 and the mechanism by which it affects the cisplatin (DDP) resistance of OC cells remain elusive. METHODS: We induced human THP-1 monocytes to differentiate into macrophages. Next, SKOV3 and TOV-112D cells were co-cultured with the macrophages, followed by incubation with increasing concentrations of DDP. The effects of CXCL16, CXCR6, and WTAP on the DDP resistance of OC cells were investigated using the CCK-8 assay, colony formation assay, flow cytometry, and TUNEL staining. CXCL16 concentrations were determined by ELISA. Quantitative real-time PCR and western blotting were used to examine related markers. RESULTS: Our results showed that after being co-cultured with TAMs, the DDP resistance of OC cells was significantly enhanced and their CXCL16 levels were elevated. Acquired DDP resistance was characterized by an increased IC50 value for DDP, the formation of cell colonies, and decreased levels of cell apoptosis, which were accompanied by reduced levels of caspase-3 and Bax expression, and increased levels of Bcl-2, PARP1, BRCA1, and BRCA2 expression. Either CXCL16 knockdown in TAMs or CXCR6 knockdown in OC cells suppressed the DDP resistance of OC cells that had been co-cultured with TAMs. Knockdown of CXCL16 affected m6A RNA methylation in OC cells, as reflected by decreased YTHDF1/WTAP expression and increased ALKBH5 expression. WTAP overexpression and knockdown promoted and suppressed the DDP resistance of OC cells, respectively. CONCLUSION: Tumor-associated macrophages promote the cisplatin resistance of OC cells by enhancing WTAP-mediated N6-methyladenosine RNA methylation via the CXCL16/CXCR6 axis.

Network-based pharmacological study of the mechanism of kaiyuzhongyutang in the treatment of tubal fimbria obstruction.

To apply a network pharmacological approach to explore the targets and possible mechanisms of Kai Yu Zhong Yu Tang (KYZYT) in the treatment of tubal fimbria obstruction. The target information of KYZYT was extracted from TCMSP and HERB database. Genes related to tubal fimbria obstruction were searched using the GENECARD database. Target protein network maps (PPI) were drawn using string database analysis and Cytoscape 3.7.1 software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and gene function analysis (GO) enrichment analysis were performed with the help of Perl language and biological program package in R language. To explore the multiple pharmacological mechanisms of action of KYZYT in the interventional treatment of tubal fimbria obstruction and to lay the foundation for further experimental validation. Through the collection and analysis of multiple databases, 355 biological targets of KYZYT were identified. 168 targets of tubal fimbria obstruction were obtained from disease database. The "drug-component" and "drug-target" networks of KYZYT were constructed, and the protein interaction network (PPI) of overlapping targets was analyzed to identify the key targets of the drug affecting the disease. In addition, KEGG pathway analysis and GO enrichment analysis were performed on the overlapping targets to explore the mechanism of KYZYT in the treatment of tubal fimbria obstruction. KYZYT has the characteristics of multi-component, multi-target and multi-pathway in the treatment of tubal fimbria obstruction, which provides new ideas and scientific basis for further clarification of the molecular mechanism.

A methylation-driven genes prognostic signature and the immune microenvironment in epithelial ovarian cancer.

Aberrant gene methylation has been implicated in the development and progression of tumors. In this study, we aimed to identity methylation-driven genes involved in epithelial ovarian cancer (EOC) to establish a prognostic signature for patients with EOC. We identified and verified 6 MDGs that are closely related to the prognosis of ovarian cancer. A prognostic risk score model and nomogram for predicting the prognosis of ovarian cancer were constructed based on the six MDGs. It can also effectively reflect the immune environment and immunotherapy response of ovarian cancer. These MDGs have great significance to the implementation of individualized treatment and disease monitoring of ovarian cancer patients.

Nomograms for Predicting Overall Survival and Cancer-Specific Survival of Young Patients with Epithelial Ovarian Cancer: Analysis Based on SEER Program.

INTRODUCTION: Currently, there is no clinical prediction model for young patients (≤ 45 years old) with epithelial ovarian cancer (EOC) based on large samples of clinical data. The purpose of this study was to construct nomograms using data extracted from the Surveillance, Epidemiology, and End Results (SEER) Program to predict the overall survival (OS) and cancer-specific survival (CSS) of patients and to further guide the choice of clinical treatment options. METHODS: Data from a total of 6376 young patients with EOC collected from 1998 to 2016 were selected from the SEER database. These patients were randomly divided (7:3) into a training cohort (n = 4465) and a validation cohort (n = 1911). Cox and least absolute shrinkage and selection operator (LASSO) analyses were used to select the prognostic factors affecting OS and CSS, and the nomograms of OS and CSS were established. The performance of the nomogram models was assessed by C-index, area under the curve (AUC), calibration curves, and decision curve analysis (DCA). Sample were chosen from patients who underwent surgery in Shengjing Hospital to set external validation. Kaplan-Meier curves were plotted to compare survival outcomes between subgroups. RESULTS: Nomograms showed good predictive power and clinical practicality. The internal and external validation indicated better performance of the nomograms than the American Joint Committee on Cancer (AJCC) staging system and tumor grade system. Significant differences were observed in the survival curves of different risk subgroups. CONCLUSIONS: We constructed predictive nomograms to evaluate the OS and CSS of young patients with EOC. The nomograms will provide an individualized evaluation of OS and CSS for suitable treatment of young patients with EOC.

Effect of modified levonorgestrel-releasing intrauterine system in human adenomyosis with heavy menstrual bleeding.

AIM: The levonorgestrel-releasing intrauterine (LNG-IUS) system is an effective primary treatment for adenomyosis; however, it has high expulsion rates. We aimed to modify the system-allowing affixion to the myometrium-and evaluate the expulsion rate, effectiveness, and side effects in patients with adenomyosis and heavy menstrual bleeding. METHODS: This study included patients with adenomyosis and heavy menstrual bleeding who underwent implantation of: a modified LNG-IUS (experimental group, n = 47); and the original system after gonadotropin-releasing hormone agonist treatment (control group, n = 47), between January 2014 and April 2016. RESULTS: In the experimental group, two device expulsions occurred 12-18 months postimplantation. In the remaining 45 patients, the system was safely removed after the 60-month validity period, and no extrauterine device movement or infection occurred. In the control group, downward displacement and expulsion of the device occurred in eight (17%) patients within 60 months. The 5-year total expulsion rates were 4.3% and 17.0% in the experimental and control groups, respectively (p = 0.045). There were significant changes in the pretreatment severity of dysmenorrhea, menstrual volume, uterine volume (cm3 ), and hemoglobin level in each group compared with after 1 year (p < 0.01 in all groups). The severity of dysmenorrhea, menstrual volume, uterine volume, and hemoglobin level after 1 year were similar between the two groups (p > 0.05 in all groups). CONCLUSIONS: Use of the modified LNG-IUS is a safe, cost-effective, and simple method for reducing the downward movement and expulsion rate in patients with adenomyosis and heavy menstrual bleeding.

Clinical Nomograms for Predicting the Overall Survival and Cancer-specific Survival of patients with Ovarian Carcinosarcoma patients after Primary Surgery.

Background: At present, there is no clinical prediction model for ovarian carcinosarcoma (OCS) that is based on a large sample of real data. This study aimed to construct nomograms using data extracted from the Surveillance, Epidemiology, and End Results (SEER) database that can be used to predict the overall survival (OS) and cancer-specific survival (CSS) of patients with OCS and further guide the choice of clinical treatment. Methods: We selected 2753 cases of OCS from the SEER database from 1998 to 2016. Patients were randomly divided in a 7:3 ratio into a training cohort (n = 1929) and a validation cohort (n = 824). Cox analysis was used to select prognostic factors for OS and CSS, and nomograms were then established. The performance of nomogram models was assessed using the concordance index, the area under the receiver operating characteristic curve, calibration curves, and by decision curve analysis. Data from 21 OCS patients at Shengjing Hospital from 2001 to 2021 were collected for external verification. Kaplan-Meier curves were plotted to compare survival outcomes between subgroups. Results: Nomograms based on independent prognostic factors showed good predictive power and clinical practicality. Internal and external validation indicated that the nomograms performed better than staging and grading systems. Significant differences were observed in the survival curves of different risk subgroups. Conclusions: The developed nomograms will enable individualized evaluation of the OS and CSS, thus guiding the treatment of patients with OCS.

RNA-binding protein IGF2BP2 enhances circ_0000745 abundancy and promotes aggressiveness and stemness of ovarian cancer cells via the microRNA-3187-3p/ERBB4/PI3K/AKT axis.

BACKGROUND: Circular RNAs (circRNAs) are increasingly recognized as important regulators in cancer including ovarian cancer (OC). This work focuses on the effects of circ_0000745 on the OC development of and molecules involved. METHODS: Expression of circ_0000745 in collected OC tissues and the acquired OC cell lines was examined by RT-qPCR. The stability of circ_0000745 in cells was examined by RNase R treatment. The target transcripts interacted with circ_0000745 were predicted using bioinformatic systems. Gain- and loss-of-function studies of circ_0000745, microRNA (miR)-3187-3p and erb-b2 receptor tyrosine kinase 4 (ERBB4) were conducted to determine their functions on proliferation, migration, invasion and stem cell property of OC cells. RESULTS: Circ_0000745 and ERBB4 were abundantly expressed while miR-3187-3p was poorly expressed in OC tissues and cells. Circ_0000745 sequestered miR-3187-3p and blocked its repressive effect on ERBB4. Downregulation of circ_0000745 reduced proliferation, aggressiveness, epithelial-mesenchymal transition, and stemness of SK-OV-3 cells, but this reduction was blocked upon miR-3187-3p inhibition or ERBB4 upregulation. By contrast, artificial induction of circ_0000745 upregulation, miR-3187-3p upregulation and ERBB4 downregulation led to inverse trends in ES-2 cells. ERBB4 promoted the phosphorylation of the PI3K/AKT signaling pathway. An RNA binding protein IGF2BP2 was found to circ_0000745 bind to and promote its expression and stability. CONCLUSION: This study demonstrated that circ_0000745 upregulated by IGF2BP2 promotes aggressiveness and stemness of OC cells through a miR-3187-3p/ERBB4/PI3K/AKT axis. Circ_0000745 may serve as a promising target for OC treatment.

Multiomics analysis identifies key genes and pathways related to N6-methyladenosine RNA modification in ovarian cancer.

Aims: To explore the pathways and target genes related to N6-methyladenosine (m6A) methylation in ovarian cancer and their effect on patient prognosis. Methods & materials: The Cancer Genome Atlas was used to screen genes related to m6A regulators in terms of gene expression, mutation and copy number variation. These genes were subjected to pathway enrichment analysis. Prognosis-related genes were screened and involved in risk signature construction. Immunohistochemistry was used for verification. Results: We obtained 1408 genes dysregulated in parallel to m6A regulators, which were mainly involved in the platelet activation pathway. The m6A-related signature was constructed based on the expression of four prognosis-related genes (RPS6KA2, JUNB, HNF4A and P2RX1). Conclusion: This work provides new insights into the mechanism of m6A methylation in ovarian cancer.

Lay abstract N6-methyladenosine (m⁶A) methylation is the most common type of modification on mRNA. m⁶A methylation can affect the biological function of cells by affecting the protein expression level of mRNA. The process of m⁶A modification is controlled by many m⁶A regulators, which are dysregulated in ovarian cancer. Our research aims to screen the genes that are related to m⁶A regulation to analyze targets and mechanisms in ovarian cancer. We screened 1408 m⁶A-related genes, which are mainly involved in the platelet activation pathway. Among them, RPS6KA2 and JUNB were significantly related to poor prognosis of patients with ovarian cancer. RPS6KA2 was positively correlated with the m⁶A regulator METTL3 in ovarian cancer. Our study provides a basis for future mechanism studies.

Inhibition of Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ) Overcomes Drug Resistance and Tumorigenicity in Ovarian Cancer.

BACKGROUND/AIMS: Cancer stem-like cells are the main cause of tumor occurrence, progression, and therapeutic resistance. However, the precise signals required for the maintenance of the stem-like traits of these cells in ovarian cancer remain elusive. We have thus worked to elucidate the functional role of Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), a gene encoding the 14-3-3ζ protein, in the regulation of multidrug resistance and stem cell-like traits in ovarian cancer. METHODS: We detected the YWHAZ levels in human ovarian cancer specimens and cell lines using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blots. MTS assays, soft agar colony formation assays, migration assays, cell cycle analysis, sphere formation assays, and flow cytometry were applied to investigate the functional role of YWHAZ in ovarian cancer. RESULTS: Our data reveals substantially increased YWHAZ expression in both cisplatin- and paclitaxel-resistant ovarian cancer cells. Silencing YWHAZ restored the sensitivity of resistant ovarian cancer cells to cisplatin and paclitaxel. Furthermore, in vitro studies showed that down-regulation of YWHAZ inhibited cell cycle progression, migration, and the expression of stem cell markers. Moreover, tumorigenicity was suppressed in tumor-bearing BALB/c nude mice following YWHAZ knockdown. Additionally, we demonstrated that the expression of YWHAZ was directly down-regulated by miR-30e in resistant ovarian cancer cells. CONCLUSION: Our results have led to new insights into the essential role of YWHAZ in the regulation of tumourigenesis, stem-like traits, and drug resistance in ovarian cancer, thereby helping to identify a potential target for ovarian cancer therapy.

Tumor-associated macrophages promote the metastasis of ovarian carcinoma cells by enhancing CXCL16/CXCR6 expression.

This study investigated the underlying mechanism by which C-X-C motif chemokine ligand 16 (CXCL16)/C-X-C motif chemokine receptor 6 (CXCR6) signaling is activated by tumor-associated macrophages and assists in regulating the metastasis of ovarian carcinoma. Specimens of ovarian carcinoma tissue and adjacent tissue were collected from 20 ovarian carcinoma patients. Human THP-1 cells were induced to differentiate into macrophages, which were then co-cultured with SKOV3 cells and low concentrations of tumor necrosis factor-α (TNF-α) to simulate the inflammatory microenvironment of ovarian carcinoma. Additionally, small interfering RNA (siRNA) targeting CXCR6 was transfected into SKOV3 cells; after which, the levels of nuclear factor kappa B p65 (NF-κB p65) protein and phosphorylated PI3K and Akt were measured. The migration and invasion abilities of the SKOV3 cells were also tested. The levels of TNF-α, interluekin-6 (IL-6), NF-κB p65, CXCL16, and CXCR6 expression in the ovarian carcinoma tissues were higher than those in the precancerous tissues. CXCR6 expression was positively correlated with TNF-α, IL-6, and CXCL16 expression. Co-culture of SKOV3 cells with macrophages significantly promoted CXCL16, CXCR6, NF-κB, and p65 expression by the SKOV3 cells, increased their levels of phosphorylated PI3K and Akt, and increased the migration and invasion abilities of SKOV3 cells. Silencing of CXCR6 or blocking the PI3K/Akt signal pathway markedly attenuated the expression of NF-κB p65 and phosphorylation of PI3K and Akt, as well as the migration and invasion abilities of SKOV3 cells. These findings demonstrate that macrophages can promote the migration and invasion of ovarian carcinoma cells by affecting the CXCL16/CXCR6 pathway.

Comparison of two kinds of orthotopic xenograft models for human ovarian cancer.

OBJECTIVE: The aim of this study was to compare the characteristics of two orthotopic xenograft models established with human epithelial ovarian cancer solid tumor tissue slices and human ovarian carcinoma cell line OVCAR-3. MATERIALS AND METHODS: Tumor tissues and cell line OVCAR3 of human epithelial ovarian cancer were grown in subcutaneous tissue and the subcutaneous tumor source was fetched and inoculated in ovarian capsule of nude mice under microscope to establish the orthotopic implantation model. At four and eight weeks after modeling, the orthotopic tumor formation rate, tumor diameter, metastasis rate outside the ovary, incidence rate of ascites, and CA125 levels in the two models were observed. RESULTS: The orthotopic tumor formation rate in the solid tumor slices group (60.0%) was significantly lower than that in the cell line group (85.0%, p < 0.05). However, the tumor diameter, metastasis rate outside the ovary, incidence rate of ascites, and CA125 levels in the solid tumor slices group (2.4 +/- 0.61 cm, 75.0%, 50.0%, and 80.13 +/- 11.26 U/ml, respectively) were remarkably higher than those in the cell line group (1.6 +/- 0.53 cm, 52.9%, 29.4%, and 36.5 +/- 6.71 U/ml, respectively) (p < 0.05, respectively). CONCLUSION: There are differences between the two orthotopic xenograft models established with human epithelial ovarian cancer solid tumor tissue slices and human ovarian carcinoma cell line OVCAR-3. The biological characteristics of the solid tumor slices model are more similar to human ovarian cancer.

Safety assessment of ovarian cryopreservation and transplantation in nude mice bearing human epithelial ovarian cancer.

OBJECTIVE: Nude mice with orthotopic transplantation of human ovarian epithelial cancer were used to investigate screening criteria for paraneoplastic normal ovarian tissue and the security of the freezing and thawing for ovarian tissue transplantation. METHODS: Expression of CK-7, CA125, P53, survivin, MMP-2/TIMP- 2 in paraneoplastic normal ovarian tissues were detected by RT-PCR as well as immunohistochemistry. The tissues of the groups with all negative indicators of RT-PCR, all negative indicators of immunohistochemistry, negative expression of CK-7, CA125 and survivin, positive expression of CK-7, CA125 and survivin, cancer tissues and normal ovarian tissues of nude mice were used for freezing and thawing transplantation, to analyze overt and occult carcinogenesis rates after transplantation. RESULTS: When all indicators or the main indicators, CK-7, CA125 and survivin, were negative, tumorigenesis did not occur after transplantation. In addition the occult carcinogenesis rate was lower than in the group with positive expression of CK-7, CA125 and survivin (P<0.01). After subcutaneous and orthotopic transplantation of ovarian tissues, rates did not change (P>0.05). There was no statistical significance among rates after transplantation of ovarian tissues which were obtained under different severity conditions (P>0.05). CONCLUSION: Negative expression of CK-7, CA125 and survivin can be treated as screening criteria for security of ovarian tissues for transplantation. Immunohistochemical methods can be used as the primary detection approach. Both subcutaneous and orthotopic transplantation are safe. The initial severity does not affect the carcinogenesis rate after tissue transplantation. Freezing and thawing ovarian tissue transplantation in nude mice with human epithelial ovarian carcinoma is feasible and safe.

Expression and correlation of Lewis y antigen and TGF-ß1 in ovarian epithelial carcinoma.

Lewis y is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Elevation of Lewis y is frequently observed in epithelial-derived cancers. This study aimed to detect the expression and clinical significance of the Lewis y antigen and TGF-ß1 (transforming growth factor ß1) in ovarian epithelial tumors, and to evaluate the correlation between them. Immunohistochemical staining was used to detect the expression of Lewis y antigen and TGF-ß1 in 60 cases of ovarian epithelial malignant tumors, 20 cases of borderline ovary tumors, 20 cases of benign ovary tumors and 10 cases of normal ovarian tissues. An immunofluorescence double labeling method was also used to detect the correlation between Lewis y antigen and TGF-ß1. The positive rates of Lewis y antigen in ovarian epithelial cancer tissues was 88.33%, significantly higher compared to those of borderline ovarian tumors (60.00%) (P<0.05), benign ovarian tumors (35.00%) (P<0.01) and normal ovarian tissues (0%) (P<0.01). Its expression was not associated with clinical parameters; the positive rates of TGF-ß1 in ovarian epithelial cancers were 78.33%, significantly higher compared to those of benign ovarian tumors (65.00%) (P<0.05) and normal ovarian tissues (40.00%) (P<0.05); the positive rates of the TGF-ß1 and Lewis y were not associated with metastasis of lymph nodes and histological types, differentiation degree and clinical stage (P>0.05). Expression of Lewis y antigen and TGF-ß1 was significantly positively associated with epithelial carcinoma. Close correlation between Lewis y, TGF-ß1 and ovarian cancer was observed. Altered expression of Lewis y antigen may cause changes in TGF-ß1 expression. Lewis y can increase the growth of ovarian cancer cells and the invasion ability by promoting TGF-ß1 abnormal expression and by promoting angiogenesis and a change in its signal transduction pathway. This study provides theoretical evidence for the development of ovarian cancer biological treatments.