Objective: To investigate the mechanism of signal transducer and activator of transcription 3 (STAT3) and cancer associated fibroblasts (CAF) jointly generate chemo-resistance in epithelial-ovarian cancer and their effect on prognosis. Methods: A total of 119 patients with high-grade ovarian serous cancer who received surgery in Cancer Hospital of Chinese Academy of Medical Sciences from September 2009 to October 2017 were collected. The clinico-pathological data and follow-up data were complete. Multivariate Cox regression model was used to analyze the prognostic factors. Ovarian cancer tissue chips of patients in our hospital were prepared. EnVision two-step method immunohistochemistry was used to detect the protein expression levels of STAT3, the specific markers of CAF activation, fibroblast activating protein (FAP), and type Ⅰ collagen (COL1A1) secreted by CAF. The relationship between the expression of STAT3, FAP, COL1A1 protein and drug resistance and prognosis of ovarian cancer patients was analyzed, and the correlation between the expression of three proteins was analyzed. These results were verified through the gene expression and prognostic information of human ovarian cancer tissues collected in the GSE26712 dataset of gene expression omnibus (GEO) database. Results: (1) Multivariate Cox regression model analysis showed that chemotherapy resistance was an independent risk factor for overall survival (OS) of ovarian cancer (P<0.001). (2) The expression levels of STAT3, FAP, and COL1A1 proteins in chemotherapy resistant patients were significantly higher than those in chemotherapy sensitive patients (all P<0.05). Patients with high expression of STAT3, FAP, and COL1A1 had significantly shorter OS than those with low expression (all P<0.05). According to the human ovarian cancer GSE26712 dataset of GEO database, patients with high expression of STAT3, FAP, and COL1A1 also showed shorter OS than patients with low expression (all P<0.05), the verification results were consistent with the detection results of ovarian cancer patients in our hospital. (3) Correlation analysis showed that the protein level of STAT3 was positively correlated with FAP and COL1A1 in our hospital's ovarian cancer tissue chips (r=0.47, P<0.001; r=0.30, P=0.006), the analysis of GEO database GSE26712 dataset showed that the expression of STAT3 gene and FAP, COL1A1 gene were also significantly positively correlated (r=0.31, P<0.001; r=0.52, P<0.001). Conclusion: STAT3 and CAF could promote chemotherapy resistance of ovarian cancer and lead to poor prognosis.
Female ; Humans ; Cancer-Associated Fibroblasts/pathology* ; Carcinoma, Ovarian Epithelial ; Ovarian Neoplasms/pathology* ; Prognosis ; STAT3 Transcription Factor/metabolism* ; Drug Resistance, Neoplasm

Objective: To investigate the expression and significance of protease activated receptor 2 (PAR2) in ovarian epithelial carcinoma. Methods: PAR2 mRNA expression levels in 410 cases of epithelial ovarian carcinoma and 88 cases of human normal ovary were analyzed from cancer Genome Atlas (TCGA) database and tissue genotypic expression database (GTEx). Immunohistochemical (IHC) staining of PAR2 protein was performed in 149 patients with ovarian cancer who underwent primary surgical treatment at Cancer Hospital of Chinese Academy of Medical Sciences. Then the relationship between mRNA/protein expression of PAR2 and clinicopathological features and prognosis was analyzed. Gene functions and related signaling pathways involved in PAR2 were studied by enrichment analysis. Results: The mRNA expression of PAR2 in epithelial ovarian carcinoma was significantly higher than that in normal ovarian tissue (3.05±0.72 vs. 0.33±0.16, P=0.004). There were 77 cases showing positive and 19 showing strong positive of PAR2 IHC staining among the 149 patients, accounting for 64.4% in total. PAR2 mRNA/protein expression was closely correlated with tumor reduction effect and initial therapeutic effect (P<0.05). Survival analysis showed that the progression free survival time (P=0.033) and overall survival time (P=0.011) in the group with high PAR2 mRNA expression was significantly lower than that in the low PAR2 mRNA group. Multivariate analysis showed tumor reduction effect, initial therapeutic effect were independent prognostic factors on both progression-free survival and overall survival (P<0.05). The progression-free survival (P=0.016) and overall survival (P=0.038) of the PAR2 protein high expression group was significantly lower than that of the low group. Multivariate analysis showed PAR2 expression, initial treatment effect and chemotherapy resistance were independent prognostic factors on both progression-free survival and overall survival (P<0.05). Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), PAR2 target genes were mainly enriched in function related to intercellular connection, accounting for 40%. Gene enrichment analysis (GSEA) showed that the Wnt/β-catenin signaling pathway (P=0.023), the MAPK signaling pathway (P=0.029) and glycolysis related pathway (P=0.018) were enriched in ovarian cancer patients with high PAR2 mRNA expression. Conclusions: PAR2 expression is closely related to tumor reduction effect, initial treatment effect and survival of ovarian cancer patients. PAR2 may be involved in Wnt/β-catenin signaling pathway and intercellular connection promoting ovarian cancer invasion and metastasis.
Female ; Humans ; Carcinoma, Ovarian Epithelial ; Receptor, PAR-2 ; Ovarian Neoplasms/pathology* ; Prognosis ; RNA, Messenger/metabolism*

Objective: To investigate the clinical significance of endothelin A receptor (ETAR) expression in high-grade serous ovarian carcinoma (HGSOC). To design ETAR carboxyl terminal (ETAR-C) amino acids derived polypeptide and to study the inhibitory effect on ovarian epithelial carcinoma cells in vitro. Methods: (1) A total of 126 patients who received surgical treatment and were diagnosed with HGSOC by postoperative pathological examination in Central Hospital of Xuzhou from January 1, 2007 to December 31, 2017 were selected. All patients had completed clinicopathological data and follow-up data. Cancer tissue samples were collected and ETAR mRNA expression in HGSOC tissues was detected by reverse transcript-PCR. The clinical significance was analyzed. (2) ETAR-C fusion polypeptide was designed based on the sequence of carboxyl terminal amino acids of ETAR, expressed and purified in vitro. The effects of ETAR-C fusion polypeptide on migration and invasion ability of ovarian cancer SKOV3 and CAOV3 cells were detected by scratch test and invasion test, respectively. The effect of ETAR-C fusion polypeptide on chemosensitivity of cisplatin-resistant ovarian cancer SKOV3/cDDP and CAOV3/cDDP cells was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The effect of ETAR-C fusion polypeptide on β-arrestin-1 expression in ovarian cancer SKOV3 and CAOV3 cells was detected by western blot. Results: (1) The relative expression level of ETAR mRNA in HGSOC tissues was 18.6±5.1. Patients with HGSOC were divided into high ETAR mRNA expression (n=76) and low ETAR mRNA expression (n=50) with 61.7% as cut-off value analyzed by X-Tile software. High expression of ETAR mRNA was significantly correlated with abdominal water volume, platinum drug resistance, and cancer antigen 125 (CA₁₂₅) value in HGSOC patients (all P<0.05), but was not related to the age of patients with HGSOC and the size of postoperative residual lesions (all P>0.05). The 5-year progression free survival rates were 18.4% and 28.0%, and the 5-year overall survival rates were 38.2% and 52.0% in HGSOC patients with high and low ETAR mRNA expression respectively, there were statistically significant differences (P=0.046, P=0.034). (2) The results of scratch test and invasion test showed that the scratch healing rate and cell invasion rate of SKOV3 or CAOV3 cells treated with endothelin-1 (ET-1) and ET-1+ETAR-C were respectively compared, and the differences were statistically significant (all P<0.05). MTT assay showed that the inhibition rates of ETAR-C fusion polypeptide treated in SKOV3/cDDP and CAOV3/cDDP cells were significantly higher than those of control cells after the addition of 4, 6, 8, 10, 12, and 24 μg/ml cisplatin (all P<0.05). Western blot analysis showed that the relative expression levels of β-arrestin-1 in SKOV3 or CAOV3 cells treated with ET-1 and ET-1+ETAR-C were 1.85±0.09 and 1.13±0.09 (SKOV3 cells), 2.14±0.15 and 1.66±0.12 (CAOV3 cells), respectively. The differences were statistically significant (all P<0.05). Conclusions: The prognosis of HGSOC patients with high expression of ETAR mRNA is significantly worse than those with low expression of ETAR mRNA. ETAR might be a new target for HGSOC treatment. The ETAR-C fusion polypeptide that interferes with the interaction of ETAR and β-arrestin-1 has good inhibitory effect on ovarian cancer cells in vitro, and might have clinical application potential.
Female ; Humans ; Amino Acids/therapeutic use* ; beta-Arrestins/therapeutic use* ; Cell Line, Tumor ; Cisplatin/pharmacology* ; Clinical Relevance ; Ovarian Neoplasms/pathology* ; Receptor, Endothelin A/therapeutic use* ; RNA, Messenger/metabolism*

Objective: To explore the effect of down-regulation of retinol binding protein 2 (RBP2) expression on the biological characteristics of ovarian cancer cells and its mechanism. Methods: Knockdown of RBP2 and cisplatin (DDP)-resistant ovarian cancer cell line SKOV3/DDP-RBP2i was established, the negative control group and blank control group were also set. Cell counting kit 8 (CCK-8) was used to detect the cell proliferation ability, flow cytometry was used to detect cell apoptosis, scratch test and Transwell invasion test were used to detect cell migration and invasion ability, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and western blot were used to detect the expressions of molecular markers related to epithelial-mesenchymal transition (EMT). The effect of RBP2 on the growth of ovarian cancer was verified through experiment of transplanted tumors in nude mice, and the relationships between RBP2 expression and tumor metastasis and patient prognosis were analyzed using the clinical data of ovarian cancer in TCGA database. Results: After down-regulating the expression of RBP2, the proliferation ability of SKOV3/DDP cell was significantly reduced. On the fifth day, the proliferation activities of SKOV3/DDP-RBP2i group, negative control group and blank control group were (56.67±4.16)%, (84.67±3.51) and (87.00±4.00)% respectively, with statistically significant difference (P<0.001). The apoptosis rate of SKOV3/DDP-RBP2i group was (14.19±1.50)%, higher than (8.77±0.75)% of the negative control group and (7.48±0.52)% of the blank control group (P<0.001). The number of invasive cells of SKOV3/DDP-RBP2i group was (55.20±2.39), lower than (82.60±5.18) and (80.80±7.26) of the negative control group and the blank control group, respectively (P<0.001). The scratch healing rate of SKOV3/DDP-RBP2i group was (28.47±2.72)%, lower than (50.58±4.06)% and (48.92±4.63)% of the negative control group and the blank control group, respectively (P<0.001). The mRNA and protein expressions of E-cadherin in the SKOV3/DDP-RBP2i group were higher than those in the negative control group (P=0.015, P<0.001) and the blank control group (P=0.006, P<0.001). The mRNA and protein expression of N-cadherin in SKOV3/DDP-RBP2i group were lower than those in the negative control group (P=0.012, P<0.001) and the blank control group (P=0.005, P<0.001). The mRNA and protein expressions of vimentin in SKOV3/DDP-RBP2i group were also lower than those in the negative control group (P=0.016, P=0.001) and the blank control group (P=0.011, P=0.001). Five weeks after the cells inoculated into the nude mice, the tumor volume of SKOV3/DDP-RBP2i group, negative control group and blank control group were statistically significant different. The tumor volume of SKOV3/DDP-RBP2i group was smaller than those of negative control group and blank control group (P=0.001). Bioinformatics analysis showed that the expression of RBP2 in patients with metastatic ovarian cancer was higher than that without metastasis (P=0.043), and the median overall survival of ovarian cancer patients with high RBP2 expression was 41 months, shorter than 69 months of low RBP2 expression patients (P<0.001). Conclusion: Downregulation of the expression of RBP2 in SKOV3/DDP cells can inhibit cell migration and invasion, and the mechanism may be related to the inhibition of EMT.
Animals ; Apoptosis ; Carcinoma, Ovarian Epithelial/genetics* ; Cell Line, Tumor ; Cell Proliferation ; Cisplatin/pharmacology* ; Drug Resistance, Neoplasm/genetics* ; Female ; Gene Silencing ; Humans ; Mice ; Mice, Nude ; Ovarian Neoplasms/pathology* ; Retinol-Binding Proteins, Cellular/metabolism*

Mucin16 (MUC16), also known as carbohydrate antigen 125 (CA125), is a glycoprotein antigen that can be recognized by the monoclonal antibody OC125 detected from epithelial ovarian carcinoma antigen by Bast et al in 1981. CA125 is not present in normal ovarian tissue but is usually elevated in the serum of epithelial ovarian carcinoma patients. CA125 is the most commonly used serologic biomarker for the diagnosis and recurrence monitoring of epithelial ovarian carcinoma. MUC16 is highly expressed in varieties of tumors. MUC16 can interact with galectin-1/3, mesothelin, sialic acid-binding immunoglobulin-type lectins-9 (Siglec-9), and other ligands. MUC16 plays an important role in tumor genesis, proliferation, migration, invasion, and tumor immunity through various signaling pathways. Besides, therapies targeting MUC16 have some significant achievements. Related preclinical studies and clinical trials are in progress. MUC16 may be a potential novel target for tumor therapy. This article will review the mechanism of MUC16 in tumor genesis and progression, and focus on the research actuality of MUC16 in tumor therapy. This article also provides references for subsequent tumor therapy studies targeting MUC16.
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CA-125 Antigen/metabolism* ; Carcinoma, Ovarian Epithelial ; Female ; Humans ; Lung Neoplasms ; Membrane Proteins/metabolism* ; Ovarian Neoplasms/pathology*

OBJECTIVE: To analyze the expression of immunoglobulin mucin molecule 3 (TIM-3) in epithelial ovarian cancer (EOC) and the effects of TIM-3 knockdown and overexpression on proliferation and migration of ovarian cancer cells. METHODS: We analyzed TIM-3 expression in EOC and normal ovarian tissues using GEPIA database. We also detected TIM-3 expression levels in 82 surgical specimens of EOC and 18 specimens of normal ovarian tissues using immunohistochemistry, and analyzed the correlation of TIM-3 expression with clinicopathological parameters and survival outcomes of the patients. The expression of TIM-3 and Wnt1 mRNA in the tissues were detected using qRT-PCR. We constructed SKOV3 cell models of TIM-3 knockdown and overexpression and examined the changes in proliferation, apoptosis, migration and invasion of the cells using MTT assay, Annexin V-FITC/PI staining, scratch test and Transwell assay. The activity of Wnt/β-catenin pathway in the transfected was detected using dual luciferase reporter assay, and the mRNA levels of TCF-7, TCCFL-2 and CD44 were detected using qPCR. The protein expressions of MMP-9, CD44, Wnt1, β-catenin and E-cad in the transfected cells were detected with Western blotting. RESULTS: The positive expression rate of TIM-3 was significantly higher in EOC tissues than in normal ovarian tissues (P < 0.05). The expression of TIM-3 was significantly correlated with FIGO stage, histological differentiation and lymph node metastasis, and was positively correlated with Wnt1 level (P < 0.05). In SKOV3 cells, TIM-3 knockdown significantly lowered the activity of Wnt/ β-catenin pathway, inhibited cell proliferation, migration and invasion, and promoted cell apoptosis. TIM-3 knockdown significantly down-regulated the mRNA levels of TCF-7, TCFL-2 and CD44 and the protein levels of MMP-9, CD44, Wnt1 and β-catenin, and significantly up-regulated the expression level of E-cad (P < 0.05). Overexpression of TIM-3 caused opposite effects in SKOV3 cells. CONCLUSION TIM-3 is highly expressed in EOC tissue to promote malignant behaviors of the tumor cells possibly by activating the Wnt/β-catenin signal pathway.
Carcinoma, Ovarian Epithelial/pathology* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Female ; Gene Expression Regulation, Neoplastic ; Hepatitis A Virus Cellular Receptor 2/metabolism* ; Humans ; Ovarian Neoplasms/metabolism*

Objective: The underlying mechanism of Ezrin in ovarian cancer (OVCA) is far from being understood. Therefore, this study aimed to assess the role of Ezrin in OVCA cells (SKOV3 and CaOV3) and investigate the associated molecular mechanisms. Methods: We performed Western blotting, reverse transcription-quantitative polymerase chain reaction, MTT, cell colony, cell wound healing, transwell migration and invasion, RhoA and Rac active pull down assays, and confocal immunofluorescence experiments to evaluate the functions and molecular mechanisms of Ezrin overexpression or knockdown in the proliferation and metastasis of OVCA cells. Results: The ectopic expression of Ezrin significantly increased cell proliferation, invasiveness, and epithelial-mesenchymal transition (EMT) in OVCA cells. By contrast, the knockdown of endogenous Ezrin prevented OVCA cell proliferation, invasiveness, and EMT. Lastly, we observed that Ezrin can positively regulate the active forms of RhoA rather than Rac-1 in OVCA cells, thereby promoting robust stress fiber formation. Conclusion Our results indicated that Ezrin regulates OVCA cell proliferation and invasiveness by modulating EMT and induces actin stress fiber formation by regulating Rho-GTPase activity, which provides novel insights into the treatment of the OVCA.
Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cytoskeletal Proteins/metabolism* ; Epithelial-Mesenchymal Transition ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Neoplasm Invasiveness ; Ovarian Neoplasms/pathology* ; Stress Fibers/metabolism* ; rhoA GTP-Binding Protein/metabolism*

Antigens, Neoplasm ; genetics ; Apoprotein(a) ; genetics ; Carrier Proteins ; genetics ; Endometriosis ; metabolism ; pathology ; Endometrium ; metabolism ; pathology ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Membrane Proteins ; genetics ; Mutation, Missense ; genetics ; Nuclear Proteins ; genetics ; Ovarian Neoplasms ; metabolism ; pathology ; Proprotein Convertase 5 ; genetics ; Salivary Cystatins ; genetics ; Ubiquitin-Protein Ligases ; genetics ; Whole Exome Sequencing

OBJECTIVE: To isolate tumor stem-like cells from human epithelial ovarian cancer SKOV3 cells and explore their role in the formation of vascularization mimicry (VM). METHODS: SKOV3 cells were passaged to the 7th generation by suspension culture in serum-free medium, and the percentages of CD133- and CD117-positive cells in the 1st, 3rd, 5th and 7th generations were analyzed using flow cytometry. The proliferative activity of the cells sorted from the 7th generation SKOV3 cells was assessed with colony formation assay. A three-dimensional cell culture model was established to compare the ability of VM formation between the sorted cells and the parental SKOV3 cells. The expression levels of matrix metalloproteinases-2 (MMP-2) and MMP-9 in the two groups were detected using real-time PCR and Western blotting. RESULTS: Some SKOV3 cells formed typical cell spheres with suspension growth in serum-free medium and were passaged to the 7th generation. Flow cytometry revealed that the percentage of CD133-positive cells increased with cell passaging. The cloning efficiency of the sorted cells was significantly higher than that of the parental SKOV3 cells (50.33% 5.33%, < 0.001). The VM formation ability of the sorted cells was stronger than that of the parental SKOV3 cells in the three-dimensional cell culture system. RT-PCR and Western blotting showed that the expression levels of MMP-2 and MMP-9 were significantly higher in the 7th passage cells than in the parental cells ( < 0.05). CONCLUSIONS The sorted cells from SKOV3 cells cultured in serum-free medium exhibit biological properties of tumor stem cells with strong VM formation ability, suggesting their role in VM formation.
Carcinoma, Ovarian Epithelial ; pathology ; Cell Line, Tumor ; Cell Movement ; Female ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplastic Stem Cells ; cytology ; Neovascularization, Pathologic ; pathology ; Ovarian Neoplasms ; pathology

The aim of this study was to investigate the effect of Wnt5a on the vincristine (VCR) resistance in human ovarian carcinoma SKOV3 cells and its possible mechanism. The drug-resistant SKOV3/VCR cells were established by stepwise exposure to VCR, and then the SKOV3/VCR cells were stably transfected with specific shRNA interference plasmid vector targeting for Wnt5a. The mRNA expression level of Wnt5a was measured by RT-PCR. CCK-8 assay was used to detect the cell viability of SKOV3/VCR cells. The apoptosis was analyzed by flow cytometry. The protein expression levels of Wnt5a, MDR1, Survivin, β-catenin, Akt, p-Akt(S473), GSK3β and p-GSK3β(Ser9) were detected by Western blot. The result showed that SKOV3/VCR cells had significantly higher protein expression levels of Wnt5a, MDR1, Survivin and β-catenin, phosphorylation levels of Akt and GSK3β, and mRNA expression level of Wnt5a, compared with SKOV3 cells (P < 0.05). WNT5A gene silencing significantly increased the sensitivity of SKOV3/VCR cells to VCR, the IC of VCR being decreased from 38.412 to 9.283 mg/L (P < 0.05), synergistically enhanced VCR-induced apoptosis of SKOV3/VCR cells (P < 0.05), down-regulated the protein expression levels of MDR1, β-catenin and Survivin (P < 0.05), and inhibited phosphorylation of Akt and GSK3β (P < 0.05). Meanwhile, LY294002 (PI3K inhibitor) decreased the protein expression levels of MDR1, β-catenin and Survivin, as well as the phosphorylation levels of Akt and GSK3β in SKOV3/VCR cells (P < 0.05). These results suggest that WNT5A gene silencing reverses VCR resistance in SKOV3/VCR cells possibly through blocking the PI3K/Akt/GSK3β/β-catenin signaling pathway, and thus down-regulating the protein expression levels of MDR1 and Survivin.
ATP Binding Cassette Transporter, Subfamily B ; metabolism ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Gene Silencing ; Glycogen Synthase Kinase 3 beta ; metabolism ; Humans ; Ovarian Neoplasms ; pathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Survivin ; metabolism ; Vincristine ; pharmacology ; Wnt-5a Protein ; metabolism