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 cytotoxic effects of induced pluripotent stem (iPS) cells of anti-mesothelin (MSLN)-chimeric antigen receptor natural killer (CAR-NK) cells (anti-MSLN-iCAR-NK cells) on ovarian epithelial cancer cells. Methods: Twenty cases of ovarian cancer patients who underwent surgical treatment at Henan Provincial People's Hospital from September 2020 to September 2021 were collected, and 20 cases of normal ovarian tissues resected during the same period due to other benign diseases were also collected. (1) Immunohistochemistry and immunofluorescence were used to verify the expression of MSLN protein in ovarian cancer tissues. (2) Fresh ovarian cancer tissues were extracted and cultured to obtain primary ovarian cancer cells. Recombinant lentiviral vectors targeting anti-MSLN-CAR-CD₂₄₄ were constructed and co-cultured with iPS cells to obtain anti-MSLN-iCAR cells. These cells were differentiated into anti-MSLN-iCAR-NK cells using cytokine-induced differentiation method. The cell experiments were divided into three groups: anti-MSLN-iCAR-NK cell group, natural killer (NK) cell group, and control group. (3) Flow cytometry and live cell staining experiment were used to detect the apoptosis of ovarian cancer cells in the three groups. (4) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B (GZMB), perforin 1 (PRF1), interleukin (IL)-6, and IL-10 in the three groups of ovarian cancer cells. Results: (1) Immunohistochemistry analysis showed that a positive expression rate of MSLN protein in ovarian cancer tissues of 65% (13/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ²=4.912, P=0.027). Immunofluorescence analysis revealed that the positive expression rate of MSLN protein in ovarian cancer tissues was 70% (14/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ²=6.400, P=0.011). (2) Flow cytometry analysis showed that the apoptotic rate of ovarian cancer cells in the anti-MSLN-iCAR-NK cell group was (29.27±0.85)%, while in the NK cell group and control group were (8.44±0.34)% and (6.83±0.26)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.01). Live cell staining experiment showed that the ratio of dead cells to live cells in the anti-MSLN-iCAR-NK cell group was (36.3±8.3)%, while in the NK cell group and control group were (5.4±1.4)% and (2.0±1.3)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.001). (3) ELISA analysis revealed that the expression levels of IFN-γ, TNF-α, GZMB, PRF1, IL-6, and IL-10 in ovarian cancer cells of the anti-MSLN-iCAR-NK cell group were significantly higher than those in the NK cell group and the control group (all P<0.05). Conclusion: The anti-MSLN-iCAR-NK cells exhibit a strong killing ability against ovarian cancer cells, indicating their potential as a novel immunotherapy approach for ovarian cancer.
Humans ; Female ; Carcinoma, Ovarian Epithelial/metabolism* ; Ovarian Neoplasms/metabolism* ; Interleukin-10/pharmacology* ; Induced Pluripotent Stem Cells/metabolism* ; Iron-Dextran Complex/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism* ; Cell Line, Tumor ; Killer Cells, Natural ; Interleukin-6

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.
.
CA-125 Antigen/metabolism* ; Carcinoma, Ovarian Epithelial ; Female ; Humans ; Lung Neoplasms ; Membrane Proteins/metabolism* ; Ovarian Neoplasms/pathology*

Objective: To study the effects of exosome secreted by ovarian cancer (OC) cell on the differentiation and metastasis of normal fibroblasts (NFs). Methods: NFs were collected from patients who underwent hysteromyoma resection in the Affiliated Hospital of Qingdao University from May to December 2019. Exosome was extracted from the culture supernatant of SKOV3 cells by using ultra-high-speed centrifugation. The NFs were co-cultured with condition medium (CM), exosome of SKOV3 (SKOV3-exo) and control medium. The expression levels of fibroblast activation protein (FAP) and α-smooth muscle actin (α-SMA) were detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. The metastatic ability of NFs was detected by Transwell array. Results: Under the transmission electron microscope, the extracellular vesicles extracted from the culture supernatant of SKOV3 were 30-100 nm in diameter with cup holder-like bilayer membrane structure, and the protein expression levels of TSG101 and HSP27 in exosomes (1.00±0.05 and 1.12±0.13) were higher than those of ovarian cancer SKOV3 cells (0.22±0.21 and 0.36±0.14, respectively, P<0.05). PKH67 fluorescently labeled exosomes could be taken up by NFs. The expression levels of α-SMA and FAP mRNA in CM group(2.91±0.15 and 3.21±0.33)and SKOV3-exo group (3.50±0.21 and 4.63±0.24, respectively) were higher than that in blank group (1.00±0.06 and 1.00±0.13, P<0.05). The protein expression levels of α-SMA and FAP in CM group and SKOV3-exo group (0.89±0.11 and 1.25±0.09, 0.81±0.09 and 1.20±0.12) were higher than those in the blank group (0.12±0.31 and 0.11±0.19, respectively, P<0.05). The migrated numbers of cells in the CM group and SKOV3-exo group [(215.01±14.80) and (389.72±19.43), respectively] were higher than that in the blank group [(113.73±4.70), P<0.05]. Conclusion: The exosome secreted by SKOV3 cells can be taken up by NFs, which makes it to differentiate into cancer associated fibroblasts (CAFs) and significantly enhances its metastatic ability, indicating that OC cells may promote the transformation of normal ovarian mesenchymal fibroblasts to CAFs through exosome pathways, and then promote the development of ovarian cancer.
Carcinoma, Ovarian Epithelial ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Coculture Techniques ; Exosomes ; Female ; Fibroblasts ; Humans ; Ovarian Neoplasms/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*

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: To investigate whether miR-125b-5p regulates biological behaviors of ovarian cancer cells by targeted regulation of CD147 expression. METHODS: RT-qPCR was used to detect the expression of miR-125b-5p and CD147 mRNA in ovarian cancer tissues and cancer cell lines. SKOV3 cells transfected with miR-125b-5p mimic and HO8910 cells transfected with miR-125b-5p inhibitor were examined for changes in proliferation, migration and invasion using CCK-8 assay, colonyforming assay and Transwell assay. Starbase was used to predict the potential binding sites between miR-125b-5p and CD147, and double luciferase reporter gene assay was used to verify the targeting relationship. In SKOV3 cells, the effects of cotransfection with miR-125b-5p mimic and pcDNA3.1-CD147 (or pcDNA3.1) plasmid on cell proliferation, migration and invasion were assessed with CCK-8 assay and Transwell assay. RESULTS: The expression of miR-125b-5p was significantly lowered and that of CD147 was increased in both ovarian cancer tissues and ovarian cancer cell lines (P < 0.05). Overexpression of miR-125b-5p in SKOV3 cells resulted in significantly suppressed cell proliferation, migration and invasion, while downregulation of miR-125b-5p in HO8910 cells promoted cell proliferation, migration and invasion. Bioinformatic analysis predicted that miR-125b-5p binds to CD147, which was confirmed by luciferase reporter gene assay. RT-qPCR and Western blotting showed that miR-125b-5p negatively regulated CD147 expression (P < 0.05). In SKOV3 cells, the inhibitory effects of miR-125b-5p mimic on cell proliferation, invasion and migration were significantly attenuated by co-transfection of the cells with pcDNA3.1-CD147 plasmid. CONCLUSION miR-125b-5p inhibits the migration and invasion of ovarian cancer cells by negatively regulating the expression of CD147.
Basigin ; Carcinoma, Ovarian Epithelial ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs/metabolism* ; Neoplasm Invasiveness/genetics* ; Ovarian Neoplasms/genetics* ; RNA, Messenger/genetics* ; Sincalide/metabolism*

Objective: To investigate the molecular mechanism of high phosphorylation levels of cofilin-1 (p-CFL-1) associated with paclitaxel resistance in epithelial ovarian cancer (EOC) cells. Methods: Cells displaying varying levels of p-CFL-1 and CFL-1 were created by plasmid transfection and shRNA interference. Cell inhibition rate indicating paclitaxel efficacy was assessed by Cell Counting Kit-8 (CCK-8) assay. Apoptosis was assessed by flow cytometry and protein levels were detected by western blotting. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of phosphokinases and phosphatases of CFL-1. Survival analysis evaluated the correlation between the prognosis of EOC patients and the levels of p-CFL-1 and slingshot-1 (SSH-1). Results: High levels of p-CFL-1 were observed in EOC cells that survived treatment with high doses of paclitaxel. SKOV3 cell mutants with upregulated p-CFL-1 showed impaired paclitaxel efficacy, as well as decreased apoptosis rates and pro-survival patterns of apoptosis-specific protein expression. Cytoplasmic accumulation of p-CFL-1 inhibited paclitaxel-induced mitochondrial apoptosis. SSH-1 silencing mediated CFL-1 phosphorylation in paclitaxel-resistant SKOV3 cells. Clinically, the high level of p-CFL-1 and the low level of SSH-1 in EOC tissues were closely related to chemotherapy resistance and poor prognosis in EOC patients. Conclusion The SSH-1/p-CFL-1 signaling pathway mediates paclitaxel resistance by apoptosis inhibition in EOC and is expected to be a potential prognostic predictor.
Antineoplastic Agents, Phytogenic/therapeutic use* ; Apoptosis ; Carcinoma, Ovarian Epithelial/metabolism* ; Cell Line, Tumor ; Cofilin 1/metabolism* ; Drug Resistance, Neoplasm ; Female ; Humans ; Ovarian Neoplasms/metabolism* ; Paclitaxel/therapeutic use* ; Phosphoprotein Phosphatases/metabolism* ; Phosphorylation

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