Ovarian Epithelial Cells Research Articles

Polyploidy, EZH2 upregulation, and transformation in cytomegalovirus-infected human ovarian epithelial cells

Human cytomegalovirus (HCMV) infection has been implicated in epithelial ovarian cancer (OC). Polyploidy giant cancer cells (PGCCs) have been observed in high-grade serous ovarian carcinoma (HGSOC); they possess cancer stem cell-like characteristics and give rise to progeny cells expressing epithelial-mesenchymal transition (EMT) markers. EZH2 plays a potential oncogenic role, correlating with high proliferative index and tumor grade in OC. Herein, we present the experimental evidence for HCMV as a reprogramming vector that elicited human ovarian epithelial cells (OECs) transformation leading to the generation of “CMV-transformed Ovarian cells” (CTO). The infection with the two high-risk clinical strains, namely HCMV-DB and BL provoked a distinct cellular and molecular mechanisms in infected OECs. EZH2 upregulation and cellular proliferation were curtailed by using EZH2 inhibitors. The HGSOC biopsies were characterized by an elevated EZH2 expression, possessing a strong positive correlation between the aforementioned marker and HCMV. From HGSOC biopsies, we isolated three HCMV clinical strains that transformed OECs generating CTO cells which displayed proliferative potentials in addition to EZH2 upregulation and PGCCs generation; these features were reduced upon EZH2 inhibition. High-risk HCMV strains transformed OECs confirming an HCMV-induced epithelial ovarian cancer model and highlighting EZH2 tumorigenic properties. Our findings might be highly relevant in the pathophysiology of ovarian tumors thereby nominating new targeted therapeutics.

MLK3 localizes mainly to the cytoplasm and promotes oxidative stress injury via a positive feedback loop.

Activation of mixed lineage kinase 3 (MLK3) by phosphorylation at Thr277/Ser281 stimulates downstream apoptotic pathways and ultimately leads to cell injury. MLK3 is reported to localize to both the cytoplasm and nucleus in human ovarian cancer cells and immortalized ovarian epithelial cells (T80 and T90 cells), and phosphorylation at Thr477 is required for the cytoplasmic retention of MLK3 in T80 cells. However, the subcellular distribution of MLK3 in other cell types has rarely been reported, and whether phosphorylation of MLK3 at Thr277/Ser281 affects its subcellular distribution is unknown. Here, our bioinformatics analysis predicted that MLK3 was mainly distributed in the cytoplasm and nucleus. In the human HEK293T embryonic kidney cell line and murine HT22 hippocampal neuronal cell line, endogenous MLK3 was more abundant in the cytoplasm and less abundant in the nucleus. In addition, overexpressed Myc-tagged MLK3 and EGFP-tagged MLK3 were also observed to localize mainly to the cytoplasm. MLK3 that was activated by phosphorylation at Thr277/Ser281 was mainly distributed in the cytoplasm, and phosphorylation deficient (T277A/S281A) and mimic (T277E/S281E) mutants both showed distributions similar to that of wild type (wt) MLK3, further proving that phosphorylation at Thr277/Ser281 was not involved in regulating MLK3 subcellular localization. In HEK293T cells, H2O2 stimulation accelerated MLK3 phosphorylation (activation), and this phosphorylation was reduced by the antioxidant N-acetylcysteine in a dose-dependent manner. Overexpressing wt MLK3 promoted the production of intracellular reactive oxygen species and increased cell apoptosis, both of which were enhanced by the phosphorylation-mimic (T277E/S281E) MLK3 variant but not by the phosphorylation-deficient (T277A/S281A) MLK3 variant. These findings provided additional evidence for the cytoplasmic and nuclear distribution of MLK3 in HEK293T cells or HT22 cells and revealed the pivotal role of MLK3 in the positive feedback loop of oxidative stress injury.

Different Impacts of Cryopreservation in Endothelial and Epithelial Ovarian Cells.

The aim of our laboratory-based study was to investigate the extent of delayed-onset cell death after cryopreservation in endothelial and epithelial cell lines of ovarian origin. We found differences in percentages of vital cells directly after warming and after cultivation for 48 to 72 h. A granulosa cell line of endothelial origin (KGN) and an epithelial cell line (OvCar-3) were used. In both DMSO-containing and DMSO-free protocols, significant differences in vitality rates between the different cell lines when using open and closed vitrification could be shown (DMSO-containing: KGN open vs. OvCar open, p = 0.001; KGN closed vs. OvCar closed, p = 0.001; DMSO-free: KGN open vs. OvCar open, p = 0.001; KGN closed vs. OvCar closed, p = 0.031). Furthermore, there was a marked difference in the percentage of vital cells immediately after warming and after cultivation for 48 to 72 h; whereas the KGN cell line showed a loss of cell viability of 41% using a DMSO-containing protocol, the OvCar-3 cell loss was only 11% after cultivation. Using a DMSO-free protocol, the percentages of late-onset cell death were 77% and 48% for KGN and OvCar-3 cells, respectively. Our data support the hypothesis that cryopreservation-induced damage is cell type and cryoprotective agent dependent.

Prognostic risk factors of serous ovarian carcinoma based on mesenchymal stem cell phenotype and guidance for therapeutic efficacy

BackgroundEpithelial ovarian cancer is the leading cause of death from gynecologic cancer, in which serous ovarian carcinoma (SOC) is the most common histological subtype. Although PARP inhibitors (PARPi) and antiangiogenics have been accepted as maintenance treatment in SOC, response to immunotherapy of SOC patients is limited.MethodsThe source of transcriptomic data of SOC was from the Cancer Genome Atlas database and Gene Expression Omnibus. The abundance scores of mesenchymal stem cells (MSC scores) were estimated for each sample by xCell. Weighted correlation network analysis is correlated the significant genes with MSC scores. Based on prognostic risk model construction with Cox regression analysis, patients with SOC were divided into low- and high-risk groups. And distribution of immune cells, immunosuppressors and pro-angiogenic factors in different risk groups was achieved by single-sample gene set enrichment analysis. The risk model of MSC scores was further validated in datasets of immune checkpoint blockade and antiangiogenic therapy. In the experiment, the mRNA expression of prognostic genes related to MSC scores was detected by real-time polymerase chain reaction, while the protein level was evaluated by immunohistochemistry.ResultsThree prognostic genes (PER1, AKAP12 and MMP17) were the constituents of risk model. Patients classified as high-risk exhibited worse prognosis, presented with an immunosuppressive phenotype, and demonstrated high micro-vessel density. Additionally, these patients were insensitive to immunotherapy and would achieve a longer overall survival with antiangiogenesis treatment. The validation experiments showed that the mRNA of PER1, AKAP12, and MMP17 was highly expressed in normal ovarian epithelial cells compared to SOC cell lines and there was a positive correlation between protein levels of PER1, AKAP12 and MMP17 and metastasis in human ovarian serous tumors.ConclusionThis prognostic model established on MSC scores can predict prognosis of patients and provide the guidance for patients receiving immunotherapy and molecular targeted therapy. Because the number of prognostic genes was fewer than other signatures of SOC, it will be easily accessible on clinic.

Promotive role of eukaryotic translation initiation factor 4A isoform 3 in ovarian cancer cell growth and aerobic glycolysis through the pyruvate dehydrogenase kinase 4 signaling.

Ovarian cancer (OC) represents one of the most detrimental gynecological malignancies. RNA-binding protein eukaryotic translation initiation factor 4A isoform 3 (EIF4A3) is well-regarded as a definitive oncogene that contributes to the development of multiple malignant tumors. This study sought to elucidate the molecular mechanism of EIF4A3 in OC growth and aerobic glycolysis by regulation of pyruvate dehydrogenase kinase 4 (PDK4) mRNA stability. We determined the EIF4A3 and PDK4 expression levels in OC cell lines and normal ovarian epithelial cells, and subsequently evaluated the cell viability and colony formation by cell counting kit-8 and colony formation assays. The degree of cell aerobic glycolysis was evaluated by measurements of lactic acid production, glucose intake, adenosine triphosphate level, extracellular oxygen consumption, and protein levels of pyruvate kinase isozymes M2 and hexokinase-2. Afterwards, we verified the binding of EIF4A3 and PDK4 mRNA via RNA immunoprecipitation, and determined the mRNA stability after actinomycin D treatment. Finally, a series of rescue experiments was performed with pcDNA3.1-PDK4. EIF4A3 and PDK4 were upregulated in OC cells. Silencing EIF4A3 obstructed cell proliferation and aerobic glycolysis, while the same was annulled by EIF4A3 overexpression. Mechanically, EIF4A3 could bind to PDK4 mRNA to stabilize its mRNA and upregulate its protein levels. PDK4 overexpression inverted the inhibitory role of silencing EIF4A3 in proliferation and aerobic glycolysis. Overall, our findings highlighted that EIF4A3 induced OC progression by stabilizing PDK4 mRNA.

Knockdown of heat shock protein family D member 1 (HSPD1) promotes proliferation and migration of ovarian cancer cells via disrupting the stability of mitochondrial 3-oxoacyl-ACP synthase (OXSM)

BackgroundHeat shock protein 60 (HSP60) is essential for the folding and assembly of newly imported proteins to the mitochondria. HSP60 is overexpressed in most types of cancer, but its association with ovarian cancer is still in dispute. SKOV3 and OVCAR3 were used as experimental models after comparing the expression level of mitochondrial HSP60 in a normal human ovarian epithelial cell line and four ovarian cancer cell lines.ResultsLow HSPD1 (Heat Shock Protein Family D (HSP60) Member 1) expression was associated with unfavorable prognosis in ovarian cancer patients. Knockdown of HSPD1 significantly promoted the proliferation and migration of ovarian cancer cells. The differentially expressed proteins after HSPD1 knockdown were enriched in the lipoic acid (LA) biosynthesis and metabolism pathway, in which mitochondrial 3-oxoacyl-ACP synthase (OXSM) was the most downregulated protein and responsible for lipoic acid synthesis. HSP60 interacted with OXSM and overexpression of OXSM or LA treatment could reverse proliferation promotion mediated by HSPD1 knockdown.ConclusionsHSP60 interacted with OXSM and maintained its stability. Knockdown of HSPD1 could promote the proliferation and migration of SKOV3 and OVCAR3 via lowering the protein level of OXSM and LA synthesis.

Abstract LB164: Entinostat restores sensitivity to olaparib in two in vitro models of PARPi resistant ovarian cancer cells

Abstract The use of Poly ADP ribose polymerase inhibitors (PARPi) has revolutionized the treatment of homologous recombination (HR) deficient ovarian cancer tumors. A subset of these tumors exhibit genetic and acquired resistance to PARPi treatment. We have previously shown histone deacetylase inhibitors (HDACi) resensitizes ovarian cancer cells to PARPi. The objective of this study is to examine the effectiveness of combination HDACi and PARPi treatment in two in vitro models of mouse ovarian cancer cells. To investigate these effects, we used the following three ID8 murine ovarian cancer epithelial cell lines: TP53− / − (HR deficient), TP53− / −/BRCA2− / − (HR proficient), and an olaparib resistant line TP53− / −/BRCA2− / −-OR (ID8_OR). We also created ovarian cancer organoids from these three lines. In short, 8-week old female C57BL/6 mice were injected with 7 million untreated cells in PBS. Following the formation of ascites, mice were sacrificed and organoids were derived from both ascites and tumors. For viability assays, cells were pretreated with entinostat (Ent, 0.25uM) for 24h, followed by 72h (2D cells) or 7 days (organoids) of Ent (0-2uM), olaparib (Ola, 0-40uM), or in combination. Cell proliferation and viability was assessed using MTS and ATP-based assays. For immunofluorescence and western blotting analysis, cells were treated with 0.5uM Ent, 10uM Ola or the combination.TP53 adherent cells and organoids treated with 0.5uM Ent + 10uM of Ola significantly reduced cell proliferation when compared to Ola alone (p= 0.0028, 0.0021 Two-Way ANOVA). When compared to Ola and Ent alone, TP53/BRCA2 adherent cells treated with 0.125uM of Ent + 2.5uM of Ola also reduced cell proliferation (p=0.0011 and p=0.0072, 0.0845). Lastly, ID8_OR adherent cells and organoids treated with 0.125uM of Ent + 2.5μM of Ola significantly reduced cell proliferation when compared to Ola alone (p=0.0018, 0.0264).Immunofluorescences analysis revealed, when treated with Ola and Ent combination, adherent TP53 (p= 0.002, n.s), TP53/BRCA2 (p=0.0033, 0.0315), and ID8_OR (n.s) cells displayed increased expression of DNA damage marker gH2AX when compared to Control and Ola alone. We also observed decreased expression of RAD51 in cells treated with combination when compared to control and Ola alone, TP53 (p= <0.0001, 0.0023) and ID8_OR (n.s). In our organoids, we found an increase in gH2AX across all three cell lines when treated with Ola+Ent compared to control and Ola alone: TP53 (p= <0.0001, 0.0057), TP53/BRCA2 (n.s), and ID8_OR (p= 0.0116, 0.0151). Similarly, we found a decrease in RAD51 when cells were treated with Ola+Ent compared to control and Ola alone: TP53 (p= n.s, 0.0418) and ID8_OR (n.s). Lastly, preliminary western blot analysis of the organoids, revealed a decreases in proliferation marker PCNA in both adherent cells and organoids when comparing the control to combination treatment. In conclusion, the combination of Ola and Ent treatment restores responsiveness of two in vitro models of genetic and acquired PARPi resistance through increasing DNA damage and reducing DNA repair and proliferation. Citation Format: Bisiayo E. Fashemi, Lilian N. van Biljon1, Tyler Woodard, Vijayalaxmi Gupta, Mary M. Mullen, Benjamin Bitler, Dineo Khabele. Entinostat restores sensitivity to olaparib in two in vitro models of PARPi resistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB164.

Oncogenic role of copper‑induced cell death‑associated protein DLD in human cancer: A pan‑cancer analysis and experimental verification.

Copper ions can bind directly to lipoylated components of the tricarboxylic acid (TCA) cycle, triggering the aggregation of mitochondrial lipoylated proteins and the destabilization of Fe-S cluster proteins, resulting in copper-dependent cell death. Dihydrolipoamide dehydrogenase (DLD) is a key protein of the TCA cycle and constitutes the E3 component of the α-ketoglutarate dehydrogenase complex, which is deeply interconnected with the mitochondrial electron transfer chain in the TCA cycle. Tumor cells demonstrate dependency on glutaminolysis fuelling to carry out the TCA cycle and essential biosynthetic processes supporting tumor growth. Therefore, DLD plays an important role in the tumor biological process. However, to the best of our knowledge, no pan-cancer analysis is currently available for DLD. Therefore, the present study first explored the DLD expression profile in 33 tumors in publicly available datasets, including TIMER2, GEPIA2, UALCAN, cBioPortal and STRING. TIMER2, GEPIA2 and UALCAN were used for exploring gene expression; survival prognosis was detected by GEPIA2; genetic alteration was analysed by cBioPortal; immune infiltration data was obtained from TIMER2; interacting proteins of DLD were detected by STRING. DLD was found to be highly expressed in colon, liver, lung, stomach, renal, corpus uteri endometrial and ovarian cancers compared with normal tissues, and its high expression was associated with poorer prognosis in ovarian cancer. To the best of our knowledge, the present study provided the first comprehensive pan-cancer analysis of the oncogenic role of DLD across different tumors types. As the expression of DLD in ovarian cancer was high, and high expression is associated with poor prognosis, experimental verification of DLD in ovarian cancer was conducted. In the present study, DLD expression was found to be high in the ovarian cancer OC3 cell line, compared with the normal ovarian epithelial IOSE80 cell line by reverse transcription-quantitative PCR analysis. After knockdown of DLD expression, it was found that DLD regulated metabolic pathways by suppressing the intracellular NAD+/NADH ratio, which then in turn suppressed tumor cell proliferation detected by MTT assay. In conclusion, the present pan-cancer analysis of DLD demonstrated that DLD expression was associated with the clinical prognosis, immune infiltration and tumor mutational burden in 33 tumor types, and experimental verification in ovarian cancer was conducted. These results may contribute to the understanding of the role of DLD in tumorigenesis.

Whole-exome mutational landscape and molecular marker study in mucinous and clear cell ovarian cancer cell lines 3AO and ES2

BackgroundOvarian cancer is one of the most lethal cancers in women because it is often diagnosed at an advanced stage. The molecular markers investigated thus far have been unsatisfactory.MethodsWe performed whole-exome sequencing on the human ovarian cancer cell lines 3AO and ES2 and the normal ovarian epithelial cell line IOSE-80. Molecular markers of ovarian cancer were screened from shared mutation genes and copy number variation genes in the 6q21-qter region.ResultsWe found that missense mutations were the most common mutations in the gene (93%). The MUC12, FLG and MUC16 genes were highly mutated in 3AO and ES2 cells. Copy number amplification occurred mainly in 4p16.1 and 11q14.3, and copy number deletions occurred in 4q34.3 and 18p11.21. A total of 23 hub genes were screened, of which 16 were closely related to the survival of ovarian cancer patients. The three genes CCDC170, THBS2 and COL14A1 are most significantly correlated with the survival and prognosis of ovarian cancer. In particular, the overall survival of ovarian cancer patients with high CCDC170 gene expression was significantly prolonged (P < 0.001). The expression of CCDC170 in normal tissues was significantly higher than that in ovarian cancer tissues (P < 0.05), and its expression was significantly decreased in advanced ovarian cancer. Western blotting and immunofluorescence assays also showed that the expression of CCDC170 in ovarian cancer cells was significantly lower than that in normal cells (P < 0.001, P < 0.01).ConclusionsCCDC170 is expected to become a new diagnostic molecular target and prognostic indicator for ovarian cancer patients, which can provide new ideas for the design of antitumor drugs.

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

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

Abstract 5023: PFKP contributes to ovarian cancer metastasis, stemness and anchorage-independent growth via α5 integrin and ERK/MMP9 signaling

Abstract Background: Ovarian cancer is one of the most aggressive malignancies threatening women’s health worldwide. Its high mortality rate is largely due to symptoms not appearing until late in the disease, usually after the cancer has metastasized. Despite current state-of-the-art treatments, the prognosis for patients remains poor. New effective treatments are urgently needed to improve patient survival. High lactate production and low glucose oxidation, independent of oxygen availability, is known as the Warburg effect and is common in cancer. Phosphofructokinase 1 (PFK-1) irreversibly converts fructose 6-phosphate to fructose 1,6-bisphosphate, the first step in the Warburg effect. The platelet isoform of PFK (PFKP) regulates glycolysis and plays roles in tumorigenesis in other human cancers, the precise mechanisms has yet to be defined. Moreover, PFKP in ovarian cancer remain unknown. We hypothesize that PFKP modulates metastasis and stemness in ovarian cancer. Methods: Expression of PFKP in clinical samples, ascites-derived tumor cells, normal ovarian epithelial cells (HOSEs) and ovarian cancer cell lines was determined by immunohistochemistry/immunofluorescence and qPCR/immunoblotting, respectively. The correlation between PFKP and clinicopathological parameters was analyzed. Ectopic expression or knockdown of PFKP followed by migration, invasion, sphere formation and soft agar assays were performed in ascites-derived tumor cells and ovarian cancer cell lines to investigate the effects on metastasis, stemness and malignant transformation respectively. Additionally, the related signaling pathway was explored by qPCR and immuoblotting. Results: Up-regulation of PFKP in clinical samples, ascites-derived tumor cells and ovarian cancer cell lines was found when compared to HOSEs. Higher PFKP was correlated to shorter overall and disease-free survival. Functionally, knockdown of PFKP by siRNA/shRNA approaches in ascites-derived tumor cells, ES-2 and HeyA8 cells decreased migration, invasion, sphere formation and colony formation in soft agar, which was accompanied by decreased expression α5 integrin, pERK and MMP9. Ectopic expression of PFKP in SKOV-3 cells showed opposite effects. Intriguingly, we found that MEK-1 inhibitor (U0126) or an anti-MMP9 neutralizing antibody could block PFKP-mediated migration/sphere formation abilities. Conclusion: PFKP was overexpressed in ovarian cancer and regulates metastasis, stemness and anchorage-independent growth. α5 integrin and ERK/MMP9 signaling were involved in such functional effects. PFKP could be a potential prognostic marker and therapeutic target for ovarian cancer. Citation Format: Ruiqian Zhang, Michelle K.Y. Siu, Xuetang Mo, Mingo M.H. Yung, Jingjing Wang, Yuxin Jiang, Annie N.Y. Cheung, Hextan Y.S. Ngan, Karen K.L. Chan. PFKP contributes to ovarian cancer metastasis, stemness and anchorage-independent growth via α5 integrin and ERK/MMP9 signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5023.

Up-regulation of oxidized low-density lipoprotein receptor 1 correlates with decreased miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, and miR-4465, higher recurrence rate, and poor prognosis in ovarian cancer.

MicroRNAs (miRNAs) are widely involved in cell metabolism, and their abnormal expression is involved in the regulation of ovarian cancer development, metastasis, and recurrence. The current study aimed to explore the potential mechanism and prognostic value of miRNAs related to the targeted regulation of oxidized low-density lipoprotein receptor 1 (OLR1) expression in ovarian cancer. A prospective study was conducted including 132 ovarian cancer patients. Patients were followed up for 36 months. The dual-luciferase reporter gene detection was used to verify the targeting relationship between miRNA and OLR1. Cell Counting Kit-8 assay, flow cytometric analysis, transwell migration, and invasion assays were performed to address the malignant biological behaviors of ovarian cancer cells. OLR1 protein and gene expression levels were significantly higher in ovarian cancer tissues and cell lines than in adjacent tissues and normal ovarian epithelial cells. OLR1 depletion facilitated apoptosis and impeded cell proliferation, migration, and invasion in ovarian cancer. Predictive software and dual-luciferase reporter assays showed that miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, and miR-4465 targeted OLR1 expression. Functionally, the introduction of miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, and miR-4465 mimics abrogated the aggressive phenotype in ovarian cancer cells. Lastly, compared with adjacent tissues, the levels of miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, and miR-4465 in cancer tissues were significantly lower (P<0.001). Compared with high miR-106b-5p, high miR-93-5p, high miR-3129-5p, high miR-199b-3p, high miR-4465 group and low OLR1 group, the low miR-106b-5p, low miR-93-5p, low miR-3129-5p, low miR-199b-3p, low miR-4465 group and high OLR1 group have significantly higher recurrence (all P<0.05) and higher mortality (all P<0.05). The identification value of recurrence assessment model [Y = 4.267+0.336*(miR-106b-5p)+0.168*(miR-93-5p)+1.847*(miR-3129-5p)+2.119*(miR-199b-3p)+0.872*(miR-4465)-3.408*(OLR1)] was high with an AUC of 0.918. The prognosis assessment model [Y = 3.914+0.143*(miR-106b-5p)+0.102*(miR-93-5p)+0.115*(miR-3129-5p)+1.369*(miR-199b-3p)+0.186*(miR-4465)-0.334*(OLR1)] also had high identification value with an AUC of 0.934. The combined detection of miR-106b-5p, miR-93-5p, miR-3129-5p, miR-199b-3p, miR-4465, and OLR1 is expected to become a molecular biomarker for the long-term prognostic assessment of ovarian cancer.

Tunicamycin induced inhibition of calpain 1 and 2 enzyme activity in ovarian cancer cells

Background: Tunicamycin (TN) is an antitumor agent and induced intracellular calcium levels in many cells, however its molecular mechanism is still needed to be explored. Calpeptin (Calp) is an inhibitor of both calpain 1 and 2 (CAPN-1/2) enzymes, and plays a fundamental role in tumor mechanism. In this study, the effects of TN and Calp were investigated on CAPN-1/2 enzyme activity in normal and ovarian cancer cells adhered to fibronectin. Methods: 24uM TN, 50uM Calp, and combined TN and Calp (TN+Calp) were applied for 1 and 12 hours to FN-bound (FN+) and non-FN-bound (FN-) normal human ovarian epithelial (IHOSE) and ovarian cancer (SKOV-3) cells. The activation of CAPN-1/2 was measured by the luminescent method and the significance of the results was analyzed with the t-test. Results: CAPN-1/2 enzyme activity (at 12 hour) was present in both cell lines, but the level of enzyme is higher in IHOSE cells compared to SKOV-3 cells. The results showed that 1 hour TN and TN+Calp applications stimulated CAPN-1/2 enzyme activity in IHOSE cells but did not show any stimulating effect in SKOV-3 cells. After 12-hour of treatment, the cells with TN, Calp or TN+Calp showed an inhibitory effect on CAPN-1/2 enzyme in both FN+IHOSE and SKOV-3 cells. At 12-hour TN+Calp administration was determined to be the most effective inhibitor in FN+ SKOV-3 since it inhibited CAPN1/2 activity statistically significantly more than both Calp and TN administrations. Conclusions: The effects of TN, Calp and TN+Calp applications on the CAPN-1/2 enzyme varied according to the cell type, normal or cancer cells, and whether the cell was bound to FN and the incubation period. 12 h administrations of TN, Calp or TN+Calp inhibited the CAPN-1/2 enzyme in both FN+ IHOSE and SKOV-3 cells.

Molecular mechanism of Wilms’ tumor (Wt1) (+/−KTS) variants promoting proliferation and migration of ovarian epithelial cells by bioinformatics analysis

Epithelial ovarian cancer (EOC) is a gynecological disease with the highest mortality. With the lack of understanding of its pathogenesis, no accurate early diagnosis and screening method has been established for EOC. Studies revealed the multi-faceted function of Wilms’ tumor (Wt1) genes in cancer, which may be related to the existence of multiple alternative splices. Our results show that Wt1 (+KTS) or Wt1 (−KTS) overexpression can significantly promote the proliferation and migration of human ovarian epithelial cells HOSEpiC, and Wt1 (+KTS) effects were more evident. To explore the Wt1 (+/−KTS) variant mechanism in HOSEpiC proliferation and migration and ovarian cancer (OC) occurrence and development, this study explored the differential regulation of Wt1 (+/−KTS) in HOSEpiC proliferation and migration by transcriptome sequencing. OC-related hub genes were screened by bioinformatics analysis to further explore the differential molecular mechanism of Wt1 (+/−KTS) in the occurrence of OC. Finally, we found that the regulation of Wt1 (+/−KTS) variants on the proliferation and migration of HOSEpiC may act through different genes and signaling pathways and screened out key genes and differentially regulated genes that regulate the malignant transformation of ovarian epithelial cells. The implementation of this study will provide new clues for the early diagnosis and precise treatment of OC.

NUF2 promotes tumorigenesis by interacting with HNRNPA2B1 via PI3K/AKT/mTOR pathway in ovarian cancer

BackgroundOvarian cancer (OC) is one of the commonest and deadliest diseases that threaten the health of women worldwide. It is essential to find out its pathogenic mechanisms and therapeutic targets for OC patients. Although NUF2 (Ndc80 kinetochore complex component) has been suggested to play an important role in the development of many cancers, but little is known about its function and the roles of proteins that regulate NUF2 in OC. This study aimed to investigate the effect of NUF2 on the tumorigenicity of OC and the activities of proteins that interact with NUF2.MethodsOncomine database and immunohistochemical (IHC) staining were used to evaluate the expression of NUF2 in OC tissues and normal tissues respectively. Normal ovarian epithelial cell lines (HOSEpiC) and OC cell lines (OVCAR3、HEY、SKOV3) were cultured. Western blot was applied to analyze the expression of NUF2 in these cell lines. Small interfering RNA (siRNA) was used to silence the expression of NUF2 in OC cell lines, SKOV3 and HEY. Gene Set Variation Analysis (GSVA), Gene Set Enrichment Analysis (GSEA), the CCK-8 method, colony formation assay and flow cytometry were conducted to analyze the biological functions of NUF2 in vitro. OC subcutaneous xenograft tumor models were used for in vivo tests. Immunoprecipitation and mass spectrometry (IP/MS) were performed to verify the molecular mechanisms of NUF2 in OC. IP, immunofluorescence, IHC staining, and Gene Expression Profiling Interactive Analysis platform (GEPIA) were used to analyze the relationship between HNRNPA2B1 and NUF2 in OC cells. SiRNA was used to silence the expression of HNRNPA2B1 in SKOV3 cells, reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay and western blot were used to detect the effect of HNRNPA2B1 on NUF2. GEPIA, The Cancer Genome Atlas (TCGA) database, GSEA and western blot were used to detect the potential signaling pathways related to the roles of HNRNPA2B1 and NUF2 in OC cells.ResultsOur results showed high NUF2 expression in OC tissues and OC cell lines, which was associated with shorter overall survival and progression-free survival in patients. NUF2 depletion by siRNA suppressed the proliferation abilities and induced cell apoptosis of OC cells in vitro, and impeded OC growth in vivo. Mechanistically, NUF2 interacted with HNRNPA2B1 and activated the PI3K/AKT/mTOR signaling pathway in OC cells.ConclusionNUF2 could serve as a prognostic biomarker, and regulated the carcinogenesis and progression of OC. Moreover, NUF2 may interact with HNRNPA2B1 by activating the PI3K/AKT/mTOR signaling pathway to promote the development of OC cells. Our present study supported the key role of NUF2 in OC and suggested its potential as a novel therapeutic target.

Promoting action of long non-coding RNA small nucleolar RNA host gene 4 in ovarian cancer.

Long non-coding RNA (LncRNA) small nucleolar RNA host gene 4 (SNHG4) has been shown to be aberrantly expressed in a variety of cancers and involved in cancer development, but its role in ovarian cancer (OC) is unclear. The purpose of this study was to explore the biological function of SNHG4 in OC and reveal its potential downstream molecular targets. OC tumor tissue and normal tissue were collected; normal human ovarian epithelial cell line (IOSE80) and human ovarian cancer cell line (A2780, SKOV-3, OV-90 and CAOV3) were selected. RT-qPCR was used to detect SNHG4, miR-98-5p, and TMED5, while western blot was used to detect the protein expression levels of TMED5, Ki67, MMP-9, Bcl-2, Bax, Gsk3β, Wnt3a, and β-catenin. The subcellular localization of SNHG4 was assessed by nucleocytoplasmic separation assay. CCK-8, colony formation assay, flow cytometry, and Transwell were used to assess the biological behavior of OC cells. The targeting relationship between SNHG4, miR-98-5p and TMED5 was verified by dual luciferase reporter assay and RIP assay. In OC, SNHG4 and TMED5 were highly expressed, and miR-98-5p was underexpressed. Knockdown of SNHG4 inhibited OC cell proliferation, migration and invasion, promoted apoptosis, and prevented Wnt/β-catenin pathway activation. The effect of knockdown of SNHG4 was reversed by knockdown of miR-98-5p or overexpression of TMED5. Mechanistically, SNHG4 competitively adsorbed miR-98-5p to mediate TMED5 expression, thereby activating the Wnt/β-catenin pathway. SNHG4 accelerates OC development via mediating the miR-98-5p/TMED5 axis and activating the Wnt/β-Catenin pathway. SNHG4 gene silencing might be a novel option for OC treatment.

Expression and Localization of Fas-Associated Factor 1 in Testicular Tissues of Different Ages and Ovaries at Different Reproductive Cycle Phases of Bos grunniens.

Fas-associated factor 1 (FAF1), a member of the Fas family, is involved in biological processes such as apoptosis, inflammation, cell proliferation and proteostasis. This study aimed to explore the biological role of FAF1 in testicular tissue at different ages (juveniles (1 and 2 years old), adults (3, 4, 6, and 7 years old) and old-aged animals (11 years old)) and ovaries during different reproductive cycle phases (follicular, luteal, and pregnancy phases). FAF1 mRNA, relative protein expression and protein expression localization were determined in testes and ovaries using real-time quantification, WB and immunohistochemistry (IHC), respectively. Real-time quantification of testis tissues showed that the relative expression of FAF1 mRNA in testis tissues at 3, 4 and 7 years of age was significantly higher than of those in other ages, and in ovarian tissues was significantly higher in luteal phase ovaries than those in follicular and pregnancy phase ovaries; follicular phase ovaries were the lowest. WB of testis tissues showed that the relative protein expression of FAF1 protein was significantly higher at 11 and 7 years of age; in ovarian tissue, the relative protein expression of FAF1 protein was significantly higher in follicular phase ovaries than in luteal and pregnancy phase ovaries, and lowest in luteal phase ovaries. The relative protein expression of FAF1 at 3, 4 and 7 years of age was the lowest. IHC showed that FAF1 was mainly expressed in spermatozoa, spermatocytes, spermatogonia and supporting cells; in ovarian tissue, FAF1 was expressed in ovarian germ epithelial cells, granulosa cells, cumulus cells and luteal cells. The IHC results showed that FAF1 mRNA and protein were significantly differentially expressed in testes of different ages and ovarian tissues of different reproductive cycle phases, revealing the significance of FAF1 in the regulation of male and female B. grunniens reproductive physiology. Furthermore, our results provide a basis for the further exploration of FAF1 in the reproductive physiology of B. grunniens.

Expression of selected nuclear receptors in human epithelial ovarian cell line Caov3 exposed to bisphenol derivatives.

Objectives. Bisphenol A (BPA) is an indispensable industrial chemical. However, as a proven endocrine disruptor, it may be associated with several health disturbances, including the reproductive functions impairment and cancer. Due to the restriction of BPA usage, many bisphenol derivatives gradually substitute BPA. However, studies have reported adverse biological effects of BPA analogs, but the specific sites of their action remain largely unknown. Nuclear receptors (NRs) appear to play significant roles in various types of cancer. In addition, they are considered relevant targets of bisphenols. In the present study, we investigated the effects of BPA and its analogs bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF) on mRNA expression of selected NRs in the human ovarian epithelial cell line Caov3. The NRs examined included retinoic acid receptor α (RARA), retinoid X receptor α (RXRA), peroxisome proliferator activating receptor β/δ (PPARD), chicken ovalbumin upstream promoter-transcription factor 2 (COUPTFII), and nuclear receptor-related protein 1 (NURR1). Methods. Caov3 cells were treated with the bisphenols at the concentrations of 1 nM, 100 nM, 10 µM and 100 µM. After 24 h and 72 h of incubation, cell viability was determined by the MTS assay, and the selected genes expression was analyzed using RT-qPCR. Results. Bisphenol treatment did not affect Caov3 cell viability, except the significant impairment after exposure to the highest BPAF dose (100 µM). At lower doses, neither bisphenol analog altered the expression of the NRs. However, at the highest concentration (100 µM), BPAF and BPA altered the mRNA levels of PPARD, COUPTFII, and NURR1 in a time- and receptor-specific manner. Conclusions. The effects of bisphenols on the specific NRs in the epithelial ovarian cancer cells were addressed for the first time by the present study. Although generally we did not find that bisphenols may provoke significant alterations in the expression of the selected NRs in Caov3 cells, they may alter mRNA expression of certain NRs at high concentrations.

MicroRNA-592 Inhibits the Growth of Ovarian Cancer Cells by Targeting ERBB3.

Objectives: Ovarian cancer is the most lethal gynecologic malignancy, and targeted therapy for different pathological types and molecular phenotypes is urgent to be studied. Studies have shown that MicroRNA-592 (miR-592) plays an important negative regulatory role in the occurrence of gastrointestinal malignancies, breast cancer, non-small cell lung cancer, and glioma, but the expression of miR-592 in ovarian cancer and the mechanism of action are still unclear. Methods: The expressions of miR-592 were examined by RT-PCR and Western Blot. Cell viability and migratory capacity were detected by CCK-8 and transwell assay. TargetScan (http://www.targetscan.org) was analyzed to predict potential targets of miR-592. Then Dual-luciferase reporter gene assay was performed to verify the targeting relationship between miR-592 and ERBB3. A mouse xenograft model was applied to confirm the effect of miR-592. Results: In our study, we found that the expression of miR-592 is reduced in epithelial ovarian cancer tissues. The exogenous expression of miR-592 inhibits the proliferation, migration, and invasion in epithelial ovarian cancer tumor cells. Furthermore, the exogenous expression of miR-592 inhibits tumor growth in the nude mouse xenograft model. Therefore, miR-592 may play a role of tumor suppressor miRNA in the occurrence and development of ovarian cancer. Further experiments demonstrated that tumor-related ERBB3 is a target gene mediated by miRNA-592. The dual-luciferase reporter system was used to identify miRNA-592 target genes; qPCR and Western Blot were used to detect the expression of ERBB3. Mechanical experiments confirmed that miRNA-592 negatively regulated ERBB3.Conclusion: Together, these findings identify a heretofore unrecognized link between miR-592 and ERBB3 and suggest that targeting on miR-592 warrants attention as a novel and potential therapeutic strategy for ovarian cancer.

Guttiferone BL from the Fruits of Allanblackia gabonensis Induces Mitochondrial-Dependent Apoptosis in PA-1 Ovarian Cancer Cells.

Despite the recent advancement of treatment strategies, cancer ranks 2nd among the causes of death globally. Phytochemicals have gained popularity as an alternate therapeutic strategy due to their nontoxic nature. Here, we have investigated the anticancer properties of guttiferone BL (GBL) along with four known compounds previously isolated from Allanblackia gabonensis. The cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The study was extended for the assessment of the effect of GBL in PA-1 cells apoptosis induction, cell cycle distribution, and change in mitochondrial membrane potential using flow cytometry, Western blot analysis, and real-time PCR. Among the five tested compounds, GBL displayed significant antiproliferative effects against all tested human cancer cells (IC50 < 10μM). Moreover, GBL exhibited no significant cytotoxicity towards normal ovarian epithelial cell line (IOSE 364) up to 50 μM. GBL induced sub-G0 cell cycle arrest and significant upregulation of cell cycle regulatory proteins of ovarian cancer cell PA-1. Furthermore, GBL induced its apoptosis as depicted by the accumulation of cells both at the early and late apoptotic phase in Annexin V/PI assay. In addition, it decreased the PA-1 mitochondrial membrane potential and promoted upregulation of caspase-3, caspase-9, and Bax and downregulation of Bcl-2. GBL also showed a dose-dependent inhibition of PA-1 migration. Altogether, this study reveals that guttiferone BL, studied herein for the first time, exhibits efficient antiproliferative activity by the induction of apoptosis through the mitochondrial-dependent pathway. Its investigation as a therapeutic agent against human cancers especially ovarian cancer should be envisaged.