Activation of the caspase-1/gasdermin D pathway via α-linolenic acid-mediated GPR120 signaling induces pyroptosis and suppresses ovarian cancer tumor growth.

Stanniocalcin 1 (STC1) is a secreted glycoprotein hormone. High expression of STC1 has been associated with several cancers including ovarian cancer, but its role in the development of ovarian cancer is not clear. We used five human ovarian epithelial cancer cell lines (OVCA420, OVCA432, OVCA433, SKOV3, and HEY), immortalized human ovarian surface epithelial cells (T29 and T80), ovarian cancer tissues from 342 patients, serum from 73 ovarian cancer patients and from58 control subjects, and 116 mice, with six or eight per group. Protein expression was assessed. Cells overexpressing STC1 protein were generated by ectopic expression of human STC1 cDNA. STC1 expression was silenced by using small interfering RNA against STC1. Cell proliferation, migration, colony formation, and apoptosis were assessed. Xenograft tumor growth in mice was studied. Neutralizing anti-STC1 antibody was used to inhibit STC1 function. All statistical tests were two-sided. STC1 protein expression was higher in all human ovarian cancer cell lines examined than in immortalized human ovarian epithelial cell lines, higher in ovarian cancer tissue than in normal ovarian tissue (P < .001), and higher in serum from ovarian cancer patients than from control subjects (P = .021). Ovarian cancer cells with STC1 overexpression, compared with corresponding control cells, had increased cell proliferation, migration, and colony formation in cell culture and increased growth of xenograft tumors in mice. These activities in normal or malignant ovarian cells with STC1 overexpression, compared with control cells, were also accompanied by increased expression of cell cycle regulatory proteins and antiapoptotic proteins but decreased cleavage of several caspases. Within 24 hours of treatment, apoptosis in cultures of HEY ovarian cancer cells treated with neutralizing anti-STC1 monoclonal antibody was higher (17.3% apoptotic cells) than that in cultures treated with mouse IgG control cells (4.4%) (12.9% difference, 95% confidence interval = 11.6% to 14.2%). STC1 protein may be involved in ovarian tumorigenesis.

The purpose of this study is to investigate underlying mechanism of how cytokine interferon-gamma (IFN-γ) regulates PD-L1 expression in ovarian cancer cells. We treated a panel of human and mouse ovarian cancer cell lines with recombinant human/mouse IFN-γ. Our data showed that IFN-γ up-regulated mRNA and protein expression of PD-L1 significantly in a majority of ovarian cancer cells. The functional IFN-γ receptor is comprised of two ligand-binding IFNGR1 chains associated with two signal-transducing IFNGR2 chains and associated signaling machinery. Here we found that the mRNA expression levels of IFNGR1 and IFNGR2 were abundant in all human ovarian cancer cell lines being tested, while their expressions were not affected by IFN-γ treatment. After knocking down the expression levels of IFNGR1 and IFNGR2 in a ovarian cancer cell line by target gene-specific siRNA, our data showed that the IFN-γ-mediated induction of PD-L1 were diminished in the ovarian cancer cells when compared to those with non-targeting scrambled siRNA controls, indicating the induction of PD-L1 by IFN-γ is dependent on the presence of IFN-γ receptors in the ovarian cancer cells. Although abundant expression of IFNGR1 and IFNGR2 were found in all human ovarian cancer cell lines being tested, the IFN-γ-mediated induction of PD-L1 was not detected in a few of the human ovarian cancer cell lines (namely IGROV-1, TOV21G and SKOV3). We further investigated the integrity of IFN-γ signaling in the human ovarian cancer cell lines by examining activation of STAT1 protein and induction of IRF-1 gene in human ovarian cancer cell lines after IFN-γ treatment. Our data showed that phosphorylated-STAT1 protein and IRF-1 gene expression were up-regulated significantly in a majority of human ovarian cancer cells after IFN-γ treatment, except IGROV-1 and TOV21G cells. These results suggested that IGROV-1 and TOV21G cells might harbor defects in intracellular JAK-STAT1 signaling. We then examined the presence of JAK1 truncating mutations in human ovarian cancer cell lines by Sanger sequencing, and confirmed that IGROV-1 and TOV21G cells, but not the others, have JAK1 truncating mutations. Since our data showed that SKOV3 cells have wild type JAK1, we further investigated other possible defects in IFN-γ signaling in SKOV3 cells. We investigated the IFN-γ-induced STAT3 protein activation in human ovarian cancer cell lines, and defects were found in Y705 STAT3 phosphorylation in SKOV3 as well as in IGROV-1 and TOV21G cells. To summarize, our results showed that IFN-γ induces PD-L1 expression in ovarian cancer cells via IFNGR-JAK-STAT pathway. The failure of IFN-γ-mediated induction of PD-L1 in a minority group of human ovarian cancer cell lines is due to defective IFN-γ signaling, including JAK1 truncating mutations and impaired STAT3 activation. This work is supported by Hong Kong Research Grants Council General Research Fund (467713 and 14109515). Citation Format: Tat-San Lau, Loucia Kit-Ying Chan, Tak-Hong Cheung, So-Fan Yim, Jacqueline Ho-Sze Lee, Joseph Kwong. Interferon-gamma induces PD-L1 expression via IFNGR-JAK-STAT pathway in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2017-648

Background: Fat represents an important source of energy for ovarian cancer (OC) cells and is supplied either through import from the tumor milieu or via de novo lipogenesis. During fast tumor growth, when nutrients are scarce, lipogenesis becomes the primary source of fatty acids. Stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in this pathway converts saturated (SFAs) into unsaturated fatty acids (UFAs) and is highly expressed in OC. The goal of this study was to determine how the balance between SFAs and UFAs tightly controlled by SCD, regulates OC cell survival and tumorigenicity. Methods: SCD was knocked down by shRNA or inhibited by using the small molecule CAY10566 and global effects on the lipidome and transcriptome were examined by targeted and untargeted lipidomics, stimulated Raman spectroscopy (SRS), and RNA sequencing. In OC cells in which SCD was blocked or knocked down, the effects of exogenous SFAs and UFAs on cell survival and endoplasmic reticulum (ER) stress pathway were assessed by using annexin V staining, XBP1 splicing assay and Western blotting of PERK/eIF2α/ATF4. Tumorigenicity was assessed by using an intraperitoneal (i.p.) xenograft model in nude mice. Results: RNA-seq analysis of SCD knockdown cells cultured under low serum conditions revealed activation of ER stress response pathways. Targeted lipidomics and SRS microscopy showed downregulation of UFAs vs. SFAs, while untargeted lipidomics discovered decreased fatty acyl chain plasticity among phosphatidylcholines. Activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes was observed in cells accumulating SFAs. Stiff and disorganized ER membrane was detected by electron microscopy and Raman spectroscopy. Annexin V staining demonstrated apoptosis of OC cells under long-term mild ER stress or short-time severe ER stress induced by increased levels of SFAs. ER stress and apoptosis were rescued by addition of UFAs. In vivo SCD knockdown suppressed tumor growth and treatment with the SCD inhibitor CAY10566 reduced the number of metastases and the volume of ascites in mice fed with SFA enriched diet, but not in mice fed a balanced diet. Conclusion: Our data support that OC cells are highly susceptible to unbalanced intracellular SFAs/UFAs, undergoing ER stress and apoptosis in the presence of excessive intracellular SFAs. SCD inhibition coupled with a diet rich in saturated fats decreased cancer progression in vivo. These results support SCD as a key regulator of cancer cell fate during metabolic stress in growing tumors and point to new treatment strategies targeting lipid metabolism. Citation Format: Guangyuan Zhao, Yuying Tan, Horacio Cardenas, David Vayngart, Yinu Wang, Christina Ferreira, Ji-Xin Cheng, Daniela Matei. Ovarian cancer cell fate is regulated by the balance between saturated and unsaturated fatty acids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5811.

Survivin, an anti-apoptosis gene that is abnormally overexpressed in a variety of human tumors, may play an important role in the carcinogenesis and drug resistance of cancer. This study was designed to explore the effects of liposome-survivin antisense oligonucleotide (Lip-ASODN) on the growth and apoptosis of human ovarian cancer cell lines, A2780 and SKOV3. To investigate the use of survivin as a therapeutic target on ovarian cancer, we carried out transfections with Lip-ASODN to induce apoptosis in ovarian cancer cell lines, A2780 and SKOV3. The expression of survivin mRNA and relative protein were evaluated separately by quantitative real-time RT-PCR and Western blot analysis. Cell proliferation inhibition was determined by methyl thiazolyl tetrazolium (MTT) assay, and the induced cell apoptosis was examined using flow cytometry (FCM) after Lip-ASODN transfection. Our results showed that the overexpression of survivin led to infinite carcino-proliferation, and survivin expression in the survivin-positive ovarian cancer cell line A2780 and SKOV3 cells was significantly and gradually reduced when transfected with Lip-ASODN at concentrations of 200, 400 and 600 nM by degrees. Lip-ASODN transfection induced greater apoptosis rates in the human ovarian cancer cell lines A2780 and SKOV3 (p<0.05). The growth inhibition and apoptotic rates of tumor cells change when treated with different concentrations of Lip-ASODN. The cell growth inhibition peak rate was reached when increasing Lip-ASODN concentration to 600 nM. Furthermore, time course evaluation showed that survivin protein expression was inhibited by Lip-ASODN within 12 h after transfection. We concluded that down-regulation of survivin by a targeted antisense oligonucleotide appears to be an effective gene therapy approach in the treatment of ovarian cancer.

Objectives Existence of side population (SP) in human ovarian cancer cell line OVCAR3 and characteristics of both stem cells and cancer cells were detected. Relation between SP phenotype and ABCG2 protein( ATP-binding cassette superfamily G member 2/breast cancer resistance protein, BCRP) was examined,and the potential to use OVCAR3 as tools for investigating the characteristics of cancer stem cells (CSCs)was estimated as well. Methods Hoechst 33342 efflux SP cells and dye-uptaking non-SP cells were separated by flow cytometry from human serous ovarian adenocarcinoma cell line OVCAR3. Cell colonyforming efficiency (CFE) of both subgroups ware evaluated. Soft agar assay was performed to compare the growth capacity of SP cells and non-SP cells. ABCG2 expression was detected by Western blotting and immunofluoreacence using an anti-ABCG2 monoclonal antibody. Results Flow cytometric analysis confirmed the presence of small subpopulations of cells excluding Hoechst dye in OVCAP,3 cells (2. 0% ) in a verapamil-sensitive manner. Hoechst dye uptake in OVCAR3 cells was enhanced by addition of verapamil, consistent with activity of ABCG2 protein. CFE of both SP and non-SP was 46.17%±23.27% and 6.17%±3.06%, respectively(P<0. 05). The Hoechst dye excluding SP cells exhibited immunoreactivity to stem cell markers ABCG2, demonstrated by Western blotting. They could grow in soft agar plate and formed cell sphere. Conclusions Human epithelial ovarian carcinoma cell line OVCAR3 contains a small subpoptdation of cells that exhibits a cancer stem cell-like phenotype. Special significance is the expression of ABCG2 in human tumor cells, a calcium-sensitive cell surface protein that acts to exclude Hoechst dye. These cells play a principal role in maintaining cancer cell line. It can be seen that cancer cell lines are potentially useful tools for investigating the characteristics of CSCs under controlled experimental conditions. Key words: Ovarian neoplasms; Tumor stem cells; ATP-binding cassette superfamily G member 2; Side-population cells; Hoechst 33342

Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers, such as ovarian cancer (OC) and testicular cancer (TC). A monoclonal antibody against CLDN6, IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. In this series of nonclinical studies, we investigated CLDN6 expression in normal and cancer tissues, as well as the localization and possible function of CLDN6 in cancer cells. Methods Expression of CLDN6 was assessed in a wide range of human tissues (eg, lung, colon, skin, ovary) and cultured cells by quantitative RT-PCR, immunohistochemistry (IHC), flow cytometry, and western blotting. To investigate the effect of dedifferentiation on CLDN6 expression, human-induced pluripotent cells were generated by transfecting foreskin fibroblasts with a reprogramming cocktail, and then CLDN6 expression was evaluated. To characterize CLDN6 as a potential novel marker to identify cancer stem cells (CSCs) in OC, coexpression of CLDN6 with known CSC surface markers were analyzed by flow cytometry, and CLDN6+ and CLDN6-negative cells were tested in colony formation and sphere formation assay. Human OC cell lines were transplanted intraperitoneally into nude mice and assessed for metastasis to investigate tumorigenecity of CLDN6+ cells. Results Except for low mRNA levels measured in placenta, testis, umbilical cord, cerebellum, and lung samples, no CLDN6 (mRNA or protein) was detected in the vast majority of normal tissues. Additionally, there was also a lack of CLDN6 protein expression in tissue zones where stem cells for tissue homeostasis would normally be found as determined by IHC with an anti-CLDN6 antibody. CLDN6 was expressed on the cell surface of several solid tumors, including ovarian, testicular, uterine, and lung cancer tissues; OC and TC samples had high level expression. CLDN6 expression was strongly activated in human-induced pluripotent stem cells generated from fibroblasts. CLDN6 showed selective coexpression with known CSC markers such as CD44, CD24, and CD90 in OC and TC cell lines. In addition, some CLDN6+ OC cells exhibited CSC-like behavior in vitro: CLDN6+ populations were clonogenic and formed well-defined spheres in low attachment conditions; these spheres had the ability to self-renew into secondary spheres. Analysis of OC metastases in mouse xenografts showed when xenografts were generated by OC cells that had &amp;lt;10% of CLDN6+ cells, the metastases were enriched in CLDN6+ cells, suggesting CLDN6+ cells had selective growth advantage. Conclusions CLDN6 is a cancer cell-specific surface molecule aberrantly expressed in several cancers, and its expression may be an identifier for cells with CSC-like traits. These characteristics make CLDN6 an attractive target candidate for tumor-specific therapeutic antibodies. Citation Format: Özlem Türeci, Meike Wagner, Claudia Paret, Maria M. Kreuzberg, Stefan Wöll, Korden Walter, Sabine C. Häcker, Ikumi Nakajo, Tomohiro Yamada, Ugur Sahin. Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1907.

OBJECTIVES: Investigation of the expression pattern and functional roles of tumor antigen CtBP-2 in ovarian cancer. BACKGROUND: Tumor-associated antigens may have mechanistic function in cancer development and serum antibodies that target the tumor-associated antigens may have value in early detection. Antibodies to C-terminal binding protein 2 (CtBP-2), a component of the Wnt signaling pathway and a transcriptional corepressor via interaction with histone deacetylases (HDACs), were identified in patient serum using an innovative reverse capture antibody array method. We investigated the expression and function of CtBP-2 in human ovarian cancer tissue and ovarian cancer cell lines. METHODS: CtBP-2 expression was studied in 105 paraffin-embedded ovarian tumors by standard immunohistochemistry (IHC). ANOVA was used to compare the mean IHC staining scores (area stained x intensity) between healthy and tumor samples. Functional assays were performed with human ovarian cancer cell lines SKOV3 and MCAS that stably integrated a CtBP2-targeting short-hairpin RNA (shRNA) construct via a Lentivirus vector. Protein expression was examined by western blotting. Cell proliferation in culture was evaluated by cell counting. Cell adhesion assays were performed using a CytoMatrix Screen kit from Millipore. Cell migration assays were performed using a Boyden chamber-based cell migration assay. B-catenin activity was measured using a TOPFLASH luciferase reporter assay. Cellular sensitivity to trichostatin A, a HDAC inhibitor, was measured using MTT assay. RESULTS: 83% of ovarian epithelial tumors stained positive with a mean score of 4.77 (borderline tumors) and 5.49 (invasive tumors) and showed a significant difference (p-value 50% and showed reduced proliferation rates. The knock-down cell lines also showed 40-80% reduction in adhesion to extracellular matrix proteins collagen I and collagen IV, 50-70% reduction in migration, suppressed B-catenin activity and enhanced sensitivity to trichostatin A. CONCLUSIONS: CtBP-2 is overexpressed in ovarian epithelial tumors and cancer cell lines. Ovarian cancer cell lines with suppressed CtBP-2 expression show a reduction in proliferation rate, cell adhesion and cell migration. The phenotype may be related to suppressed B-catenin activity. The knockdown cell lines are also more sensitive to HDAC inhibitor trichostatin A, suggesting that the expression of CtBP-2 might modulate cellular response to HDAC inhibitor in the clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2734.

To investigate the biological effect of long non-coding ribonucleic acid (lncRNA) lung cancer-associated transcript 1 (LUCAT1) in the development of ovarian cancer. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to detect the expression levels of lncRNA LUCAT1 in three human ovarian cancer cell lines (CaoV-3, SK-OV-3 and HO-8910) and the normal human ovarian surface epithelial cell line (IOSE80). Small interfering RNAs against lncRNA LUCAT1 (si-LUCAT1) were transfected into SK-OV-3 cells. Transfection efficiency of si-LUCAT1 was verified via RT-qPCR. Cell Counting Kit-8 (CCK-8) and colony formation assays were performed to test the effect of silencing lncRNA LUCAT1 on SK-OV-3 cell proliferation. The apoptosis was measured by flow cytometry. The miRcode database was searched to predict potential microRNAs (miRNAs) binding lncRNA LUCAT1. It was found that lncRNA LUCAT1 contained a highly conserved binding site of miR-199a-5p in the 3'-untranslated region (3'-UTR). Subsequently, the targeting relationship between them was determined through Dual-Luciferase reporter gene assay and RT-qPCR analysis. LncRNA LUCAT1 was highly expressed in three human ovarian cancer cell lines compared to that in normal ovarian surface epithelial cell line (p<0.05). The cell proliferation rate in SK-OV-3 cells with lncRNA LUCAT1 knockdown was remarkably lower in comparison to that in control group. Moreover, colony formation assay also revealed that the number of cell clones decreased significantly after knockdown of lncRNA LUCAT1 compared to that in control group (p<0.05). In addition, the apoptosis rate was distinctly elevated in the lncRNA LUCAT1 silencing group (p<0.05). Furthermore, a highly conserved binding site of miR-199a-5p was found in the 3'-UTR of lncRNA LUCAT1. Dual-Luciferase reporter gene assay exhibited that the Luciferase activity of LUCAT1-wt was significantly reduced after overexpression of miR-199a-5p (p<0.05), while that of LUCAT1-mut was unchangeable. Further analysis via RT-qPCR suggested that miR-199a-5p overexpression significantly decreased the expression level of lncRNA LUCAT1 (p<0.05). LncRNA LUCAT1 is overexpressed in ovarian cancer cells, which may target miR-199a-5p to exert its effects on driving the malignant development of ovarian cancer.

Ovarian cancer is the most frequent cause of death from gynecological cancer. Even though there are a lot of options in treating gynecological malignancies, the therapeutic effect of ovarian cancer nowadays is still unfavourable, especially in treatment of the patients diagnosed with late stage disease. The development of chemotherapeutic drug resistance during the treatment of ovarian cancer is thought to cause treatment failure and the high mortality rate. Thus, searching for alternative agents to overcome chemoresistance during the treatment of ovarian cancer is essential. Salinomycin, a polyether ionophore antibiotic that has recently been shown not only to kill human breast cancer stem cell-like cells, but also to induce apoptosis and overcome multiple mechanisms of resistance to apoptosis in human cancer cells. These results strongly suggested that salinomycin should be regarded as an anticancer compound. This study aims at exploring the anticancer effects and mechanism of salinomycin on cisplatin-resistant human ovarian cancer cell line in vitro and in vivo. Concentration (0.01-200µM)- and time (24-72 h)- dependent growth inhibitory effects of salinomycin were observed in 6 ovarian cancer cell lines (OV2008, C13, A2780, A2780-cp, SKOV3 and OVCAR3) by measuring cell viability using the resazurin reduction assay. IC50 (24 h) range of salinomycin on these 6 cell lines was 1.7-7.4 µM. After cisplatin-resistant C13 cells were treated with salinomycin, the percentages of apoptotic cells determined by flow cytometry were significantly increased, showing a concentration- and time-dependent manner. But no cell cycle arrest in the G1/G0, S and G2/M phases was detected between salinomycin-treated cells and control cells. Bio-Plex phosphoprotein 5-plex assay (Akt, IκB-α, ERK1/2, JNK and p38 MAPK) revealed a marked time- and concentration-dependent increase in the phosphorylation of p38 MAPK following salinomycin treatment. Pretreatment with SB202190, an inhibitor of p38 MAPK, markedly inhibited salinomycin-induced apoptosis. Moreover, salinomycin significantly suppressed the growth of tumors in tumor (C13) xenograft model. The tumor cell apoptosis in situ analyzed by immunohistochemistry staining of cleaved caspase-3 indicated that more apoptotic cells were observed in tumor treated with salinomycin than control tumor. The results of this research demonstrated that salinomycin is a potent compound against cisplatin-resistant human ovarian cancer cell line C13 in vitro and indicates significant in vivo efficacy in tumor (C13) xenograft model. Salinomycin can inhibit the growth of cisplatin-resistant human ovarian cancer cell line C13 efficiently through induction of apoptosis, which might be associated with activation of p38 MAPK. Salinomycin shows substantial promise for further development as a potential agent for treating drug-resistant ovarian cancer.

Ovarian cancer (OC) is a commonly occurring malignant tumor in women with a high mortality rate. Early detection remains challenging, and therapeutic options for advanced OC are severely limited. Therefore, there is an urgent need to identify novel biomarkers and therapeutic targets to improve outcomes for patients. Contactin-1 (CNTN1), a member of the immunoglobulin superfamily, functions as a modulator of cancer progression, yet its specific role in OC remains undefined. The present study aimed to investigate the clinical significance and regulatory role of CNTN1 in OC proliferation and metastasis. This study examined CNTN1 expression in 40 paired OC tissue samples and cell lines using real-time quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Colony formation assays assessed proliferative capacity, while transwell assays measured invasive and migratory potential. Additionally, a tumor xenograft model in nude mice was employed to verify in vivo proliferative effects. The results demonstrated that CNTN1 expression was markedly elevated in OC tissues and positively associated with advanced tumor stage, metastasis, and poor prognosis. Silencing CNTN1 significantly inhibited proliferation, migration, and invasion in OC cell lines. Bioinformatics analysis combined with luciferase assays identified a regulatory interaction between CNTN1 and presenilin-1 (PSEN1), with CNTN1 expression positively correlating with PSEN1 levels in patient samples. Notably, PSEN1 overexpression reversed the impaired proliferation, migration, and invasion induced by CNTN1 suppression. These findings suggest that CNTN1 promotes OC progression through PSEN1 regulation and may represent a prognostic biomarker for OC.

To explore the regulatory mechanism of lncRNA NORAD on proliferation and invasion of ovarian cancer cells through miR-199a-3p. Eighty-six ovarian cancer tissues and 86 tissues adjacent to cancer, human ovarian cancer cell lines SKOV3, HO-8910, A2780, OVCAR-3, and human normal ovarian epithelial cell line IOSE80 were collected. MiR-199a-3p-mimics, miR-199a-3p-inhibitor, miR-NC, si-NORAD, Sh-NORAD, and NC were transfected into HO-8910 and A2780 cells, the expression levels of lncRNA NORAD and miR-199a-3p in ovarian cancer tissues and cells were detected by qRT-PCR, and the expression levels of N-cadherin, E-cadherin, and vimentin in cells were detected by WB. Cell Counting Kit-8 (CCK-8), transwell, and cell scratch tests were used to detect proliferation, invasion, and migration of cells, and the relationship between lncRNA NORAD and miR-199a-3p was confirmed by the Dual-Luciferase reporter assay. LncRNA NORAD was highly expressed and miR-199a-3p was lowly expressed in ovarian cancer, and the expression levels of LNCRNARAD and miR-199a-3p were negatively correlated. Cell experiments showed that inhibiting the expression of lncRNA NORAD or up-regulating the expression of miR-199a-3p could inhibit the proliferation, invasion, migration, and EMT of ovarian cancer cells, while up-regulating the expression of lncRNA NORAD or inhibiting the expression of miR-199a-3p could promote their proliferation, invasion, migration, and EMT. Dual-Luciferase reporter assay confirmed that there was a regulatory relationship between lncRNA NORAD and miR-199a-3p. LncRNA NORAD was highly expressed in ovarian cancer tissues, while silencing lncRNA NORAD expression could inhibit the proliferation, invasion, migration, and EMT of ovarian cancer cells by regulating miR-199a-3p, which might be a new target for the diagnosis and treatment of ovarian cancer.

To study the role of long-chain non-coding RNA (lncRNA) DUXAP8 in ovarian cancer (OCa) and the underlying potential mechanism. The expression pattern of DUXAP8 in ovarian cancer was analyzed using the GEPIA database. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to determine the expression of DUXAP8 in OCa tissues; at the same time, OCa cell lines were cultured to complete functional experiments, including cell counting kit-8 (CCK-8), plate cloning experiments and transwell experiments to evaluate the effects of DUXAP8 on the proliferative and migration ability of OCa cell lines. Bioinformatics analysis and Dual-Luciferase reporter genes were used to determine the binding and expression of DUXAP8 to its downstream key gene microRNA-29a-3p in OCa cells. In addition, co-transfection technology and cell function recovery experiments were used to verify the important role of the DUXAP8/microRNA-29a-3p regulatory network in OCa. DUXAP8 was abnormally highly up-regulated in OCa tissues and cell lines, besides, its expression was related to poor prognosis of patients. CCK-8 and plate cloning experiments showed that knockdown of DUXAP8 in OCa cells can significantly inhibit the proliferation of OCa cells. Transwell results suggested that knockdown of DUXAP8 can significantly inhibit OCa cell migration. In addition, it was found that DUXAP8 can bind and negatively regulate the expression of microRNA-29a-3p in OCa. Functional experiments in OCa cells also revealed that microRNA-29a-3p was a key downstream gene that mediated the regulation of DUXAP8 on OCa function. DUXAP8 has abnormally high expression in OCa and can lead to malignant progression of the tumor.

The influences of Vinculin on many cancers were blurry, including ovarian cancer. Thus, we concentrated on the efficient role of Vinculin in ovarian cancer and explored the potential mechanism(s). Expression of Vinculin in ovarian cancer tissues and cell lines was investigated by real-time polymerase chain reaction, immunohistochemistry, and Western blot. The Kaplan-Meier manner with the logrank was performed to assess overall survival. We further evaluated the relations between Vinculin expression and clinicopathological features of ovarian cancer. Moreover, Vinculin was overexpressed or silenced by respectively transfection with pcDNA-Vinculin or small interfering (si-Vinculin) into human ovarian cancer cell line Caov3 or human ovarian epithelial cell line (HOEpiC). Thereafter, cell viability, cell apoptosis, and migration were checked by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, flow cytometer, and scratch assay, respectively. Likewise, the apoptosis- and migration-related proteins were distinguished by Western blot. Compared to the nontumor tissues or HOEpiC cells, Vinculin was significantly lower expressed in the ovarian cancer tissues and cells. Furthermore, we found out that Vinculin was primarily distributed at the cell membrane and cytoplasm. Moreover, Vinculin was negatively associated with International Federation of Gynecology and Obstetrics stage, grade, and distant metastasis. Overexpression of Vinculin dramatically weakened cell viability and migration and stimulated apoptosis. Conversely, suppression of Vinculin showed opposite results. Vinculin presents unfavorable prediction in ovarian cancer and inhibits ovarian cancer proliferation and migration.

This study aimed at investigating the biological functions of long non-coding RNAs (lncRNAs) hox transcript antisense intergenic RNA (HOTAIR) in resistant ovarian cancer cells, exploring the regulation effect of HOTAIR on HOXA7, and investigating their influence on the chemosensitivity of ovarian cancer cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied for the verification of HOTAIR expression in resistant and sensitive groups. How HOTAIR downregulation affected cell proliferation, migration and invasion, and apoptosis were determined using the MTT assay and the colony formation assay, the Transwell assay and flow cytometry analysis, respectively. Immunohistochemistry was used to inspect the protein expression of HOXA7 in resistant and sensitive ovarian cancer tissues. The regulation relationship between HOTAIR and HOXA7 was investigated by qRT-PCR and Western blot. The effect of HOTAIR and HOXA7 on tumor growth was confirmed by the tumor xenograft model of nude mice. By knocking down HOXA7, HOTAIR downregulation restrained the ovarian cancer deterioration in functional experiments. Silencing of HOTAIR and HOXA7 could effectively inhibit tumor growth and increase chemosensitivity of ovarian tumors in nude mice. Downregulation of HOTAIR negatively affected the survival and activity of resistant ovarian cancer cells, and suppressed the expression of HOXA7. Silencing of HOTAIR and HOXA7 could increase the chemosensitivity of ovarian cancer cells, thus suppressing tumor development.

The anti-proliferative and anti-metastatic effects of ONC206 are potentiated by olaparib in serous ovarian cancer