Normal Ovarian Epithelial Cell Line Research Articles

Stearoyl-CoA desaturase 1 inhibition induces ER stress-mediated apoptosis in ovarian cancer cells

Ovarian cancer is a leading cause of death among gynecologic tumors, often detected at advanced stages. Metabolic reprogramming and increased lipid biosynthesis are key factors driving cancer cell growth. Stearoyl-CoA desaturase 1 (SCD1) is a crucial enzyme involved in de novo lipid synthesis, producing mono-unsaturated fatty acids (MUFAs). Here, we aimed to investigate the expression and significance of SCD1 in epithelial ovarian cancer (EOC). Comparative analysis of normal ovarian surface epithelial (NOSE) tissues and cell lines revealed elevated SCD1 expression in EOC tissues and cells. Inhibition of SCD1 significantly reduced the proliferation of EOC cells and patient-derived organoids and induced apoptotic cell death. Interestingly, SCD1 inhibition did not affect the viability of non-cancer cells, indicating selective cytotoxicity against EOC cells. SCD1 inhibition on EOC cells induced endoplasmic reticulum (ER) stress by activating the unfolded protein response (UPR) sensors and resulted in apoptosis. The addition of exogenous oleic acid, a product of SCD1, rescued EOC cells from ER stress-mediated apoptosis induced by SCD1 inhibition, underscoring the importance of lipid desaturation for cancer cell survival. Taken together, our findings suggest that the inhibition of SCD1 is a promising biomarker as well as a novel therapeutic target for ovarian cancer by regulating ER stress and inducing cancer cell apoptosis.

Unraveling the molecular mechanisms of lymph node metastasis in ovarian cancer: focus on MEOX1

BackgroundLymph node metastasis (LNM) is a major factor contributing to the high mortality rate of ovarian cancer, making the treatment of this disease challenging. However, the molecular mechanism underlying LNM in ovarian cancer is still not well understood, posing a significant obstacle to overcome.ResultsThrough data mining from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, we have identified MEOX1 as a specific gene associated with LNM in ovarian cancer. The expression of MEOX1 was found to be relatively high in serous ovarian adenocarcinoma, and its higher expression were associated with increased tumor grade and poorer clinical prognosis for ovarian cancer patients. Bioinformatics analysis revealed that MEOX1 exhibited the highest mRNA levels among all cancer types in ovarian cancer tissues and cell lines. Furthermore, gene set enrichment analysis (GSEA) and pathway analysis demonstrated that MEOX1 was involved in various LNM-related biological activities, such as lymphangiogenesis, lymphatic vessel formation during metastasis, epithelial-mesenchymal transition (EMT), G2/M checkpoint, degradation of extracellular matrix, and collagen formation. Additionally, the expression of MEOX1 was positively correlated with the expression of numerous prolymphangiogenic factors in ovarian cancer. To validate our findings, we conducted experiments using clinical tissue specimens and cell lines, which confirmed that MEOX1 was highly expressed in high-grade serous ovarian cancer (HGSOC) tissues and various ovarian cancer cell lines (A2780, SKOV3, HO8910, and OVCAR5) compared to normal ovarian tissues and normal ovarian epithelial cell line IOSE-80, respectively. Notably, we observed a higher protein level of MEOX1 in tumor tissues of LNM-positive HGSOC compared to LNM-negative HGSOC. Moreover, our fundamental experiments demonstrated that suppression of MEOX1 led to inhibitory effects on ovarian cancer cell proliferation and EMT, while overexpression of MEOX1 enhanced the proliferation and EMT capacities of ovarian cancer cells.ConclusionsThe results of our study indicate that MEOX1 plays a role in the lymph node metastasis of ovarian cancer by regulating multiple biological activities, including the proliferation and EMT of ovarian cancer, lymphangiogenesis, and ECM remodeling. Our findings suggest that MEOX1 could serve as a potential biomarker for the diagnosis and treatment of ovarian cancer with LNM.

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.

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.

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.

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.

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.

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.

The potential benefits of dinitrophenol combination with chemotherapy in the treatment of ovarian cancer.

2,4-dinitrophenol (DNP), an uncoupling mitochondrial agent, has been identified as a source of oxidative stress and linked to the pathogenesis of ovarian cancer. In this study, we determine the cytotoxic effect of DNP alone or in combination with chemotherapies in ovarian cancer cells. We utilized human ovarian cancer cell lines SKOV-3 and MDAH-2774 with their chemoresistant counterparts. Cancer stem cells (CSCs) were isolated from SKOV-3 utilizing magnetic-activated cell sorting technique for CD44+/CD117+ cells. Human normal primary ovarian epithelial (NOEC) and HOSEpiC cell lines were used as a control. Cells were treated with and without chemotherapy (Taxotere 0.3μM or cisplatin 50 μM), with or without increasing doses of DNP (0.125, 0.25, or 0.5 mM) for 24 hours followed by evaluation of cell viability and IC50 utilizing MTT assay. For determination of synergism, Facombination index plots were created using the CompuSyn software. All data were run in triplicates and analyzed by t-test. DNP treatment of ovarian cancer and chemoresistant ovarian cancer cell lines as well as CSCs resulted in decreased cell viability in a dose dependent manner with no effect on normal cells. Combination of DNP with chemotherapy synergistically enhances cytotoxicity of chemotherapeutics in all ovarian cancer cells as compared to chemotherapy alone. Our data indicates the potential of the addition of DNP to the arsenal of drugs available to treat ovarian cancer, whether alone or in combination with chemotherapies. The synergistic effects of DNP in reducing the required amount of chemotherapy, is critical for the alleviation of harmful side effects.

The Expression and Role Analysis of Methylation-Regulated Differentially Expressed Gene UBE2C in Pan-Cancer, Especially for HGSOC.

Simple SummaryDNA methylation has attracted a great deal of scientific interest as an early biomarker and potential therapeutic target. HGSOC result in high mortality due to the absence of reliable biomarkers for early diagnosis and prognosis. In this study, we performed an integrated bioinformatic analysis and found that UBE2C was hypomethylation and overexpression in ovarian cancer, which was associated with advanced cancer stages and poor prognoses. Meantime, this finding was also confirmed in pan-cancer analysis. Furthermore, the experimental validation of the expression and role of UBE2C was performed on HGSOC tissues and cancer cell lines. Importantly, demethylation could upregulate the expression of UBE2C. Taken together, methylation-regulated UBE2C may be a novel biomarker for diagnosis and prognosis, not only for ovarian cancer but a variety of cancers.High-grade serous ovarian cancer (HGSOC) is the most fatal gynecological malignant tumor. DNA methylation is associated with the occurrence and development of a variety of tumor types, including HGSOC. However, the signatures regarding DNA methylation changes for HGSOC diagnosis and prognosis are less explored. Here, we screened differentially methylated genes and differentially expressed genes in HGSOC through the GEO database. We identified that UBE2C was hypomethylation and overexpression in ovarian cancer, which was associated with more advanced cancer stages and poor prognoses. Additionally, the pan-cancer analysis showed that UBE2C was overexpressed and hypomethylation in almost all cancer types and was related to poor prognoses for various cancers. Next, we established a risk or prognosis model related to UBE2C methylation sites and screened out the three sites (cg03969725, cg02838589, and cg00242976). Furthermore, we experimentally validated the overexpression of UBE2C in HGSOC clinical samples and ovarian cell lines using quantitative real-time PCR, Western blot, and immunohistochemistry. Importantly, we discovered that ovarian cancer cell lines had lower DNA methylation levels of UBE2C than IOSE-80 cells (normal ovarian epithelial cell line) by bisulfite sequencing PCR. Consistently, treatment with 5-Azacytidine (a methylation inhibitor) was able to restore the expression of UBE2C. Taken together, our study may help us to understand the underlying molecular mechanism of UBE2C in pan-cancer tumorigenesis; it may be a useful biomarker for diagnosis, treatment, and monitoring, not only of ovarian cancer but a variety of cancers.

LncRNA SNHG20 promotes migration and invasion of ovarian cancer via modulating the microRNA-148a/ROCK1 axis

BackgroundOvarian cancer (OC) is characterized by early metastasis and poor prognosis, which threatens the health of women worldwide. Small nucleolar RNA host gene 20 (SNHG20), a long noncoding RNA (lncRNA), has been verified to be significantly up-regulated in several tumors, including OC. MicroRNA-148a (miR-148a)/rho-kinase1 (ROCK1) axis plays an important role in the modulation of tumor development. However, whether SNHG20 can regulate OC progression through miR-148a/ROCK1 axis remains unclear. Normal human ovarian epithelial cell line and four OC cell lines were adopted for in vitro experiments. Real-time PCR was performed to assess the levels of SNHG20 and miR-148a. OC cell proliferation, apoptosis, invasion and migration were detected using clone formation, flow cytometry, transwell, and wound healing assays, respectively. Tumor xenograft assay was applied to evaluate the effect of SNHG20 on tumor growth in vivo.ResultsSignificant higher expression of SNHG20 was observed in OC cell lines. SNHG20 markedly promoted the invasion, migration, proliferation and inhibited the apoptosis of OC cells. SNHG20 enhanced ROCK1 expression by sponging miR-148a, and the direct binding between SNHG20/ROCK1 and miR-148a was identified.ConclusionSNHG20 promoted invasion and migration of OC via targeting miR-148a/ROCK1 axis. The present research may provide a novel insight for the therapeutic strategies of OC.

Ultrasound‑targeted microbubble destruction‑mediated overexpression of Sirtuin 3 inhibits the progression of ovarian cancer.

Ultrasound-targeted microbubble destruction (UTMD) has recently been developed as a promising noninvasive tool for organ- and tissue-specific gene or drug delivery. The aim of the present study was to explore the role of UTMD-mediated Sirtuin 3 (SIRT3) overexpression in the malignant behaviors of human ovarian cancer (HOC) cells. Reverse transcription-quantitative PCR was performed to detect SIRT3 mRNA expression levels in normal human ovarian epithelial cells and HOC cell lines; low SIRT3 expression was found in HOC cell lines, and the SKOV3 cell line was used in the following experiments. The SIRT3-microbubble (MB) was prepared, and the effects of ultrasound-treated SIRT3-MB on biological processes of SKOV3 cells were determined. The proliferation, migration, invasion and apoptosis of SKOV3 cells were measured after SIRT3 upregulation by UTMD. Xenograft tumors in nude mice were induced to observe tumor growth in vivo. Upregulation of SIRT3 inhibited the malignant behaviors of SKOV3 cells, whereas UTMD-mediated SIRT3 upregulation further inhibited proliferation, epithelial-mesenchymal transition, invasion and migration, and induced apoptosis of SKOV3 cells, and it also inhibited tumor formation and growth in vivo. Moreover, the present study identified hypoxia inducible factor-1α (HIF-1α) as a target of SIRT3. The present study provided evidence that UTMD-mediated overexpression of SIRT3 may suppress HOC progression through the inhibition of HIF-1α.

A pancancer overview of FBN1, asprosin and its cognate receptor OR4M1 with detailed expression profiling in ovarian cancer.

Ovarian cancer affects >295,000 women worldwide and is the most lethal of gynaecological malignancies. Often diagnosed at a late stage, current research efforts seek to further the molecular understanding of its aetiopathogenesis and the development of novel biomarkers. The present study investigated the expression levels of the glucogenic hormone asprosin [encoded by fibrillin-1 (FBN1)], and its cognate receptor, olfactory receptor 4M1 (OR4M1), in ovarian cancer. A blend of in silico open access The Cancer Genome Atlas data, as well as in vitro reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry and immunofluorescence data were used. RT-qPCR revealed expression levels of OR4M1 and FBN1 in clinical samples and in ovarian cancer cell lines (SKOV-3, PEO1, PEO4 and MDAH-2774), as well as the normal human ovarian surface epithelial cell line (HOSEpiC). Immunohistochemical staining of a tissue microarray was used to identify the expression levels of OR4M1 and asprosin in ovarian cancer samples of varying histological subtype and grade, including clear cell carcinoma, serous ovarian cancer and mucinous adenocarcinoma. Immunofluorescence analysis revealed asprosin expression in SKOV-3 and HOSEpiC cells. These results demonstrated the expression of both asprosin and OR4M1 in normal and malignant human ovarian tissues. This research invokes further investigation to advance the understanding of the role of asprosin and OR4M1 within the ovarian tumour microenvironment.

MiR-198 inhibits proliferation, invasion and migration of ovarian cancer cells by regulating the PI3K/Akt signaling pathway.

The specific objective of this investigation is to explore the impact of miR-198 on proliferation, migration as well as invasion of ovarian cancer (OC) cells. OC tissue and adjacent normal tissue samples from OC patients were collected, and normal human ovarian epithelial cell IOSE80 and OC cell lines SKOV3, Caov3, A2780 and OVCAR3 were selected in this study for investigation. MiR-198 expression level was assessed using RT-qPCR. MTT, colony formation assay, Transwell and wound healing assay, and flow cytometry were adopted to analyze the role of miR-198 in OVCAR cell proliferation, invasion, migration, as well as apoptosis. Meanwhile, the levels of P13K/Akt signaling pathway-related proteins were determined by western blotting. A significant decrease in miR-198 level was revealed in the OC tissues and cells, contributing to the promotion of OVCAR3 cells in terms of proliferation, migration, invasion, and inhibition of apoptosis. MiR-198 overexpression had an opposite effect on these biological processes of OVCAR3 cells. Further study found that down-regulation of miR-198 caused a significant increase in the activity of PI3K/Akt signaling pathway in the OVCAR3 cells. In contrast, overexpressed miR-198 led to inhibition of this pathway's activity. MiR-198 may possess an ability to inhibit activation of the P13K/Akt pathway, thus suppressing the OC cell proliferation, migration, as well as invasion.

MicroRNA-664a-3p inhibits the proliferation of ovarian granulosa cells in polycystic ovary syndrome and promotes apoptosis by targeting BCL2A1.

BackgroundTo investigate whether micro ribonucleic acid-664a-3p (miR-664a-3p) targeting BCL2A1 affects the proliferation and apoptosis of ovarian granulosa cells.MethodsReal-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-664a-3p in granulosa cells, granular tumor cell lines (KGN), and normal ovarian epithelial cell lines (IOSE80) in the polycystic ovary syndrome (PCOS) group and the control group. After overexpressing miR-664a-3p or inhibiting its expression in KGN cells, qRT-PCR and Western blotting were used to detect the messenger RNA (mRNA) and protein levels of related genes. At the same time, a cell counting kit-8 (CCK-8) and flow cytometer were used to detect cell proliferation and apoptosis. The TargetScan website was used to predict the potential binding sites of miR-664a-3p and B-cell lymphoma 2-related protein A1 (BCL2A1), which was further verified by qRT-PCR, Western blotting, and the luciferase reporter gene method.ResultsThe expression of miR-664a-3p was significantly decreased in both PCOS tissues and KGN cells (both P<0.05), and the overexpression of miR-664a-3p inhibited the proliferation of KGN cells and induced their apoptosis. Moreover, our results confirmed that miR-664a-3p directly targets BCL2A1 (P<0.05), and the inhibitory effect of miR-664a-3p on KGN cells was reversed by BCL2A1 overexpression (both P<0.05). The up-regulation of BCL2A1 promotes cell proliferation and reduces cell apoptosis by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway (both P<0.05).ConclusionsThe up-regulation of miR-664a-3p inhibits the proliferation of KGN cells and increases apoptosis by down-regulating the expression of BCL2A1 and blocking the MAPK/ERK signaling pathway.

Phytochemical Profile, Antioxidant and Anti Proliferative Studies in Different Extracts of Artocarpus kemando Miq. Bark

In this study, the stem bark of Artocarpus kemando was used to find alternative antioxidants from natural sources with fewer side effects. A. kemando wasextracted successively using hexane, chloroform and methanol solvents, and evaluated for antioxidant, cytotoxic, and antiproliferative activities. The extracts were investigated for determination of their total phenolic content (TPC) and total flavonoid content (TFC). Then, the antioxidant activities were evaluated using chemical based assays such as ferric reducing antioxidant power (FRAP), total antioxidant capacity, radical scavenging of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2’-azino-bis (3-ethylbenzothia zoline-6-sulphonic) (ABTS), β-carotene-linoleic acid (BC) assay, oxygen radical absorbance capacity (ORAC), and cell based assay. The cytotoxic study was done using four different cell lines namely human estrogen receptor positive (ER+) breast cancer cell line (MCF7), human ovarian cancer cell line (CAOV-3), human promyelocytic leukemia cell line (HL60), and normal immortalised human ovarian surface epithelial cell line (TI074), and were evaluated using microculture tetrazolium salt (MTT) before morphological change study was done on CAOV-3 cell. In this study, methanol extract displayed the most promising antioxidant activity compared to other extracts when tested with DPPH, FRAP, ABTS, TAOC, BC,ORAC, and cytoprotective assays. The remarkable activity showed by the methanol extract might be due to its high content of phenolic and flavonoid compounds at 855.5 ± 0.01 GAE μg/mL and 145.45 ± 0.06 QAE μg/mL, respectively. Nevertheless, the chloroform extract displayed better scavenging activity compared to other extracts with IC50 value of 618 ± 0.04 μg/mL in DPPH assay. Each extract was analysed using Gas Chromatography Mass Spectrophotometry and the chemical constituents obtained were then analysed. In the cytoprotective activity, the methanol extract showed a comparable cytoprotection with ascorbic acid against the free radicals at the lowest effective concentration (EC50) value of 21.48 μg/mL. However, in the cytotoxicity study, only chloroform extract displayed significant toxicity against the cancer cells with IC50 value of 27.9 ± 0.03, 24.1 ± 0.02 and 9.0 ± 0.04 μg/mL after treatment at 24, 48, and 72 h, respectively. The chloroform extract of A. kemando was found capable of inducing apoptosis as shown with cell membrane blebbing, chromatin condensation and formation of apoptotic bodies. The results obtained from the study showed that A. kemando bark could be a potential antioxidant and antitumor agents particularly on human ovarian cancer cells.

Regulator of Ribosome Synthesis 1 Influences the Proliferation, Migration, and Invasion of Ovarian Cancer Cells

We investigated the effects of silencing the regulator of ribosome synthesis 1 (RRS1) gene on the proliferation, migration, and invasion of ovarian carcinoma cells, and its possible role in modulating signal transduction in these cells. Normal ovarian epithelial cell line IOSE80 was used as a control. We examined the mRNA and protein level of RRS1 using qRT-PCR and western blot in control and ovarian carcinoma cells (SKOV-3, SW626, and CAOV3). RNA interference technology was used to knockdown RRS1 expression in CAOV3 cells. MTT was used to examine the proliferation of these cells, while a Transwell assay was used to assay the cells’ migration and invasion abilities. Western blot was used to measure the levels of CyclinD1, P21, MMP-2, MMP-9, p-JAK2 and p-STAT3 proteins. In comparison with normal ovarian epithelial cells (IOSE80), RRS1 mRNA and protein levels were increased in ovarian carcinoma cells (SKOV-3, SW626 and CAOV3) (P &lt; 0.05). Because RRS1 levels were highest in CAOV3 cells, these cells were used for subsequent experiments. RRS1 gene expression was knocked down in CAOV3 cells, and in comparison with the negative control group, siRNA-RRS1 cells exhibited decreased proliferation in the MTT assay after 48 h and 72 h (P &lt; 0.05). These cells also exhibited reduced migration and invasion (P &lt; 0.05). Further, siRNA-RRS1 cells exhibited reduced expression of CyclinD1, MMP-2, MMP-9, P-JAK2 and P-STAT3 proteins (P &lt; 0.05), while P21 protein levels were increased (P &lt; 0.05). Silencing RRS1 expression inhibits the proliferation, migration, and invasion of ovarian carcinoma cells. This effect may be mediated by the inhibition of the STAT3 signaling pathway in these cells.

Elevated expression of Tweety homologue 3 predicts poor clinical outcomes in ovarian cancer.

Objective: To define the alteration of tweety homolog (TTYH) expression in patients with ovarian carcinoma (OC) and its correlation to prognosis.Methods: Kaplan-Meier (KM) plotter was used to evaluate the association between TTYHs expression and clinical outcomes of OC patients. The distribution of 20-year overall survival (OS) and progression-free survival (PFS) was estimated using KM survival plots. The mRNA expression of TTYHs in OC and normal ovarian tissues was confirmed by the Oncomine database. Then, using immunohistochemistry assay, the expression of TTYH1 and TTYH3 proteins in serous OC and normal ovarian tissues was detected. In addition, the protein and mRNA levels of TTYH1 and TTYH3 in human OC cell lines ES-2, A2780 and SKOV3 and normal ovarian epithelial cell lines IOSE80 were assessed by western blotting and real-time quantitative polymerase chain reaction (qRT-PCR).Results: TTYH1 possessed meaningful significance in predicting better prognosis in the serous, advanced stage, and well-differentiated OC patients, while TTYH3 expression predicted worse prognosis in serous, late-stage, and poorly differentiated OC patients. High expression of TTYH1 displayed an association with favorable PFS in OC patients with TP53 mutation. However, enhanced TTYH3 was related to an adverse clinical outcome in TP53-mutated OC patients. In addition, TTYH1 was related to a better clinical outcome in OC patients with platinums-based chemotherapy, but only indicated improved overall survival in OC patients who received taxol or platin + taxol chemotherapy. The up-regulated expression of TTYH3 predicted worse survival in OC patients receiving platin, taxol, or platin + taxol chemotherapy regimen. The levels of TTYH3 mRNA and protein were higher in OC cells and tissues when compared to normal ovarian cells and tissues.Conclusions: TTYH3 was a potential predictor for poor clinical outcome in OC patients, particularly in patients with serous, late-stage, poorly differentiated, TP53-mutation or the patients treated with chemotherapy regimens (platin, taxol, or platin + taxol).

Histone Methyltransferase KMT2D Regulates H3K4 Methylation and is Involved in the Pathogenesis of Ovarian Cancer

To investigate the function of histone-lysine N-methyltransferase 2D (KMT2D) on the methylation of H3 lysine 4 (H3K4) in the progression of Ovarian cancer (OV). KMT2D, ESR1 and H3K4me expressions in surgical resected tumors and tumor adjacent tissues of OV from 198 patients were determined using immunohistochemistry (IHC). Human OV cell lines including SKOV3, HO-8910 cells and normal ovarian epithelial cell line IOSE80 were employed for in vitro experiment, and BALB/C female nude mice were used for in vivo study. qRT-PCR and Western blotting were implemented for measuring the KMT2D, ESR1, PTGS2, STAT3, VEGFR2, H3K4me and ELF3 levels. Chromatin immunoprecipitation (ChIP) analysis was used for studying the binding between ESR1 and H3K4me. Edu staining assay was executed to determine cell viability, and colony formation and cell invasion assay. The immunofluorescence method was utilized for the visualization of protein expression and distribution in cells. In this study, KMT2D, ESR1 and H3K4me were found upregulated in OV progression. Mutated H3K4me could inhibit the proliferation, colony formation and invasion ability of OV cells. Mutated H3K4me could also hinder the ESR1 in SKOV3 expressions and HO-8910 cells, which would further mediate PTGS2/STAT3/VEGF pathway. In vivo studies also demonstrated that mutated H3K4me inhibited OV progression via targeting ESR1. All the ChIP-PCR analysis indicated the moderator effect of H3K4me on ESR1. Our findings indicated that ESR1 played an important role in the OV progression. Besides, H3K4me could promote cell proliferation and inhibit apoptosis of OV cells. Meanwhile, it could also targets the ESR1 production to enhance the migration and invasion of OV cells, which was through the activation of ESR1-ELF3-PTGS2-STAT3-VEGF cascade signaling pathway.