Normal Human Ovarian Epithelial Cells Research Articles

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.

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.

Upregulation of ryanodine receptor-1 dependent calcium signaling contributes to the malignance of high-grade serous ovarian cancer (HGSOC).

Ca2+ signaling plays crucial roles in cancer metastasis and progression. We analyzed RNAseq data from TCGA database and our patient samples; and identified over-expression of ryanodine receptor 1 (RYR1), the target of the FDA-approved drug dantrolene, is associated with poor survival of HGSOC patients. RYR1 expression is also markedly higher in several HGSOC cell lines compared to normal human ovarian surface epithelial cells (HOSE) and fallopian tube epithelial cells. Suppression of RYR1 expression/activity by shRNA or dantrolene significantly inhibited proliferation and migration of HGSOC cells (OVCA432, OVCA433, OV90). Ca2+ fluorescence measurement showed that the resting [Ca2+] and Ca2+ release triggered by the RYR-agonist 4-chloro-m-cresol (4-cmc) were significantly higher in HGSOC cells than the HOSE cells. Store-operated Ca2+ entry (SOCE) induced by 4-cmc was also enhanced in HGSOC cells. The elevated resting [Ca2+], the 4-cmc-induced Ca2+ release and SOCE in the HGSOC cells were reduced to the control level by shRNA against RYR1; and were significantly potentiated by over-expression of RYR1. RYR-dependent endoplasmic reticulum (ER)-mitochondrial Ca2+ transfer was examined by expressing the ER specific Ca2+ biosensor G-CEPIA1er and mitochondria Ca2+ biosensor CEPIA2mt in HGSOC cells. Activation of RYR by 4-cmc triggered a large reduction in ER [Ca2+], which was associated with a robust increase in mitochondrial [Ca2+]. Activation of RYRs by photorelease of caged cADP-ribose in a subcellular region caused an immediate transient local reduction in ER [Ca2+] and a fast transient local increase in mitochondria [Ca2+]. In contrast, RyR-dependent ER-mitochondrial Ca2+ transfer was not observed in the HOSE cells. These observations are the first direct evidence of enhanced RYR-mediated ER-mitochondrial Ca2+ transfer in ovarian cancer cells, and upregulation of RYR1-dependent Ca2+-signaling pathway contributes to the malignant phenotypes of HGSOC cells.

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.

MIR503HG impeded ovarian cancer progression by interacting with SPI1 and preventing TMEFF1 transcription.

MIR503 host gene (MIR503HG) acts as an important tumor suppressor in many human cancers, but its role and regulatory mechanism in ovarian cancer need to be further studied. In this study, lower expressed MIR503HG was observed in ovarian tumor tissues and cells than in adjacent normal tissues and normal human ovarian epithelial cells. MIR503HG overexpression impaired the proliferative, invasive and EMT properties, and facilitated cell apoptosis in ovarian cancer cells. Nuclear and cytoplasmic separation test suggested that MIR503HG was mainly expressed in the nucleus. RNA immunoprecipitation and RNA pull-down assays confirmed that MIR503HG could bind to transcription factor SPI1 (Spi-1 proto-oncogene), and dual luciferase reporter gene and Chromatin immunoprecipitation assays verified that SPI1 could bind to TMEFF1 (Transmembrane protein with EGF like and two follistatin like domains 1) promoter, suggesting that MIR503HG suppressed TMEFF1 expression by competitively binding SPI1 and blocking transcriptional activation of TMEFF1. Moreover, interference with TMEFF1 reversed the promotion effect of MIR503HG silence on the malignant behaviors of ovarian cancer cells. Moreover, MIR503HG knockdown activated the MAPK and PI3K/AKT pathways by increasing the expression of TMEFF1. In addition, overexpression of MIR503HG in vivo suppressed the tumorigenic ability in nude mice. In conclusion, MIR503HG acted as a tumor suppressor lncRNA in ovarian cancer by suppressing transcription factor SPI1-mediated transcriptional activation of TMEFF1.

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.

Talcum powder induces malignant transformation in normal human primary ovarian epithelial cells.

Several studies have linked perineal use of talcum powder to increased risk of ovarian cancer (OC). Here, we determined that exposure to talcum powder induces malignant transformation in human normal ovarian cells. Human primary ovarian epithelial cells (HPOE), ovarian epithelial cells (HOSEpiC), and primary fibroblasts (NF) were treated with either 100 or 500 μg/mL of talcum powder or titanium dioxide (TiO<inf>2</inf>) as a particulate control for 72 hours before assessment with a cell transformation assay and p53 and Ki-67 immunohistochemistry. Treatment with talcum powder resulted in formation of colonies, indicating cell malignant transformation in a dose dependent manner in ovarian cell lines. No colonies formed in the untreated ovarian cells or control ovarian cells (TiO<inf>2</inf> treated) at either dose. There were no colonies formed in talc treated NF cells. Transformed ovarian cells were increased by 11% and 20% in HPOE and 24% and 40% in HOSEpic cells for talcum powder 100 and 500 μg/mL doses, respectively (P<0.05). There were no detectible transformed cells when cells were treated with TiO<inf>2</inf>. Importantly, p53 mutant type as well as increased expression of Ki-67 were detected in HPOE and HOSEpic cells when exposed to talcum powder. Exposure to talcum powder induces malignant transformation in ovarian epithelial cells but not in NF cells. These findings represent a direct effect of talcum powder exposure that is specific to normal ovarian cells and further supports previous studies demonstrating an association between the genital use of talcum powder and an increased risk 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.

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.

MicroRNA-378a-3p contributes to ovarian cancer progression through downregulating PDIA4.

ObjectiveMicroRNAs, as essential players in tumorigenesis, have been demonstrated to have a revolutionary effect on human cancer research. Ovarian cancer is the primary reason of death among gynecologic malignancies. In view of this, it is significant to identify prognostic and predictive markers for treatment of ovarian cancer. The aim of this study was to probe into the effects of miR‐378a‐3p and protein disulfide‐isomerase A4 (PDIA4) on the biological functions of ovarian cancer cells.MethodsmiR‐378a‐3p expression and PDIA4 messenger RNA expression in human ovarian cancer cells, normal human ovarian epithelial cells, and serum of both ovarian cancer patients and healthy people were detected by reverse transcription‐quantitative polymerase chain reaction, and the PDIA4 protein expression was tested by Western blot analysis. Ovarian cancer OVCAR3 and SKOV3 cells were transfected or cotransfected with miR‐378a‐3p mimic or pcDNA3.1‐PDIA4 or their negative control plasmids to explore their roles in biological functions in ovarian cancer cells. Luciferase activity and RIPA assays were implemented to validate the interaction between miR‐378a‐3p and PDIA4. Western blot analysis was utilized to detect phosphatidylinositol‐3 kinase/serine/threonine kinase (PI3K/AKT) signaling pathway‐related protein expression and their phosphate expression levels.ResultsmiR‐378a‐3p was elevated and PDIA4 was decreased in ovarian cancer cells and serum. In addition, miR‐378a‐3p mimic induced ovarian cancer cell growth, while miR‐378a‐3p inhibitor and pcDNA3.1‐PDIA4 presented an inverse trend. pcDNA3.1‐PDIA4 partially eliminated the capabilities of miR‐378a‐3p mimic on ovarian cancer progression. Meanwhile, miR‐378a‐3p was found to negatively regulate PDIA4, and miR‐378a‐3p mimic increased the phosphorylation levels of AKT and PI3K, while pcDNA3.1‐PDIA4 exhibited an opposite tendency. Furthermore, pcDNA3.1‐PDIA4 largely eliminated the functions of miR‐378a‐3p mimic on phosphorylation levels of AKT and PI3K.ConclusionThis study provides evidences that miR‐378a‐3p activates PI3K/AKT signaling pathway by modulating PDIA4 expression, thereby playing a role in promoting the growth of ovarian cancer cells. This study provides novel directions for targeted therapy of ovarian cancer.

Upregulation of bromodomain PHD finger transcription factor in ovarian cancer and its critical role for cancer cell proliferation and survival.

Bromodomain PHD finger transcription factor (BPTF) is a core subunit of the nucleosome-remodeling factor (NURF) complex, which plays an important role in the development of several cancers. However, it is unknown whether BPTF regulates the progression of ovarian cancer (OC). To investigate this, we measured the relative expression levels of BPTF in OC cell lines and tissues using Western blot and immunohistochemistry, respectively, and the results were analyzed using the χ2 test. We also examined the effects from BPTF knockdown on the proliferation, migration, invasiveness, and apoptosis of OC cell lines. Mechanistic studies revealed that these effects were achieved through simultaneous modulation of multiple signaling pathways. We found that BPTF was highly expressed in OC cell lines and tissues compared with a normal human ovarian epithelial cell line and non-cancerous tissues (P < 0.05). These results are also supported by the public RNA-seq data. BPTF overexpression was correlated with a poor prognosis for OC patient survival (P < 0.05). In vitro experiments revealed that the downregulation of BPTF inhibited OC cell proliferation, colony formation, migration, and invasiveness, and induced apoptosis. BPTF knockdown also affected the epithelial-mesenchymal transition (EMT) signaling pathways and induced the cleavage of apoptosis-related proteins. Consequently, BPTF plays a critical role in OC cell survival, and functions as a potential therapeutic target for OC.

LncRNA MIAT Regulates Cell Growth, Migration, and Invasion Through Sponging miR-150-5p in Ovarian Cancer.

Background: MIAT (myocardial infarction-associated transcript) regulates cell proliferation, apoptosis, and metastasis in several cancers. In this study, the authors aimed to explore the role of MIAT in ovarian cancer. Materials and Methods: The expression of MIAT in ovarian cancer subtypes, normal human ovarian surface epithelial and ovarian cancer cell lines was measured by qualitative real-time polymerase chain reaction (qRT-PCR). OVCAR3 and SKOV3 cells were transfected with MIAT overexpression plasmid or siMIAT. The cell growth ability was then evaluated by CCK-8 and colony formation assays. The cell migration and invasion rate were separately measured by wound-healing and transwell assays. The levels of epithelial-mesenchymal transition (EMT)-associated markers were evaluated by Western blotting. MIAT sponging miR-150-5p was predicted by starBase and confirmed by dual-luciferase reporter assays. The expression of miR-150-5p in OVCAR3 and SKOV3 cells with MIAT overexpression or knockdown, and in ovarian cancer subtypes was also measured by qRT-PCR. Further analyses confirmed the role of MIAT sponging miR-150-5p in ovarian cancer cells. Results: MIAT was highly expressed in mesenchymal subtype ovarian cancer tissues and ovarian cancer cells. In OVCAR3 and SKOV3 cells, overexpression of MIAT promoted, and knockdown of MIAT suppressed the cell growth, migration, invasion, and EMT. miR-150-5p was sponged and regulated by MIAT. miR-150-5p was downregulated in mesenchymal subtype ovarian cancer. Suppression of cell migration, invasion, and EMT caused by miR-150-5p overexpression was rescued by MIAT overexpression. Conclusions: MIAT acts as an oncogene in ovarian cancer cells through sponging miR-150-5p. MIAT or miR-150-5p expression might be a potential prognostic biomarker for ovarian cancer patients. MIAT and miR-150-5p are potential therapeutic targets in treatment of ovarian cancer.

LncRNA LUCAT1 promotes proliferation of ovarian cancer cells by regulating miR-199a-5p expression.

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.

&lt;p&gt;LncRNA-MALAT1 regulates proliferation and apoptosis of ovarian cancer cells by targeting miR-503-5p&lt;/p&gt;

ObjectiveOvarian cancer (OC) is a common female disease with a poor prognosis. But the possible mechanism of OC tumor progression remains an active area of research. This study is intended to explore the effect of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on proliferation and apoptosis of OC and its mechanism.Materials and methodsMALAT1 and miR-503-5p expressions in human OC cell lines and normal human ovarian epithelial (HOSE) cell line were measured using qRT-PCR. OC cell line SKOV3 is divided into 4 groups: pcDNA3.1 group, pcDNA3.1-MALAT1 group, si-NC group, and si-MALAT1 group. MTT assay and 5-ethynyl-2ʹ-deoxyuridine (EdU) assay were applied for the detection of cell proliferation. Relationship of MALAT1 with miR-503-5p was verified using luciferase assay and RNA pull-down. The luciferase activity in cells was normalized to RNA concentrations determined by Bradford assays.ResultsMALAT1 expression in OC cells was elevated compared with HOSE cells. MTT assay and EdU assay supported that si-MALAT1 could inhibit cell proliferation in OC cells. Treatment of si-MALAT1 results in increased cell apoptosis rate in both SKOV3 cells and OVCAR3 cells. The expression of lncRNA-MALAT1 was negatively associated with the expression of miR-503-5p in OC cells, while luciferase assay and RNA pull-down together supported the direct binding of MALAT1 with miR-503-5p. Knockdown of MALAT1 was able to inhibit the activation of JAK2/STAT3 signal pathway, and MALAT1 overexpression was accompanied by activation of these factors.ConclusionlncRNA-MALAT1 can negatively target miR-503-5p expression to further promote proliferation and depress apoptosis of OC cells through the JAK2-STAT3 pathway.

Promoter Methylation Down-regulates Osteoprotegerin Expression in Ovarian Carcinoma.

Previous studies have documented that osteoprotegerin (OPG) is involved in the development and progression of several human malignancies. However, OPG has also been shown to act as a tumor suppressor. The aim of this study was to examine the expression status of OPG in ovarian carcinoma cells and investigate the underlying mechanism responsible for alterations in OPG expression. The expression levels of OPG mRNA and protein were assessed in human ovarian carcinoma cell lines. The methylation status of the OPG promoter region was determined using the bisulfite pyrosequencing technique. The effects of demethylation on OPG expression were also analyzed. The human ovarian carcinoma cell lines, SW 626, OVCAR-3, ES-2, TOV-112D, and TOV-21G, expressed significantly lower levels of OPG mRNA and protein than the normal human ovarian epithelial cell line, HS823.Tc. Moreover, three CpG sites in the OPG promoter region were highly methylated in the SW 626, OVCAR-3, ES-2, and TOV-112D ovarian carcinoma cell lines compared to normal control cells. Furthermore, in all the examined ovarian carcinoma cell lines, treatment with the demethylating agent, 5-aza-2-deoxycytidine, resulted in significantly increased expression levels of OPG mRNA and protein compared to the respective pre-treatment levels. OPG expression was down-regulated in the studied ovarian carcinoma cells compared to the normal control cells, while demethylation significantly restored OPG expression in the OPG-down-regulated cell lines. Our results suggest that OPG down-regulation in ovarian carcinoma occurs, at least partly, through epigenetic repression, suggesting its involvement in ovarian carcinogenesis.

Overexpression of SMYD3 in Ovarian Cancer is Associated with Ovarian Cancer Proliferation and Apoptosis via Methylating H3K4 and H4K20.

Background: Epigenetic regulation has been verified as a key mechanism in tumorigenesis. SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase, is a promising epigenetic therapeutic target and is overexpressed in numerous human tumors. SMYD3 can promote oncogenic progression by methylating lysines to integrate cytoplasmic kinase signaling cascades or by methylating histone lysines to regulate specific gene transcription. However, the exact role of SMYD3 in the progression of ovarian cancer is still unknown.Methods: Immunohistochemistry was employed to test SMYD3 expression in ovarian cancer tissues from clinical patients. CCK-8 assay, Real-time cell analysis (RTCA), colony formation assay, cell cycle and apoptosis tested by Flow cytometer were employed to test the effects of SMYD3 on cell proliferation and apoptosis in ovarian cancer cell lines. A PCR array was used to identify the downstream targets of SMYD3. And, PCR and Western blot were used to verify their expression. The binding of SMYD3 on the promoter of target genes were tested by ChIP assays. We also use nude mice subcutaneous tumor model and patient-derived xenograft (PDX) model to investigate the tumor promotive function of SMYD3 in vivo.Results: SMYD3 expression was higher in ovarian cancer tissues and cell lines than in normal ovarian epithelial tissue and human ovarian surface epithelial cells (HOSEpiC). After silencing SMYD3, the proliferation of ovarian cancer cells was significantly inhibited in vitro. In addition, the SMYD3-specific small-molecule inhibitor BCI-121 suppressed ovarian cancer cell proliferation. Downregulation of SMYD3 led to S phase arrest and increased the cell apoptosis rate. Furthermore, a PCR array revealed that SMYD3 knockdown caused the upregulation of the cyclin-dependent kinase (CDK) inhibitors CDKN2A (p16INK4), CDKN2B (p15INK4B), CDKN3 and CDC25A, which may be responsible for the S phase arrest. In addition, the upregulation of CD40LG and downregulation of BIRC3 may explain the increased cell apoptosis rate after silencing SMYD3. We also discovered that SMYD3 bound on the promoter of CDKN2A and down-regulated its expression by triple-methylating H4K20. In addition, SMYD3 bound on the promoter of BIRC3 and up-regulated its expression by triple-methylating H3K4. Finally, knocking down SMYD3 could inhibit ovarian cancer growth in nude mice subcutaneous tumor model and PDX model.Conclusion: Our results demonstrated that SMYD3 was overexpressed in ovarian cancer and contributes to the regulation of tumor proliferation and apoptosis via SMYD3-H4K20me3-CDKN2A pathway and SMYD3-H3K4me3-BIRC3 pathway. Thus, SMYD3 is a promising epigenetic therapeutic target for ovarian cancer.

Overexpression and cytoplasmic localization of caspase-6 is associated with lamin A degradation in set of ovarian cancers

BackgroundIn most women with ovarian cancer, the diagnosis occurs after dissemination of tumor cells beyond ovaries. Several molecular perturbations occur ahead of tumor initiation including loss of lamin A/C. Our hypothesis was that the loss of nuclear structural proteins A type lamins (lamin A/C) transcribed from LMNA gene and substrate for active caspase-6 maybe one of the molecular perturbations. Our objective is to investigate the association between the loss of lamin A/C and the overexpression of caspase-6 in ovarian cancer cells.MethodWestern blotting and immunofluorescence were used to analyze the expression of lamin A/C and active caspase-6 in normal human ovarian surface epithelial (HOSE) cells, immortalized human ovarian surface epithelial cells and a set of seven ovarian cancer cell lines (including OVCAR3, OVCAR5, and A2780). The activity of caspase-6 was measured by densitometry, fluorescence and flow cytometry. Immunohistochemistry was used to evaluate the expression of caspase-6 in set of ovarian cancer tissues previously reported to have lost lamin A/C.ResultsThe results showed that HOSE cells expressed lamin A/C and no or low level of active caspase-6 while cancer cells highly expressed caspase-6 and no or low level of lamin A/C. The inhibition of caspase-6 activity in OVCAR3 cells increased lamin A but has no effect on lamin C; active caspase-6 was localized in the cytoplasm associated with the loss of lamin A.ConclusionOverexpression and cytoplasmic localization of caspase-6 in ovarian cancer cells may be involved in lamin A degradation and deficiency observed in some ovarian cancer cells.

Low expression level of HMBOX1 in high-grade serous ovarian cancer accelerates cell proliferation by inhibiting cell apoptosis

Homeobox-containing 1 (HMBOX1) has been described as a transcription factor involved in the occurrence of some tumors, but its roles in ovarian cancer have never been reported. Here we aimed to investigate the roles of HMBOX1 on high-grade serous ovarian carcinoma (HGSOC). In this present study, HMBOX1 expression was decreased in HGSOC tissues and ovarian cancer cell lines (HO8910 and A2780) compared with ovarian surface epithelial tissues or normal human ovarian surface epithelial cell line (HOSEpiC). The cell proliferation of HOSEpiC was weaker than ovarian cancer cell lines. By altering the expression of HMBOX1 in A2780 and HOSEpiC, we demonstrated that HMBOX1 inhibited the cell proliferation and promoted the cell apoptosis. Furthermore, our study revealed that HMBOX1 downregulated the expression of anti-apoptotic proteins (Bcl-2, Bcl-xL), raised the expression of pro-apoptotic-regulated proteins (Bad, Bax), apoptotic executionior (Caspase3), and P53. In conclusion, HMBOX1 played important roles in occurrence of HGSOC through regulation of proliferation and apoptosis, which implied that HMBOX1 might serve as a new therapeutic target for HGSOC.