Normal Ovarian Epithelial Cell Line Research Articles

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.

MiR-29c-3p regulates proliferation and migration in ovarian cancer by targeting KIF4A

BackgroundIncreasing evidence suggested that microRNA and kinesin superfamily proteins play an essential role in ovarian cancer. The association between KIF4A and ovarian cancer (OC) was investigated in this study.MethodsWe performed bioinformatics analysis in the GEO database to screen out the differentially expressed miRNAs (DEmiRNAs) associated with ovarian cancer prognosis. Upstream targeting prediction for KIF4A was acquired by using the mirDIP database. The potential regulatory factor miR-29c-3p for KIF4A was obtained from the intersection of the above all miRNAs. The prognosis of KIF4A and target-miRNA in OC was obtained in the subsequent analysis. qRT-PCR and Western blot detected KIF4A expression level in IOSE80 (human normal ovarian epithelial cell line). In the meantime, the gene expression level was detected in A2780, HO-8910PM, COC1, and SKOV3 cell lines (human ovarian carcinoma cell line). MTT and colony formation assays were used to detect cell proliferation of SKOV3 cell line. The following assays detected cell migration through the use of transwell and wound heal assays. Targeted binding relationship between KIF4A and miRNA was detected by using the dual-luciferase reporter assay.ResultsBoth high expression of KIF4A and lower expression of miR-29c-3p could be used as biomarkers indicating poor prognosis in OC patients. Cellular function tests confirmed that when KIF4A was silenced, it inhibited the proliferation and migration of OC cells. In addition, 3′-UTR of KIF4A had a direct binding site with miR-29c-3p, which indicated that the expression of KIF4A could be regulated by miR-29c-3p. In subsequent assays, the proliferation and migration of OC cells were inhibited by the overexpression of miR-29c-3p. At the same time, rescue experiments also confirmed that the promotion of KIF4A could be reversed by miR-29c-3p.ConclusionIn a word, our data revealed a new mechanism for the role of KIF4A in the occurrence and development of OC.

LncRNA HEIH accelerates cell proliferation and inhibits cell senescence by targeting miR-3619-5p/CTTNBP2 axis in ovarian cancer.

Epithelial ovarian cancer is the most lethal malignancy in gynecology. Numerous studies have confirmed that long noncoding RNAs (lncRNAs) are abnormally expressed in ovarian cancer and are closely associated with the cell proliferation and senescence in cancers. However, the role and underlying molecular mechanism of long noncoding RNA high expression in hepatocellular carcinoma (HEIH) in ovarian cancer remain unknown. Experiments including Real-time quantitative polymerase chain reaction, RNA immunoprecipitation, luciferase reporter, Fluorescence in situ hybridization, western blot, colony formation assays, β-galactosidase senescence assay, cell apoptosis, proliferation, invasion, and migration assays were applied to investigate the role of HEIH in ovarian cancer. The data were expressed as the mean ± standard deviation. Student t test was used to compare the data between two groups. The one-way analysis of variance was applied to compare the data among multiple groups with Tukey post hoc test. All experiments were repeated three times. P < 0.05 was considered statistically significant. Herein, HEIH expression was found to be up-regulated in ovarian cancer tissues (n = 25; twofold higher than normal tissues, P < 0.05) and cell lines (sixfold higher than normal ovarian epithelial cell line on average, P < 0.05), and high HEIH expression predicted poor prognosis (survival rate is about 25% after 40 mo; P < 0.05). Moreover, we found that HEIH accelerated proliferation, migration, and invasion, whereas inhibited cell senescence in ovarian cancer (P < 0.05). In mechanism, HEIH was confirmed to serve as a sponge for miR-3619-5p, and miR-3619-5p counteracted HEIH-mediated regulation of ovarian cancer (P < 0.05). Besides, cortactin-binding protein 2 (CTTNBP2) was found to be the downstream target of miR-3619-5p. Rescue assays validated that CTTNBP2 up-regulation significantly reversed the inhibitory effects of HEIH knockdown on ovarian cancer progression (P < 0.05). Furthermore, we found that HEIH facilitated tumor growth in vivo by regulating CTTNBP2 expression (P < 0.05). In conclusion, our research revealed that HEIH accelerated cell proliferation, migration and invasion, whereas inhibited cell senescence in ovarian cancer via targeting the miR-3619-5p/CTTNBP2 axis. These findings may be valuable for finding new therapeutic targets to improve ovarian cancer treatment.

The Overexpression of Keratin 23 Promotes Migration of Ovarian Cancer via Epithelial-Mesenchymal Transition.

Background Keratin 23 (KRT23) is a new member of the KRT gene family and known to be involved in the development and migration of various types of tumors. However, the role of KRT23 in ovarian cancer (OC) remains unclear. This study is aimed at investigating the function of KRT23 in OC. Methods The expression of KRT23 in normal ovarian and OC tissues was determined using the Oncomine database and immunohistochemical staining. Reverse transcription quantitative polymerase chain reaction assay was used to analyze the expression of KRT23 in normal ovarian epithelial cell lines and OC cell lines. Small interfering RNA (siRNA), wound healing assay, and transwell assay were conducted to detect the effects of KRT23 on OC cell migration and invasion. Further mechanistic studies were verified by the Gene Expression Profiling Interactive Analysis platform, Western blotting, and immunofluorescence staining. Results KRT23 was highly expressed in OC tissues and cell lines. High KRT23 expression could regulate OC cell migration and invasion, and the reduction of KRT23 by siRNA inhibited the migration and invasion of OC cells in vitro. Furthermore, KRT23 mediated epithelial-mesenchymal transition (EMT) by regulating p-Smad2/3 levels in the TGF-β/Smad signaling pathway. Conclusions These results demonstrate that KRT23 plays an important role in OC migration via EMT by regulating the TGF-β/Smad signaling pathway.

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 SNHG17 promotes proliferation and invasion of ovarian cancer cells through up-regulating FOXA1.

This study was designed to investigate the specific mechanism through which long non-coding RNA (lncRNA) SNHG17 promotes the proliferative capacity and invasiveness of ovarian tumor cells. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) detected the expressions of SNHG17 and FOXA1 in 30 pairs of ovarian cancer tissue specimens and corresponding adjacent ones. Meanwhile, in ovarian cancer cell lines (A2780, OVCAR3, SKOV3, CAOV3) and normal ovarian epithelial cell line (IOSE80), SNHG17 and FOXA1 mRNA levels were also examined. In in vitro experiment, si-SNHG17, si-FOXA1, and their corresponding negative controls were transfected into ovarian cancer cell lines, respectively. After that, Cell Counting Kit-8 (CCK-8) and plate cloning experiments were carried out to examine cell proliferation ability, while transwell assay was performed for cell invasiveness detection. Lastly, the interplay between SNHG17 and FOXA1 was further assessed via qRT-PCR and Western blot. qRT-PCR results indicated that SNHG17 expression was remarkably enhanced in ovarian cancer tissue samples compared with that in adjacent ones. In addition, ovarian cancer cells also contained higher expression of SNHG17 than the normal ovarian epithelial cells. However, down-regulating SNHG17 attenuated the cell proliferation and invasive ability. At the same time, compared with that in adjacent tissue samples, FOXA1 also showed a higher expression in ovarian cancer tissues, which was positively correlated with SNHG17. Silencing SNHG17 markedly downregulated FOXA1 expression at both mRNA and protein levels. Furthermore, downregulation of FOXA1 expression was found to be able to inhibit cell proliferation and invasion as well. LncRNA SNHG17 can promote ovarian tumor cell proliferative ability and invasiveness by upregulating FOXA1, and serve as a potential therapeutic target for ovarian cancer.

Vigilin interacts with ER-β to protect against palmitic acid-induced granulosa cells apoptosis via inhibiting calcineurin-mediated Drp1 signaling pathway

BackgroundHypercholesterolemia is one of the causes of female infertility, and as a common fatty acid in follicular fluid, palmitic acid (PA) level plays a vital role in granule cell which is closely related to the developmental potential of follicle. MethodsThe ovarian granulosa cell-like human granulosa (KGN) cell line and the immortalized normal ovarian surface epithelial cell line (IOSE80) were used to verify the effect of PA on cell viability and apoptosis by MTT and flow cytometry assay, respectively. Then mitochondria damage was confirmed by mitochondrial membrane potential, mitochondrial ROS detection assay and western blot in KGN cells. Thorough luciferase reporter assay and RIP-qPCR, the relationship between vigilin and ER-β was investigated. ResultsIn our study, PA induced mitochondrial damage-mediated cell apoptosis of KGN cells was dose-dependently, while PA shown no effects on in IOSE80 cells. Then the role of calcineurin (CnA)-mediated Drp1 signaling pathway on KGN cells was confirmed by treating with Mdivi-1 or FK506T. In addition, the changed level of vigilin and ER-β was observed in cell apoptosis of KGN cells induced by PA. By transfecting vigilin vector or ER-β vector into KGN cells, respectively, vigilin and ER-β were demonstrated to regulate the apoptosis of KGN cells. And vigilin was a binding protein of ER-β mRNA. ConclusionVigilin could interact with ER-β mRNA to promote ER-β expression. And Vigilin/ ER-β relieve the mitochondrial damage and cell apoptosis induced by PA through regulating CnA-mediated Drp1 signaling pathway, which revealed the mechanism and strategy of hypercholesterolemia in female infertility.

MicroRNA-196b promotes cell growth and metastasis of ovarian cancer by targeting ZMYND11.

The purpose of this study was to explore the mechanism by which microRNA-196b exerts a tumor promotion effect on ovarian cancer (OCa). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression of microRNA-196b in 60 pairs of tumor samples and paracancerous ones collected from patients with ovarian cancer, meanwhile, microRNA-196b expression in human ovarian cancer cell lines and normal ovarian epithelial cell lines were also analyzed by qRT-PCR. Bioinformatics methods suggested that ZMYND11 was predicted the target gene of microRNA-196b and its binding relationship was verified by dual luciferase reporter gene experiment. Then cell counting kit (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assay were performed to analyze the influence of microRNA-196b overexpression on OCa cell proliferation. QRT-PCR results showed that microRNA-196b level in OCa tissues was remarkably higher than that in normal ovarian tissues, which was closely relevant to the poor prognosis of tumors. The dual luciferase reporter gene experiments confirmed that microRNA-196b could directly bind to the 3'-UTR of ZMYND11. Overexpression of microRNA-196b remarkably enhanced the proliferation, invasiveness and migratory ability of OCa cells. Meanwhile, overexpression of microRNA-196b significantly decreased ZMYND11 mRNA and protein expression. In addition, ZMYND11 level was also significantly increased in ovarian cancer cells. Compared with the miRNA-NC group, microRNA-196b-mimics + ZMYND11-OE treatment reversed the effect of microRNA-196b-mimics on OCa cell functions. MicroRNA-196b was highly expressed in OCa tissues, which can promote the proliferation, invasiveness and migratory ability of OCa cells by targeting ZMYND11. In addition, the expression disorder of microRNA-196b was associated with the malignant development of OCa.

Long non-coding RNA NPBWR1-2 affects the development of ovarian cancer via multiple microRNAs.

Ovarian cancer has a high incidence rate and mortality in gynaecologic malignancies. Epithelial ovarian cancer (EOC) accounts for >95% of ovarian cancer cases. Most of the patients with EOC are difficult to diagnose in early stage. The aim of the present study was to compare the long non-coding (lnc)RNA expression profiles of five ovarian cancer cell lines (IGROV1, A2780, SKOV3, ES2, and Hey) and an ovarian epithelial cell line (IOSE80) in order to identify differentially expressed lncRNAs and their associated microRNAs (miRNAs). The expression profiles of lncRNAs and mRNAs in these cell lines were determined by microarray gene analysis and reverse transcription-quantitative PCR. lncRNA neuropeptides B and W receptor 1–2 (NPBWR1-2) overexpression was induced in the SKOV3 cell line. Cell viability, proliferation, migration, invasion and apoptosis were evaluated using MTT, colony-formation, Transwell and flow cytometry assays, respectively. The microarray results indicated that several lncRNAs were differentially expressed in the five ovarian cancer cell lines compared with the normal ovarian epithelial cell line. Compared with IOSE80, lncRNA NPBWR1-2 was downregulated by more than two-fold in all five ovarian cancer cell lines. Moreover, NPBWR1-2 overexpression in the SKOV3 cell line decreased cell viability, inhibited proliferation, migration and invasion, and promoted apoptosis compared with the control cells. A total of 20 miRNAs, which are involved in tumorigenesis and development, were predicted to be associated with NPBWR1-2 by bioinformatics analysis. The results of the present study suggest that lncRNA NPBWR1-2 affects the occurrence and development of ovarian cancer via multiple miRNAs, providing a theoretical basis for the development of novel clinical treatments.

Effects of Wnt/β-Catenin Signal Pathway Regulated by miR-342-5p Targeting CBX2 on Proliferation, Metastasis and Invasion of Ovarian Cancer Cells.

ObjectiveThis study aimed to investigate the effect of Wnt/β-catenin signal pathway mediated by miR-342-5p targeting CBX2 gene on the proliferation, metastasis, invasion and apoptosis of ovarian cancer cells, and to explore its related regulatory mechanism.MethodsHuman normal ovarian epithelial cell line IOSE80, human ovarian cancer cell line SKOV3 and OVCAR3 were the subjects. Software were used to predict the binding site of miR-342-5p targeting CBX2 gene. The proliferation rate of ovarian cancer cells was detected by MTT method; the cell viability of each group was observed by colony formation test; the apoptosis of cells in each group was detected by flow cytometry; the invasive ability of cells was determined by transwell test, and the migration ability of cells was detected by scratch test. The mRNA expression levels of miR-342-5p, CBX2, Wnt1, β-catenin, C-myc and Cyclin D1 were measured by qRT-PCR. Also, Western blot was used to determine the protein expression levels of CBX2, Wnt1, β-catenin, C-myc and Cyclin D1.ResultsCBX2 was identified as the target gene of miR-342-5p. MTT test results showed that miR-342-5p could significantly inhibit the proliferation of SKOV3 and OVCAR3 cells, colony formation assay results indicated that the viability of SKOV3 and OVCAR3 cells transfected with miR-342-5p decreased significantly, and flow cytometry results suggested that miR-342-5p could promote the apoptosis of SKOV3 and OVCAR3 cells. Also, the results of transwell showed that miR-342-5p could significantly inhibit the invasive ability of SKOV3 and OVCAR3 cells, and the results of scratch assay suggested that miR-342-5p could significantly inhibit the migration of SKOV3 and OVCAR3 cells. Moreover, qRT-PCR and Western blot results indicated that the mRNA and protein expression levels of CBX2, Wnt1, β-catenin, C-myc and Cyclin D1 decreased in SKOV3 and OVCAR3 cells transfected with miR-342-5p, while the mRNA expression levels of miR-342-5p increased significantly (P<0.05).ConclusionMiR-342-5p targeted gene is CBX2, which can significantly reduce the proliferation, invasion, migration and viability of ovarian cancer cell lines SKOV3 and OVCAR3, and promote their apoptosis. The mechanism may be related to the mediation of Wnt/β-catenin signal pathway and down-regulation of the related genes expression.

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.

Mir-30b-3p affects the migration and invasion function of ovarian cancer cells by targeting the CTHRC1 gene

BackgroundThe aim of this study was to investigate the effect role and mechanism of miR-30b-3p on ovarian cancer cells biological function.MethodsThe expression of miR-30b-3p was detected in ovarian cancer cell lines and normal ovarian epithelial cell line by qRT-PCR. Mir-30b-3p mimic was transfected into OVCAR3 cells. Cell-counting kit-8 (CCK-8) assay was conducted to explore the effect of mir-30b-3p on the OVCAR3 cells’ proliferation. Cell cycle and apoptosis were detected by Flow cytometry. Cell invasion ability was detected by Transwell test. The regulation of putative target of miR-30b-3p was verified by double luciferase reporter assays and Western blot.ResultWe found that miR-30b-3p was downregulated in OVCAR3 cells. Overexpression of miR-30b-3p suppressed proliferation, promoted apoptosis, slowed cell cycle and inhibited migration and invasion of OVCAR3 cells. Bioinformatics analysis identified 3′-untranslated region (3′UTR) of Collagen triple helix repeat-containing 1 (CTHRC1) as the presumed binding site for miR-30b-3p. Detection of double luciferase reporter and Western-Blot result confirmed that CTHRC1 was the target gene of miR-30b-3p. Furthermore, E-cadherin, β-cadherin and Vimentin protein expression level were changed after transfection of miR-30b-3p.ConclusionmiR-30b-3p function as an anti-cancer gene. Overexpression of miR-30b-3p can inhibit the biological function of ovarian cancer cells. MiR-30b-3p targets CTHRC1 gene plays an important role in epithelial–mesenchymal transformation (EMT), and supports miR-30b-3p as a potential biological indicator for ovarian cancer in the future.

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.

Regulatory mechanism of lncRNA NORAD on proliferation and invasion of ovarian cancer cells through miR-199a-3p.

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.

&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.

MiR-96-5p promotes the proliferation and migration of ovarian cancer cells by suppressing Caveolae1

BackgroundOvarian cancer (OC) is the second most common gynaecological malignancy. MicroRNAs (miRNAs) have been found to be aberrantly expressed in OC tissue and have been proposed as biomarkers and therapeutic targets for OC.ResultsIn this study, we found that miR-96-5p was up-regulated in OC tissues and OC cells compared to normal ovarian tissues and epithelial cell line. And, miR-96-5p was also up-regulated in the serum samples from OC patients compared to health participants. In addition, there was a positive correlation of miR-96-5p levels between OC tissues and serum samples. At the cellular level, overexpression of miR-96-5p promoted cell proliferation and migration in OC cells. Moreover, we further validated Caveolae1 (CAV1) as the direct target of miR-96-5p in OC cells through luciferase activity assays and western blot. CAV1 was obvious low expression in OC tissues. The overexpression of CAV1 abrogated the promotion of miR-96-5p on the OC cells proliferation and migration. Finally, we found that AKT signaling pathway was involved in this process. MiR-96-5p inhibited the phosphorylation of AKT and expression of down-stream proteins Cyclin D1 and P70 by targeting CAV1.ConclusionsThe above findings suggested that targeting miR-96-5p may be a promising strategy for OC treatment.

LncRNA MEG3 modified epithelial-mesenchymal transition of ovarian cancer cells by sponging miR-219a-5p and regulating EGFR.

This study was aimed to verify whether there existed any associations between long noncoding RNA MEG3/miR-219a-5p/EGFR axis and the development of ovarian cancer (OC). As a whole, we gathered 317 pairs of OC tissues and surgical marginal normal tissues and simultaneously acquired four OC cell lines (ie, A2780, Caov-3, OVCAR-3, and SKOV-3) and human normal ovarian surface epithelial cell line. Moreover, pcDNA3.1-MEG3, si-MEG3, miR-219a-5p mimic, miR-219a-5p inhibitor, pcDNA3.1-EGFR, and si-EGFR were, respectively, transfected into the OC cells, and their impacts on viability, proliferation, apoptosis, invasion, and migration of OC cells were assessed via conduction of MTT assay, colony formation assay, flow cytometry assay, transwell assay, and scratch assay. Ultimately, dual-luciferase reporter gene assay was performed to testify the targeted relationships among maternally expressed gene 3 (MEG3), miR-219a-5p, and estimated glomerular filtration rate (EGFR). It was indicated that underexpressed MEG3 and miR-219a-5p were significantly associated with unfavorable prognosis of patients with OC when compared with overexpressed MEG3 and miR-219a-5p (P < .05). In addition, the OC cells transfected with si-MEG3 or miR-219a-5p inhibitor exhibited stronger viability, proliferation, invasion, and migration than untreated cells (P < .05). Correspondingly, the apoptotic percentage of OC cells was reduced observably under treatments of si-MEG3 and miR-219a-5p inhibitor (P < .05). Moreover, MEG3 exerted modulatory effects on the expression of miR-219a-5p (P < .05), and there was a sponging relationship between them (P < .05). Finally, EGFR expression was modified by both MEG3 and miR-219a-5p significantly (P < .05), and raising EGFR expression could changeover the impacts of MEG3 and miR-219a-5p on the above-mentioned activity of OC cells (P < .05). Conclusively, MEG3 could serve as a promising biomarker for diagnosis and treatment of OC, considering its involvement with OC etiology via regulation of miR-219a-5p/EGFR axis.

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.

MicroRNA let‑7d‑5p rescues ovarian cancer cell apoptosis and restores chemosensitivity by regulating the p53 signaling pathway via HMGA1.

Ovarian cancer (OC) is the gynecological malignancy type with the highest mortality rate in females. The regulatory effect of microRNAs(miRs) on their target genes serves a key role in tumor development. Therefore, in the present study, whether miR let‑7d‑5p targeting high mobility groupA1(HMGA1) regulated biological characteristics and chemosensitivity of OC cells by mediating the p53 signaling pathway was investigated. The let‑7d‑5p level was detected in OC tissues and adjacent normal tissues, followed by detection in OC cell lines SKOV3, A2780, OVCAR‑3 and CaOV3, and human normal ovarian epithelial cell line(IOSE‑80), in order to select the OC cell line for the following experiments. Subsequently, OC cells were treated with the let‑7d‑5p mimic, siHMGA1 and Tenovin‑1. The targeting association between let‑7d‑5p and HMGA1 was then examined, and the OC cell viability, migration, cycle and apoptosis were evaluated. Subsequently, the chemosensitivity of OC cells to cisplatin was verified. Finally, expression levels of let‑7d‑5p, HMGA1, p21, B‑cell lymphoma‑2(Bcl‑2)‑associatedX (Bax), p27, p53 wild‑type(p53wt), p53 mutated (p53mut), proliferating cell nuclear antigen(PCNA), cyclin‑dependent kinase2(CDK2), matrix metallopeptidase(MMP)2, MMP9 and Bcl‑2 were determined. As demonstrated in the results, let‑7d‑5p expression was low in OC tissues and had an increased reduction in the OVCAR‑3 cell line. HMGA1 was confirmed as a target of let‑7d‑5p, and its expression was also silenced by let‑7d‑5p. let‑7d‑5p repressed OC cell viability, migration, cell cycle progression and apoptosis, while it promoted the chemosensitivity of OC cells to cisplatin by targeting HMGA1. The expression of let‑7d‑5p, p21, Bax, p27 and p53wt was increased, while that of HMGA1, p53mut, PCNA, CDK2, MMP2, MMP9 and Bcl‑2 was reduced following cell transfection. The results in the present study provided evidence that let‑7d‑5p may suppress proliferation, and facilitate apoptosis and cisplatin chemosensitivity of OC cells by silencing HMGA1 via the p53 signaling pathway.