Ovarian Cancer Cell Line SKOV3 Research Articles

Knockdown of ribosomal protein S6 suppresses proliferation, migration, and invasion in epithelial ovarian cancer

BackgroundOvarian cancer typically is diagnosed late because insensitivity and lack of specificity of current biomarkers prior to its clinical detection. Ribosomal protein S6 (RPS6) is a ribosomal protein involved in the ribosomal 40S subunit, but its biological role in epithelial ovarian cancer (EOC) is still unknown.ResultsRPS6 was elevated in EOC compared to normal ovarian tissues and adenomas. Higher expression of RPS6 predicted worse prognosis in EOC. The level of RPS6 was correlated with clinical stage, histological type and pathological grade. Knockdown of RPS6 reduced the proliferation of ovarian cancer cell lines SKOV-3 and HO8910, and inhibit the migration and invasion ability. It revealed that cells arrested at G0G1 phase after knockdown of RPS6, and the expressions of CyclinD1, Cyclin E, CDK2, CDK4, CDK6 and pRb were also reduced.ConclusionsRPS6 is involved in EOC and knockdown of RPS6 could inhibit the proliferation, invasion and migration ability of EOC in vitro by inducing G0/G1 phase arrest. RPS6 is expected to be a novel biomarker and molecular target to the EOC.

Knockout of EGFL6 by CRISPR/Cas9 Mediated Inhibition of Tumor Angiogenesis in Ovarian Cancer.

Tumor angiogenesis plays an important role in the progression and metastasis of ovarian cancer. EGFL6 protein is highly expressed in ovarian cancer and has been proposed to play an important role in promoting tumor angiogenesis. Here, a CRISPR/Cas9 system was used to knockout the EGFL6 gene in the ovarian cancer cell line SKOV3, using specific guide RNA targeting the exons of EGFL6. The knockout of EGFL6 markedly inhibited the proliferation, migration, and invasion of SKOV3 cells, as well as promoted apoptosis of tumor cells. In the nude mouse model of ovarian cancer, knockout of EGFL6 remarkably inhibited tumor growth and angiogenesis. The transcript profile assays detected 4,220 differentially expressed genes in the knockout cells, including 87 genes that were correlated to proliferation, migration, invasion, and angiogenesis. Moreover, Western blotting confirmed that EGFL6 knockout downregulated the FGF-2/PDGFB signaling pathway. Thus, the results of this study indicated that EGFL6 could regulate cell proliferation, migration, and angiogenesis in ovarian cancer cells by regulating the FGF-2/PDGFB signaling pathway.

Abstract 968: Distinct pathways of DC-induced CD8+T cell differentiation revealed by single-cell mRNA sequencing analysis

Abstract Purpose of Study: Dendritic Cells (DC) maturing in the conditions of acute/early versus late/chronic inflammation (such as alpha-type-1-polarized DCs [αDC1s] and “standard” nonpolarized DCs; sDCs) deliver differential “signal 3” to resting T cells, resulting in their different induction of effector functions in tumor-specific CD8+ T cells. Here, using single-cell mRNAseq (scRNAseq), a unique technology allowing for unbiased analysis of gene expression (GEX), we evaluated the GEX patterns and cellular heterogeneity of CD8+ T cells expanded by cancer-cell loaded αDC1s or sDCs. Procedure: Freshly isolated autologous (compared to DCs) CD8+ T cells were in vitro sensitized for 1 week, using either αDC1s loaded with UV/gamma-radiation-killed ovarian cancer (OvCa) cell lines OVCAR or SKOV3, or sDCs loaded with the same antigens. αDC1s and sDCs not loaded with exogenous antigen were used as controls. The expanded CD8+ T cells were harvested, captured at the single-cell level and immobilized in a vertical flow array chip (VFAC), which contain 100-microchambers packed with 105 beads immobilizing 1010 oligo(dT) probes with unique cell-ID, UMI, and PCR-tags. Each cell was lysed and its mRNA was individually tagged with unique cell ID sequences prior to library preparation and targeted scRNAseq using a custom 94-gene inflammation RNA-seq panel was performed. Post-clustering (Scanpy), unsupervised cell-type identification (louvain), and downstream analyses were performed on GEX data to determine impact of αDC1s versus sDCs (loaded with OVCAR or SKOV3 versus not loaded with antigens) on the effector and memory pathways of differentiation of the expanded T cells and their unique functional status. Results: PCA and unsupervised clustering revealed sample groups with distinct phenotypic signatures associated with loaded versus unloaded aDC1s, with differential GEX subclusters identified for the αDC1- activated T cells (independently on DC loading with either OVCAR or SKOV3 cells) to include at least two populations with differential expression of genes typical of central memory, effector memory and effector cells (killer molecules and chemokine receptors typical of both lymphoid and peripheral homing potential). In sharp contrast, sDC1 activated T cell showed a more uniform gene distribution patterns lacking the effector T cell component. These differences correlated with the differential ability of αDC1- and sDC-activated T cells to respond to migrate to different chemokines and to recognize and kill cancer cells. Conclusion: scRNAseq was able to identify distinct single-cell T-cell phenotypes caused by induction of αDC1s or sDCs (loaded with OVCAR or SKOV3), consistent with the differential ability of these two DC types to induce effector, effector memory and central memory CD8+ T cells in response to different tumor targets and with their differential ability to modulate peripheral tissue homing patterns. Citation Format: Bowen Dong, Sarabjot Pabla, Vincent Giamo, Weijian Jiang, Kiyomi Taniguchi, Sean T. Glenn, Masataka Shirai, Pawel Kalinski. Distinct pathways of DC-induced CD8+T cell differentiation revealed by single-cell mRNA sequencing analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 968.

The effects of miR-140-5p on the biological characteristics of ovarian cancer cells through the Wnt signaling pathway.

Ovarian cancer is usually not diagnosed until the late stage, and it is resistant to platinum and other standard chemotherapy drugs, resulting in high mortality. To investigate the effects of miR-140-5p on cell proliferation, apoptosis, invasion, and migration capability in the SKOV3 and OVCAR3 ovarian cancer cell lines through Wnt signaling pathway. Expression levels of miR-140-5p were checked using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of miR-140-5p was upregulated by transfecting cells with a miR-140-5p mimic or a mimic negative control (NC). Cell proliferation was assessed using a CCK8 assay, and cell cycle distribution and apoptosis percentage were detected with flow cytometry. A transwell invasion assay was employed to evaluate cell migration and invasion ability. The target complementary relationship between miR-140-5p and WNT1 was verified using a dual-luciferase reporter assay while â-catenin in the nuclei was observed using immunofluorescence. The expression of Wnt signaling pathway-related proteins was examined using western blot and qRT-PCR. The relative expression level of miR-140-5p in SKOV3 and OVCAR3 cells was obviously decreased compared with that in the IOSE80 cells (p < 0.05). Besides, upregulated miR-140-5p effectively suppressed cell proliferation and increased the apoptosis ratio of SKOV3 and OVCAR3 cells (p < 0.05). In addition, the invasion and migration capability of SKOV3 and OVCAR3 cells in miR-140-5p mimic group was largely suppressed compared with the NC group (p < 0.05). What is more, the target complementary relationship between miR-140-5p and the WNT1 gene was revealed; upregulated miR-140-5p suppressed the expression of Wnt signaling-related genes, and restrained nuclear transfer of â-catenin (p < 0.05). The overexpression of miR-140-5p restricted the proliferation, migration and invasion abilities, and accelerated cell apoptosis in ovarian cancer cell lines SKOV3 and OVCAR3 through the Wnt signaling pathway.

Platelet count as a biomarker for monitoring treatment response and disease recurrence in recurrent epithelial ovarian cancer

ObjectivesWe sought to determine the impact of pretreatment plasma platelet levels, dimerized plasmin fragment (D-dimer) and fibrinogen in recurrent epithelial ovarian cancer (EOC) and the impact of platelet levels on SKOV3 cell lines growth and responsiveness to chemotherapy.MethodsUnder approval of ethical committee, we identified 104 women with recurrent EOC who underwent treatment between January 2010 and February 2015. Reviewing clinical, laboratory, and pathologic records from this retrospective cohort, we analyzed the correlation between pretreatment plasma D-dimer, fibrinogen, platelet levels and clinicopathological parameters, progression free survival (PFS) and overall survival (OS). Inco-culture experiments human ovarian cancer SKOV3 cell lines were used to test the effect of platelet levels on tumor growth and responsiveness to docetaxel.ResultsOf the 104 recurrent EOC, thrombocytosis at diagnosis and the decrease of platelet count by less than 25% after primary therapy were associated with worse median progression free survival (P = 0.003;P = 0.021) and median overall survival (P = 0.009;P = 0.009). Mean platelet levels declined at the end of primary therapy(P < 0.001) and rose at recurrence(P = 0.007). In multivariate analysis, elevated platelet levels at primary therapy and the decrease of platelet count less than 25% after primary therapy were unfavorable prognostic factor for PFS(P = 0.022; P = 0.015) and OS(P = 0.013;P = 0.007) in recurrent EOC, but elevated plasma D-dimer and fibrinogen were not. In SKOV-3 ovarian cancer cell lines, suitable concentration platelet co-culture protected against apoptosis (P < 0.05).ConclusionsPlatelet count during treatment could be used as a biomarker used for monitoring the disease recurrence and predicting treatment response. And platelet with suitable concentration co-culture protected against apoptosis in SKOV3 cell line, which may explain clinical observations.

Exosomal miR‑34b inhibits proliferation and the epithelial‑mesenchymal transition by targeting Notch2 in ovarian cancer

Exosomal microRNA (miR) can affect signaling pathways in various physiological and pathological conditions, including ovarian cancer (OC). miR-34b, the first microRNA targeted in a human clinical trial for cancer treatment, exhibited decreased expression in several cancer types. However, the biological function of exosomal miR-34b in OC has not been elucidated. In the present study, using reverse transcription-quantitative PCR, it was reported that exosomal miR-34b is downregulated in OC cells. Exosomal miR-34b reduced cell proliferation and epithelial-mesenchymal transition (EMT) in the OC cell line SKOV3. In addition, it was confirmed that Notch2, which is upregulated in SKOV3 cells, is a target of miR-34b. Moreover, exosomal miR-34b and Notch2 levels were found to be negatively correlated. The present data highlights the importance of exosomal miR-34b-mediated inhibition of cell proliferation and EMT, suggesting that exosomal miR-34b has value as a diagnostic biomarker and a potential molecular target for the treatment of OC.

Polyvinylpyrrolidone Loaded-MnZnFe2O4 Magnetic Nanocomposites Induce Apoptosis in Cancer Cells Through Mitochondrial Damage and P53 Pathway

MnZnFe2O4 ferrite was successfully synthesized using metallic chlorides and polyvinylpyrolidone, by Co-precipitate method. Deferent concentrations of PVP (0.1, 0.3, 0.5, 0.7, and 0.9) wt%, were used as capping agent to stabilize the particles and prevent them from agglomeration. The PVP content had a vital influence on enhancing the properties of the ferrite nanoparticles. From X-ray data the degree of crystallinity and the Lattice constants (a) of the nanoparticles increased from (8.92 nm) and (8.344 A) to (19.94 nm) and (8.395 A) respectively with increasing PVP concentrations. The morphology and average particle size of the MnZn ferrite nanoparticles were evaluated by scanning electron microscopy (SEM), the values were in a good agreement with the XRD results. Fourier transform infrared spectroscopy (FT-IR) confirmed the cubic structure with octahedral and tetrahedral, also indicated the formation of bond between PVP and surface of metallic hydroxide of ferrite nanoparticles. Vibrating sample magnetometer (VSM) analysis was performed for all samples at room temperature, results showed that the specimens exhibited a paramagnetic behavior in the absence of PVP while in the presence of PVP they have super paramagnetic characteristics. The values of saturation magnetization (Ms) (1.78–17.67 emu/g), remnant magnetization (Mr) (0.0022–0.0696 emu/g) also increased with increasing pvp concentration. Polyvinylpyrrolidone loaded-MnZnFe2O4 magnetic nanocomposites were used it alone or as a combination therapy with NIR laser, and alternating magnetic field (AMF) as anti-proliferative agent against breast cancer cell lines AMJ-13, MCF-7,and ovarian cancer cell line SKOV-3 as well as against normal cell line HBL. While, its capability to induce apoptosis was detected using different techniques which is acridine orange/ethidium bromide (AO/EtBr) staining and mitochondrial membrane potential (MMP), and flow cytometry assay membrane potential (MMP). q-PCR was used to investigate the changes in the expression of P53 gene. The influence of Polyvinylpyrrolidone loaded-MnZnFe2O4 magnetic nanocomposites in viability of breast and ovarian cancer cells alone or when they used as a combination therapy with laser photo-thermal therapy and alternating magnetic field (AMF) was also tested using MTT assay. Our results in the present study demonstrated that the inhibition activity of Polyvinylpyrrolidone loaded-MnZnFe2O4 magnetic nanocomposites against treated cancer cell lines increased when Polyvinylpyrrolidone loaded-MnZnFe2O4 nanocomposites used with NIR laser, while highly increased cytotoxic activities were observed after exposure of Polyvinylpyrrolidone loaded-MnZnFe2O4 nanocomposites to induction heating with AMF. Treated cancer cells with polyvinylpyrrolidone loaded-MnZnFe2O4 magnetic nanocomposites significantly increased ROS synthesis, with subsequent reduction of the MMP. The results of the current study show that tested compounds suppressed cancers cells’ proliferation and has a growth inhibitory effect on different cancer cells, resulting in apoptosis as a novel pathway that involves mitochondrial damage mechanism via activated P53. Taken together the present data suggest that the Polyvinylpyrrolidone loaded-MnZnFe2O4 magnetic nanocomposites could be promising therapy protocol for cancer cells.

BNIP3 contributes to cisplatin-induced apoptosis in ovarian cancer cells.

BNIP3 is a proapoptotic protein that mediates apoptosis, necrosis and autophagy. However, the involvement of BNIP3 in cisplatin‐induced apoptosis in ovarian cancer is not clear. In this study, we examined the role of BNIP3 in ovarian cancer during cisplatin treatment and its correlation with clinical outcomes. We first measured cisplatin cytotoxicity and BNIP3 levels before and after cisplatin exposure for ovarian cancer cell lines A2780, SKOV3, OVCAR4, OV2008, ES2 and HO8910. BNIP3 was observed to be differentially expressed in these cell lines, and cisplatin induced a significant increase in BNIP3 levels in A2780 and OVCAR4. BNIP3 knockdown with siRNA in A2780 and OVCAR4 significantly reduced cisplatin cytotoxicity in these two cell lines and alleviated cisplatin‐induced apoptosis. We searched the online databases Gene Expression Omnibus and The Cancer Genome Atlas to analyze the correlation between BNIP3 level and overall survival and progression‐free survival in patients with ovarian cancer. Pooled analyses showed that higher BNIP3 level was correlated with poorer overall survival (95% confidence intervals; hazard ratio = 1.18, 1.04–1.34; P = 0.013) and progression‐free survival (95% confidence intervals; hazard ratio = 1.26, 1.10–1.43; P = 0.00049). However, the results of individual datasets and stratification analyses of histology, FIGO (Federation Internationale de Gynecolgie et d’Obstetrique) stage, chemotherapy regimen and P53 mutation status varied. These findings indicate that cisplatin‐induced apoptosis is dependent on BNIP3 level in ovarian cancer cell lines. Targeting BNIP3 may therefore be a potential way of restoring cisplatin sensitivity.

FTO accelerates ovarian cancer cell growth by promoting proliferation, inhibiting apoptosis, and activating autophagy

ObjectiveFat mass and obesity-associated protein (FTO) is identified as a critical demethylase involved in various physiological processes. Despite efforts have been made to study the biological functions of FTO in certain cancers, the role of FTO in ovarian cancer is largely unknown. In this study, we sought to investigate the function of FTO on proliferation, apoptosis and autophagy of ovarian cancer cells. MethodsQuantitative real-time PCR was performed to detect FTO expression in ovarian tumor tissues and ovarian cancer cell lines OVCAR-3, SKOV-3, COC1, HO-8910 and A2780. SKOV-3 cells were constructed with FTO overexpression and A2780 cells were constructed with FTO knockdown. CCK-8 assay was used to examine cell viability and flow cytometry was used to detect cell apoptosis. Activity assay kits were applied to detect caspase-3 and caspase-9 levels. Western blot was performed to measure the expressions of FTO, PCNA, Bax, Bcl-2, LC3, ATG5, P62, p-AKT and AKT. Stable FTO-overexpression SKOV-3 cells or FTO-depletion A2780 cells were injected subcutaneously into male Balb/c-nu mice. Xenografted tumors were assayed by H&E staining. Immunohistochemistry was subjected to measure FTO and Ki67 expressions. ResultsFTO was up-regulated in ovarian tumor tissues compared with non-cancerous ovarian tissues. FTO overexpression markedly increased viability and autophagy function, but decreased apoptosis of ovarian cancer cells. In addition, FTO overexpression promoted AKT phosphorylation. In contrast, FTO silence showed the opposite effect. ConclusionFTO accelerated ovarian cancer cell growth by promoting proliferation, inhibiting apoptosis, and activating autophagy.

Hsa-microRNA-411-5p regulates proliferation, migration and invasion by targeting the hyaluronan mediated motility receptor in ovarian cancer.

The mortality rate of ovarian cancer is the highest out of all gynecological malignancies worldwide. Therefore, it is important to understand the mechanisms of ovarian cancer, identify new biomarkers and develop targeted drugs. The role and molecular mechanisms of hsa-microRNA (miR)-411-5p in ovarian cancer have not been fully elucidated. The present study investigated the ovarian cancer cell lines OVCAR-8 and SKOV3. After transfection with miRNA mimics, cell proliferation was monitored by a proliferation assay. Furthermore, cell migration was measured by a cell wound healing assay and cell invasion was measured by Matrigel invasion assays. A miRNA luciferase reporter assay was used to analyze the relationship between miRNAs and the target gene HMMR, which was then further evaluated by gene differential analysis. In the current study, hsa-mir-411-5p was identified as a miRNA regulator of the hyaluronan mediated motility receptor, which negatively regulated the activity of ERK1/2 and ultimately inhibited ovarian cancer cell proliferation and motility. Although hsa-mir-411-5p may have different roles in other types of cancer, the present study suggested that miR-411-5p functions as a negative tumor regulator in ovarian cancer cells, displaying the potential of miR-411-5p as a biomarker for ovarian cancer.

Ursolic acid protects against cisplatin‑induced ototoxicity by inhibiting oxidative stress and TRPV1‑mediated Ca2+‑signaling.

Cisplatin (CDDP) is widely used in clinical settings for the treatment of various cancers. However, ototoxicity is a major side effect of CDDP, and there is an associated risk of irreversible hearing loss. We previously demonstrated that CDDP could induce ototoxicity via activation of the transient receptor potential vanilloid receptor 1 (TRPV1) pathway and subsequent induction of oxidative stress. The present study investigated whether ursolic acid (UA) treatment could protect against CDDP-induced ototoxicity. UA is a triterpenoid with strong antioxidant activity widely used in China for the treatment of liver diseases. This traditional Chinese medicine is mainly isolated from bearberry, a Chinese herb. The present results showed that CDDP increased auditory brainstem response threshold shifts in frequencies associated with observed damage to the outer hair cells. Moreover, CDDP increased the expression of TRPV1, calpain 2 and caspase-3 in the cochlea, and the levels of Ca2+ and 4-hydroxynonenal. UA co-treatment significantly attenuated CDDP-induced hearing loss and inhibited TRPV1 pathway activation. In addition, UA enhanced CDDP-induced growth inhibition in the human ovarian cancer cell line SKOV3, suggesting that UA synergizes with CDDP in vitro. Collectively, the present data suggested that UA could effectively attenuate CDDP-induced hearing loss by inhibiting the TRPV1/Ca2+/calpain-oxidative stress pathway without impairing the antitumor effects of CDDP.

MicroRNA-221 (Mir-221) Influences Ovarian Cancer Cell Proliferation and Apoptosis Through Regulating Suppressors of Cytokine Signaling 3-Janus Kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) Pathway

Suppressors of cytokine signaling 3 (SOCS3) regulates JAK-STAT signaling. Bioinformatics analysis showed a targeted relationship of Mir-221 with SOCS3 mRNA. Our study assessed whether Mir221 regulates SOCS3 expression and affects ovarian cancer cells. Ovarian cancer tissues were collected and compared with adjacent tissues to detect Mir-221 and SOCS3 expression. Ovarian cancer cell line SKOV3 cells were separated into miR-NC group and Mir-221 inhibitor group followed by analysis of Mir-221, SOCS3, p-JAK2, and p-STAT3 expression, cell apoptosis and proliferation. Compared with adjacent tissues, Mir-221 expression in tumor tissues was significantly elevated and SCOS3 mRNA level was decreased. There is a targeted relationship between miR-203 and SOCS3 mRNA. Compared with IOSE80 cells, ovarian cancer A2780 and SKOV3 cells presented significantly elevated Mir-221 level and decreased SOCS3 expression. Mir-221 inhibitor transfection significantly upregulated SOCS3, downregulated p-JAK2, p-STAT3 protein, promoted cell apoptosis and inhibited proliferation. The increased Mir-221 and decreased SOCS3 expression are related to the pathogenesis of ovarian cancer. Mir-221 downregulates SOCS3, affects the activity of JAK-STAT signaling, and regulates ovarian cancer cell proliferation and apoptosis.

The Novel Zinc Finger Protein 587B Gene, ZNF587B, Regulates Cell Proliferation and Metastasis in Ovarian Cancer Cells in vivo and in vitro.

BackgroundThe zinc finger protein 587B (ZNF587B) is a novel cisplatin-sensitive gene that was identified in our previous research by using a genome-scale CRISPR-Cas9 knockout library in ovarian cancer (OC) cell lines. ZNF587B belongs to the C2H2-type zinc finger protein (ZFP) family. Many ZFP protein could inhibit tumor development and malignancy. However, the function of ZNF587B remains unknown.MethodsQuantitative PCR (qPCR) was utilized to compare ZNF587B mRNA expression levels in OC and normal ovarian cell lines. The small interfering RNA (siRNA) and full-length ZNF587B eukaryotic expression plasmid were constructed and transfected into OC cells later. Colony formation, 5-ethynyl-2′-deoxyuridine (EdU) assay, transwell assay, and xenograft experiment were conducted to evaluate the effect of ZNF587B on OC cells.ResultsZNF587B was downregulated by approximately 43% and 17% in the OC cell lines SKOV3 and A2780, respectively, compared with that in the normal ovarian cell line IOSE80. Overexpression of ZNF587B reduced cell proliferation, colony formation, migration, and invasion, which could be reversed by knockdown of ZNF587B via siRNA. Xenograft experiments also confirmed that ZNF587B could suppress tumor growth. Survival data of OC patients in the SurvExpress database showed that with respect to overall survival, low-risk patients grouped by the prognostic index had a higher expression of ZNF587B and a better prognosis than high-risk group (HR = 1.77, 95% CI: 0.55–0.70, p = 0.023). Moreover, overexpression of ZNF587B promoted OC cells apoptosis when pretreated with cisplatin.ConclusionZNF587B is a novel potential tumor suppressor of OC and may be a therapeutic target for OC.

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.

ERBB2 gene expression silencing involved in ovarian cancer cell migration and invasion through mediating MAPK1/MAPK3 signaling pathway.

To explore possible mechanism of ERBB2 gene expression silencing mediating mitogen-activated protein kinase 1/mitogen-activated protein kinase 3 (MAPK1/MAPK3) signaling pathway on proliferation, migration, and invasion of ovarian cancer cells. A total of 240 cancer specimens were collected in patients with epithelial ovarian cancer intraoperatively in our hospital from January 2015 to January 2018. Expressions of ERBB2, MAPK1, and MAPK3 in tissues were detected by immunohistochemistry. Following the culture of ovarian cancer cell lines, target cell line with high expression of ERBB2 was screened by qRT-PCR. Cell grouping was performed with four groups after transfection, including Blank group, negative control (NC) group, ERBB2 shRNA group, and ERBB2 overexpression group (shorted as ERBB2 group). The expression levels of ERBB2, MAPK1, MAPK3, vascular endothelial growth factor (VEGF), metalloproteases-2 (MMP-2), and tissue inhibitor of metalloproteases-2 (TIMP-2) were detected by qRT-PCR in different transfection groups, followed by the detection of protein expressions with Western blot. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to test the proliferation activity of each group after transfection, while transwell assay and scratch test explored cell invasion and migration in each group, respectively. Immunohistochemistry showed that the positive rates of ERBB2, MAPK1, and MAPK3 in ovarian cancer tissues were significantly increased than those in adjacent normal epithelial tissues. In the cell experiment, ERBB2 gene was highly expressed in SKOV3 ovarian cancer cell line. There was no significant difference in each index between Blank group and NC group (p > 0.05). Compared with Blank group and NC group, the expression levels of ERBB2, MAPK1, MAPK3, VEGF, and MMP-2 in ERBB2 shRNA group decreased significantly, TIMP-2 increased markedly, and proliferation, invasion, and migration abilities of cells decreased markedly after transfection, showing statistically significant differences (All p < 0.05). By contrast, the expression levels of ERBB2, MAPK1, MAPK3, VEGF, and MMP-2 increased remarkably in ERBB2 group, while TIMP-2 decreased significantly, and cell proliferation, invasion, and migration ability increased evidently after transfection, with statistically significant differences (All p < 0.05). Silencing ERBB2 gene expression may inhibit the activation of MAPK1/MAPK3 signaling pathway and thus suppress the proliferation, invasion, and migration of ovarian cancer cells. Overexpression of ERBB2 gene can reverse those trends, which in turn support the role of ERBB2 gene expression silencing in molecular targeted therapy of ovarian cancer.

MiR-6086 inhibits ovarian cancer angiogenesis by downregulating the OC2/VEGFA/EGFL6 axis

miRNAs have emerged as a pivotal component of gene regulatory networks, mediating cytokines secretion, cell cycle, and differentiation regulation. However, how miRNAs collaborate with transcription factors and downstream effector proteins that determine the fate of ovarian cancer cells remains to be understood, especially regarding to mechanism of tumor angiogenesis regulation. Based on the qRT-PCR and IHC analysis, we found that miR-6086 was maintained a very low level both in ovarian cancer cell lines and tissues. Further, we identified OC2 and EGFL6 as the direct targets of miR-6086 by luciferase assay and we observed an inverse relationship between the expression of miR-6086 and the OC2/VEGFA/EGFL6 axis. The Western blotting analysis suggested that OC2 could directly upregulate VEGFA and indirectly up-regulate EGFL6 through VEGFA. Moreover, miR-6086 could indirectly downregulate VEGFA through OC2. In addition, miR-6086, siOC2 and siEGFL6 could negatively regulate the tumor growth and angiogenesis of ovarian cancer (Skov3) in the animal studies, with the inhibition rates of 77.07%, 69.89%, and 73.62%, respectively (**p < 0.01). Moreover, the tumor cell proliferation, migration, and invasion of ovarian cancer cell lines (Caov3 and Skov3) and vascular formation (HUVECs) were significantly suppressed in vitro, by decreasing the AKT/MAPK pathways (*p < 0.05). Taken together, our results reveal that miR-6086 can suppress the angiogenesis networks in ovarian cancer by down-regulating the OC2/VEGFA/EGFL6 axis, directly or indirectly, which may provide potential targets for tumor therapeutics.

Calcium phosphate nanoneedle based gene delivery system for cancer genetic immunotherapy

Ovarian cancer has become one of the most common gynecological cancers with a high mortality. However, conventional surgery together with combination chemotherapy is difficult to achieve ideal therapeutic effect. Although genetic immunotherapy is applied to active immune responses against cancer, the absence of efficient in vivo gene delivery technique is still an obstacle in clinical application. To overcome these problems, a minicircle DNA vector encoding humanized anti-EpCAM/CD3 bispecific antibody (BsAbEPH) has been constructed. Moreover, different shapes of calcium phosphate (CaPO) biomaterials were prepared. Specifically, the CaPO-nanoneedle-mediated “cell perforation” transfection technology achieves high levels of gene expression in peritoneal cavity. In an intraperitoneal xenograft model with human ovarian cancer cell line SKOV3, the CaPO-nanoneedle/minicircle DNA system expressed BsAbEPH resulted in significant retardation of cancer growth and extension of mouse life-span with limited toxicity. And this system can be made as off-the-shelf and easy-to-use products. Therefore, CaPO-nanoneedle based non-viral gene delivery technology will have great potential in clinical application.

Degradation of HOX Transcript Antisense RNA Provoked Apoptosis and Necrosis in Human Ovarian Cancer Cells

Ovarian cancer (OC) is one of the common malignancies with poor survival rates in women. Long noncoding RNAs (lncRNAs) are a class of regulatory RNA via gene expression regulation and dysregulation results in several disorders such as cancer. LncRNA HOX transcript antisense RNA (HOTAIR) is reported to be upregulated in OC. It plays a major role in cell proliferation and metastasis. The aim of this study is to investigate the effect of HOTAIR blockage in OC cells proliferation and death. We blocked HOTAIR in OC cell line SKOV3 by Antisense LNA GapmeRs. The qPCR and Annexin V/propidium iodide staining assay were performed at three time points after transfection. Moreover, we examined STAT3 and MAPK8 expression levels as cancer proliferation-associated agents under the influence of GapmeRs using qPCR at 24, 48 and 72 h after transfection. The rate of apoptotic cells increased in Antisense LNA GapmeRs-transfected group compared with the two other groups at all three time points. The ratio of the necrotic cell was also higher in Antisense LNA GapmeRs-treated group in contrary to the other groups in a time-dependent manner. However, among all selected genes only MAPK8 was significantly downregulated at 24 h, followed by upregulation at 48 and 72 h. STAT3 was remarkably increased in all three time points. These data demonstrated that HOTAIR can act as an onco-lncRNA and its inhibition significantly boosted apoptosis and necrosis in the SKOV3 cell line. Our findings can pave the way for antisense-based therapy as a potentially effective approach in the treatment of OC.

Antiproliferative effect of the Red Sea cone snail, &lt;i&gt;Conus geographus&lt;/i&gt;

Purpose: To investigate the antiproliferative effect of the Red Sea cone snail, Conus geographus, against 4 MCF-7 (breast), MDA-MB-231 (epithelial human breast), HepG2 (hepatocellular) and SKOV-3 (ovarian) cancer cell lines.&#x0D; Methods: Extraction of Red Sea cone snail sample with a mixture of CH2Cl2 and CH3OH (1:1, v/v) yielded 0.55 g of a green viscous material. The cytotoxic effects of the organic extract against the cancer cell lines were determined using cell proliferation (MTT) assay, and the half-maximal concentration (IC50) values measured. The effect of the crude extract on the cell cycle of the HepG-2 was determined by flow cytometry.&#x0D; Results: The extract produced significant inhibitory effects against SKOV-3, MDA-MB-231, MCF-7 and HepG2, with IC50 values of 22.7 ± 2.2, 68.7 ± 6.2, 47 ± 4.2 and 19 ± 2.1 μg/mL, respectively. Cell cycle analysis revealed that the extract enhanced accumulation of HepG2 cells in the Go/G1 phase, at a level of 23.4 and 24.1 % at IC50 (19 μg/mL) and ½ IC50 (9.5 μg/mL), respectively, when compared to the untreated cells.&#x0D; Conclusion: These results indicate that C. geographus extract exhibits potent cytotoxic effect against HepG2 cells via a mechanism involving G0/G1 cell cycle arrest. Thus, C. geographus is a potential source of a new anti-cancer agent.&#x0D; Keywords: Conus geographus, Marine invertebrate, HepG2, Antiproliferation

RIP1 promotes proliferation through G2/M checkpoint progression and mediates cisplatin-induced apoptosis and necroptosis in human ovarian cancer cells.

Receptor-interacting protein 1 (RIP1, also known as RIPK1) is not only a tumor-promoting factor in several cancers but also mediates either apoptosis or necroptosis in certain circumstances. In this study we investigated what role RIP1 plays in human ovarian cancer cells. We showed that knockout (KO) of RIP1 substantially suppressed cell proliferation, accompanied by the G2/M checkpoint arrest in two human ovarian cancer cell lines SKOV3 and A2780. On the other hand, RIP1 KO remarkably attenuated cisplatin-induced cytotoxicity, which was associated with reduction of the apoptosis markers PARP cleavage and the necroptosis marker phospho-MLKL. We found that RIP1 KO suppressed cisplatin-induced ROS accumulation in both SKOV3 and A2780 cells. ROS scavenger BHA, apoptosis inhibitor Z-VAD or necroptosis inhibitor NSA could effectively suppress cisplatin’s cytotoxicity in the control cells, suggesting that ROS-mediated apoptosis and necroptosis were involved in cisplatin-induced cell death. In addition, blocking necroptosis with MLKL siRNA effectively attenuated cisplatin-induced cytotoxicity. In human ovarian cancer A2780 cell line xenograft nude mice, RIP1 KO not only significantly suppressed the tumor growth but also greatly attenuated cisplatin’s anticancer activity. Our results demonstrate a dual role of RIP1 in human ovarian cancer: it acts as either a tumor-promoting factor to promote cancer cell proliferation or a tumor-suppressing factor to facilitate anticancer effects of chemotherapeutics such as cisplatin.