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@#[Abstract] Objective: To explore the anti-tumor activity of MUC16-targeted chimeric antigen receptor modified NK-92 (CARNK-92) cells against ovarian cancer. Methods: The expression of MUC16 in surgically resected tumor tissues of 15 patients with ovarian cancer treated in the Department of Obstetrics and Gynecology of Qingyang Hospital of Traditional Chinese Medicine and 4 ovarian tumor cell lines was detected by Immunohistochemistry and Flow cytometry. MUC CAR sequence was synthesized by gene synthesis, and its lentivirus expression vector were constructed. CARNK-92 cells targeting MUC16 (MUC-BBz) were obtained by lentivirus infection. The expression of CD107a in MUC-BBz was detected by Flow cytometry. The activation of MUC-BBz cells and its cytotoxicity against SKOV3 target cells were characterized by the release of LDH assay. The xenograft nude mouse model of SKOV3 cells was established to verify the in vivo anti-tumor activity of MUC-BBz cells. Results: MUC16 was highly expressed in ovarian cancer tissues and human ovarian cancer cells. MUC-BBz was successfully constructed by infecting NK-92 cells with lentivirus, with a positive rate of (42.79±2.58)%. MUC-BBz could be specifically activated by MUC16 over-expressing tumor cells. After co-incubation of effector cells and target cells, the expression of CD107a on MUC-BBz was upregulated significantly (P<0.01), and the ability of MUC-BBz secreting cytokines IFN-γ and perforin also increased (all P<0.01). The LDH test indicated that with the increase of effector-target ratio, the cytotoxicity of MUC-BBz against 4 ovarian cancer cells (hey, COC1, SKOV3 and A2780) also significantly enhanced. The results of transplanted tumor model showed that transfusion of MUC-BBz could significantly inhibit the growth of SKOV3 xenograft in mice (P<0.01). Conclusion: The CARNK-92 cells can significantly inhibit the growth of ovarian cancer cells in vitro and in vivo, which provides an important basis for further evaluation of its clinical application.
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@# Objective: To explore the action mechanism of miR-139-5p inhibiting proliferation and invasion of epithelial ovarian cancer (EOC) cells by targetedly regulatingNotch1.Methods: A total of 24 pairs of EOC tissues and its corresponding para-cancerous tissues from patients, who underwent surgical resection in the DepartmentofGynecology,Nanyang Central Hospital of Henan Province, were collected for this study; in addition, human ovarian cancer cell lines (SKOV3, ES2, HEY-T30) and human ovarian epithelial cell line IOSE80 were also collected. Real-time quantitative PCR (qPCR) was applied to detectmRNAexpressionofmiR-139-5pandNotch1 in EOC tissues and cell lines. The miR-139-5p over-expression vector and recombinant plasmid pLV-Notch1 were transfected into SKOV3 cells. Blank control group (Ctrl group) and negative control group (NC group) were set up. Dual luciferase reporter gene assay was applied to verify the targeting relationship between miR-139-5p and Notch1 3'-UTR. CCK-8, Transwell and Scratch healing experiments were applied to detect cell proliferationinvasionandmigration, respectively. Western blotting was applied to detect expressions of proliferation and migration related proteins in cells. Results: Compared with para-cancerous tissues and IOSE80 cells, the expression of miR-139-5p was significantly decreased in EOC tissues and cell lines, while the expression of Notch1 mRNA was significantly increased (all P<0.01). The results of Dual luciferase reporter showed that Notch1 was the downstream target gene of miR-139-5p. Compared with NC group, cell proliferation, invasion and migration ability, expression levels of Notch1, NICD, Cyclin D1, Cyclin A1, Snail1, β-catenin and N-cadherin were all significantly decreased on 3 d in miR-139-5p mimic group (all P<0.01), while expression of E-cadherin was significantly increased (P<0.01); meanwhile, over-expression of Notch1 could reverse the inhibitory effect of miR-1395p on proliferation, invasion and migration of SKOV3 cells. Conclusion: miR-139-5p can targetedly regulate Notch1 to inhibit proliferation, invasion and migration of EOC cells, which may be related to its down-regulation of NICD, Cyclin D1, Cyclin A1, Snail1, βcatenin and N-cadherin, and up-regulation of E-cadherin.
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@#[Abstract] Objective: To investigate the effect of HMGB1 gene on the growth of human epithelial ovarian cancer xenografts in nude mice, and to lay a foundation for finding new targets for the treatment of ovarian cancer. Methods: Human epithelial ovarian cancer SKOV3 cells in logarithmic growth phase were selected to establish a human epithelial ovarian cancer xenograft model in nude mice. Nude mice with successful model establishment were randomly divided into control group and HMGB1-siRNA group. On the 7th, 9th, 11th, 14th, and 16th days after cell inoculation, the same amount of saline and HMGB1-siRNA were respectively injected into two groups of mice under the armpit.After 3 weeks, the nude mice were sacrificed by cervical dislocation, the tumor tissues were separated, and the volume of the tumor was measured. The apoptosis of transplanted tumor cells was detected by Tunnel staining. The expressions of HMGB1, STAT3 and p-STAT3 were detected by Western blotting. The expression of vascular endothelial growth factorA(VEGF-A) and microvascularization were detected by immunohistochemistry. Results: Compared with the control group, the growth of tumor volume slowed down in HMGB1 siRNA group, and on the 21st day, the tumor volume of HMGB1-siRNA group was significantly smaller than that of the control group (P<0.05). HMGB1-siRNA successfully knocked down the expression of HMGB1 mRNA in transplanted tumor tissue. The apoptosis rate of tissue cells in HMGB1-siRNA group was significantly increased ([34±8]% vs [6±2]%, P=0.04), and the expressions of HMGB1 and p-STAT3 were significantly reduced (P<0.05). The expression of VEGF-Aand the number of microvessels were significantly lower than those of the control group (both P<0.05). Conclusion: Knockdown of HMGB1 gene reduces the expression of VEGF-A and microvessel formation possibly by inhibiting the HMGB1/STAT3 signaling pathway, thereby promoting the apoptosis of tumor tissues and slowing the growth of xenografts.
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@#[Abstract] Objective:To explore the targeting relationship between long-chain noncoding RNA HOXA-AS2 (lncRNA HOXA-AS2) and microRNA-520a-3p (miR-520a-3p) and their effects on the proliferation, migration and invasion of ovarian cancer SKOV3 cells. Methods： ：qPCR was used to detect the expression levels of lncRNA HOXA-AS2 and miR-520a-3p in various ovarian cancer cell lines (SKOV3, HO8910, OVCAR3 cells) and normal ovarian epithelial cell line HOSE. Bioinformatics methods were used to predict the targeting relationship between HOXA-AS2 and miR-520a-3p, which was then verified by Dual luciferase reporter gene assay. si-HOXA-AS2, miR-520a-3p mimic, anti-miR-520a-3p and corresponding control fragments were transfected into SKOV3 cells separately or in combination. MTT, Transwell and Western blotting were used to detect the proliferation, migration, invasion and expressions of related proteins (CyclinD1, p21, p27, MMP-2, MMP-9, MMP-14) of SKOV3 cells in each group. Results: Compared with HOSE cells, HOXA-AS2 was over-expressed while miR-520a-3p was under-expressed in ovarian cancer cell lines (all P<0.05). HOXA-AS2 could targetedly down-regulate the expression of miR-520a-3p. Compared with the NC group, the proliferation, migration and invasion of SKOV3 cells in the si-HOXA-AS2 and miR-520a-3p mimics groups were significantly reduced (all P<0.01), and the protein expressions of p21 and p27 were significantly increased, while protein expressions of CyclinD1, MMP-2, MMP-9, MMP-14 were significantly reduced (all P<0.01). The proliferation, migration and invasion of SKOV3 cells in the si-HOXA-AS2+antimiR-520a-3p group were significantly enhanced compared with those in si-HOXA-AS2 and si-HOXA-AS2+anti-miR-NC groups (all P<0.05). Conclusion: lncRNA HOXA-AS2 enhances the proliferation, migration and invasion of ovarian cancer SKOV3 cells by targetedly inhibiting the expression of miR-520a-3p.
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OBJECTIVE：To investigate the effects and m echanism of oleanolic acid on inhibiting the proliferation ，invasion and metastasis of human ovarian cancer SKOV 3 cells. METHODS ：CCK-8 assay was used to detect the effects of different concentrations of oleanolic acid （10，20，40，60，80，100 μmol/L）on the proliferation of ovarian cancer SKOV 3 cells at 12，24， 36 and 48 h. The effects of low-dose and high-dose of oleanolic acid （20，40 μmol/L）on the metastasis and invasion ability of SKOV3 cells for 24 h were observed in Transwell assay. Western blotting assay was used to detect the effects of low-dose and high-dose of oleanolic acid on the protein expression of NF-κB p65，PRL-3，TNF-α，IL-6 and E-cadherin in SKOV 3 cells. Through LPS induction and NF-κB p65 plasmid transfection ，Western blotting and RT-qPCR assay were used to investigate the effects of low-dose and high-dose oleanolic acid on the expression of NF-κB/PRL-3 pathway related proteins and their mRNA. RESULTS ： With the increase of the concentration and action time of oleanolic acid ，the proliferation capacity of ovarian cancer SKOV 3 cells was decreased ，the surval rates of administration groups were significantly lower than that of the control group （P＜0.05 or P＜ 0.01）. Low-dose and high-dose of oleanolic acid could significantly reduce the number of migrating and invading cells （P＜0.05 or P＜0.01）. The protein relative expression of NF-κB p65，PRL-3，TNF-α and IL-6 in SKOV 3 cells were significantly decreased ， while the protein relative expression of E-cadherin was significantly increased （P＜0.05 or P＜0.01）. After LPS induction ，protein and mRNA relative expression of NF-κB p65，PRL-3，TNF-α and IL-6 were increased significantly in LPS model group ，while protein and mRNA relative expression of E-cadherin were significantly decreased （P＜0.05 or P＜0.01）. The protein and mRNA relative expression of NF-κ B p65，PRL-3，TNF-α and IL-6 were significantly decreased ，and protein and mRNA relative expression of E-cadherin were significantly increased in low-dose and high-dose of oleanolic acid group （P＜0.05 or P＜0.01）. In SKOV3 cells with over-expressed NF-κB p65，low-dose and high-dose of oleanolic acid c ould significantly down-regulat the proteinexpression of NF-κ B p65，PRL-3，TNF-α and IL-6，while upregult the protein relative expression of E-cadherin （P＜0.05 E-mail：122821905@qq.com or P＜0.01）. CONCLUSIONS ： Oleanolic acid can inhibit SKOV3 cells proliferation，invasion and metastasis by regulating NF-κB/PRL-3 signaling pathway.
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@#Objective: To investigate the potential effects of miR-455-3p on proliferation, invasion and epithelial-mesenchymal transition (EMT) process of ovarian cancer cells, and explore its molecular mechanism. Methods: The IOSE80, SKOV-3 and A2780 cells were transfected with miR-455-3p mimics and negative controls (NC) by using LipofectamineTM 2000. Quantitative polymerase chain reaction (qPCR) assay was performed to detect the mRNA expressions of miR-455-3p and fatty acid-binding protein 4 (FABP4) in IOSE80, SKOV-3 and A2780 cells. The expression levels of FABP4 and EMT-associated proteins were detected by Wb. CCK-8 assay was applied to measure cell proliferation. Cell migration was analyzed by using Transwell assay. Bioinformatics analysis was used to predict the potential target of miR-455-3p, and the targeting effect of miR-455-3p on FABP4 was verified by the dual-luciferase reporter assay system. Results: The expression of miR-455-3p was declined (all P<0.05), while the expression of FABP4 was elevated (all P< 0.05) in ovarian cancer cells (SKOV-3 and A2780) in comparison with normal ovarian IOSE80 cells. Additionally, over-expression of miR-455-3p obviously inhibited cell proliferation and migration capacity of SKOV-3 cells (all P<0.05). Furthermore, over-expression of miR-455-3p impeded EMT progress by up-regulating E-cadherin expression and down-regulating N-cadherin and vimentin expression (all P<0.05). Importantly, the dual-luciferase reporter system, qPCR and Wb validated that FABP4 was a specific target gene of miR-455-3p, and miR-455-3p showed specific binding with FABP4 3’-UTR and negatively regulated the expression of FABP4 at both mRNA and protein levels. Mechanistically, over-expression of FABP4 apparently reversed the inhibitory effects of miR-455-3p on cell proliferation and migration of SKOV-3 cells (all P<0.05). Conclusion: miR-455-3p, acting as a tumor suppressor protein, can inhibit ovarian cancer cell proliferation, migration and EMT process by targeting FABP4, suggesting that miR-455-3p may be a new potential therapeutic target for ovarian cancer treatment.
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Objective To establish ovarian cancer cell line SKVO3 that can stably express human ADP ribosylation factor-4 (ARF4). Methods A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was constructed and transfected into SKOV3 cells after verifying by DNA sequencing. The expression of ARF4 mRNA was verified by real-time quantitative PCR (qRT-PCR). Then, the recombinant plasmid with lentiviral packaging plasmids were co-transfected into SKOV3 cells for packaging. The recombinant lentiviral particles LV-ARF4 were collected and transfected into SKOV3 cells, and the stable transfected SKOV3 cell line was screening by culture with puromycin. The expression of ARF4 gene was detected by qRT-PCR and Western Blot. Results A eukaryotic expression vector pCDH-CMV-MCS-EF1-Puro/ARF4 was successfully constructed. The vector could significantly up-regulate the expression of ARF4 mRNA in SKOV3 cells and be successfully packaged into recombinant lentiviral particles in HEK-293T cells. Compared with the control group, the relative expression level of ARF4 mRNA and protein in SKOV3 cells was significantly increased after the infection with LV-ARF4 (all P<0.001). Conclusion The recombinant plasmid pCDH-CMV-MCS-EF1-Puro/ARF4 and lentiviral vector LV-ARF4 were successfully constructed. The establishment of stably infected SKOV3 cell line with LV-ARF4 provides an experimental foundation for further studies on the biological function of ARF4 in ovarian cancer.
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Objective:To investigate the radiosensitizing effect of three flavonoids on ovarian cancer SKOV3 cells under hypoxia. Methods:The SKOV3 cells were divided into normoxic group and hypoxic group. The hypoxic SKOV3 cellular model in vitro was es-tablished and tested by measuring the expression profile of HIF-1αprotein in SKOV3 cells. Colony-forming assay was used to detect the radiosensitivity of normoxic and hypoxic SKOV3 cells. The cytotoxicity and radiosensitizing effects of flavonoids were evaluated on the basis of cell death and MTT assay. Results:The Western blot results showed that the gray intensity ratio of HIF-1α/β-Actin in hypoxia group was significantly higher than that in normoxia group (1. 068>0. 117). Radiosensitivity of hypoxic SKOV3 cells in hypoxia group was significantly lower than that in normoxia group. The survival rate of SKOV3 cells was decreased with the increase of concentration. When the concentration was increased, D0 and Dqin chrysin group and quercetin group were significantly decreased (P0. 05). The overall radiobiological parameters of hypoxia group were higher than those of normoxia group. Conclusion: Hypoxia can induce the expression of HIF-1α in SKOV3 cells, which results in the decrease of radiosensitivity. Chrysin and quercetin can enhance the radiosensitivity of SKOV3 cells, and the enhancement is significant under hypoxia, while breviscapine is without such effect. The radiosensitizing effect may be achieved by the level decrease of HIF-1α in SKOV3 cells and inhibition of DNA damage repair.
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Objective To discuss the impact of zedoary turmerie oil(ZTO) injection over oncosis index and Bcl-2 expression of human ovarian cancer SKOV3 cell.Methods After ZTO injection acted on human ovarian cancer SKOV3 cells,the changes in the nucleus were observed by fluorescence microscope,oncosis index was counted by projection electron microscope,the situation of cell DNA breakage was observed by agarose gel electrophoresis,and cell Bcl-2 gene expression was observed by immunohistochemical method.Results After treated by ZTO injection for 48 hours,human ovarian cancer SKOV3 cells shows that oncosis cell was swelling in fluorescence microscope,the volume increased,the membrane area narrowed,the nuclear chromatin expansed.The oncosis index increased with dose-effect relationship,DNA electrophoresis showed diffuse type and Bcl-2 gene expression was down-regulated.Conclusion ZTO injection can increase oncosis index of human ovarian cancer SKOV3 cell with the concentration,and lower Bcl-2 gene expression,which may be one of the mechanisms of ZTO injection caused oncosis of SKOV3 cell.
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Objective To explore the effect of down-regulating epidermal growth factor receptor (EGFR) expression by RNA interference on the proliferation of ovarian carcinoma SKOV3 cells. Methods The recombinant eukaryotic expression plasmids pGenSil-HK,pGenSil-EGFR1 and pGenSil-EGFR2 were constructed and transfected into SKOV3 cells respectively. The mRNA and protein expressions of EGFR in SKOV3 cells were detected by RT-PCR and Western blotting,respectively. The cell cycle and apoptosis were evaluated by flow cytometry. The proliferation of SKOV3 cells was determined by clone formation assay and MTT assay. Results We successfully constructed the recombinant eukaryotic expression plasmids pGenSil-HK,pGenSil-EGFR1 and pGenSil-EGFR2 and transfected into SKOV3 cells. Three cell clones were screened by G418. Compared with untransfected SKOV3 cells and SKOV3 cells transfected with pGenSil-HK,the expressions of EGFR in SKOV3 cells transfected with pGenSil-EGFR1,pGenSil-EGFR2 were inhibited significantly at both mRNA and protein levels,with an inhibitory rate of 41.87% and 68.07% for EGFR mRNA and of 45.21% and 70.25% for EGFR protein respectively. Compared with untransfected SKOV3 cells and SKOV3 cells transfected with pGenSil-HK,the cell apoptotic rate was significantly increased significantly,the cell cycle was arrested in G1 phase and S phase decreased significantly in pGenSil-EGFR2 SKOV3 cells (P
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Background and purpose:Ovarian cancer is one of three common malignant tumors in the female reproductive system,whose mortality is the highest in all of the gynecological cancers.The genetic instability and hypermutation rate of tumor cells were found to be associated with chemotherapeutic resistance,and accounted for the high recurrence rate and the failure of current treatment.Invasion and metastasis of tumor are closely related to angiogenesis.Experiments in vivo and in vitro have confirmed that there are many kinds of promoter and suppressor of angiogenesis in primary malignant tumor cells.Angiogenesis could be a potential molecular target for cancer treatment.Endostatin is a specific inhibitor of vessel endothelium.Animal experiments have confirmed that Endostatin could inhibit tumor growth through anti-angiogenesis and result in the decrease of invasion and metastasis of cancer,and it would not easily induced drug resistance in the cells.Thus Endostatin could be safely used for a variety of cancer treatments,being able treat any patho-angiogensis.Recently it has been reported that Endostatin may significantly enhance anti-tumor effects when it is combined with traditional treatment methods such as surgery,chemotherapy,radiotherapy and immunotherapy.Although the study of Endostatin was done in vitro,the primary results were very encouraging and provide the rationale for future clinical trials.In this study,we aim to evaluate the inhibitory effect of Endostatin on SKOV_(3) cell and to investigate the possible mechanism of the inhibition.Methods:1.The effects of Endostatin on SKOV_(3) cells proliferation was studied by means!of MTT.2.The cell apoptosis and cell cycle were detected by transmission electron microscope and cell flow cytometry.3.bcl-2 and bax expression were determined in SKOV_(3) cell by immunocytochemistry,RT-PCR and Western blot analysis.Results:Endostatin inhibited SKOV_(3) cell proliferation,and the value of A_(490) of 15?g/ml 72 h Endostation group(0.454) was much lower than that of PBS control group(1.369)(P
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Objective To study the effect of arsenic trioxide(As 2O 3) combin ed w ith radiation on the killing of ovarian cancer cells. Methods Using MTT and FCM to detect the cytotoxic and apoptosis at different As 2O 3 concentrations combined with 2 an d 8 ?Gy radiation on ovarian cancer cells(SKOV-3). Radiation survival curves were det ermined by cloning assay with 5?mol/L As 2O 3 combined with 2, 3, 4, 6, 8 and 12?Gy radiation. Curve was used to evaluate the effect of cell killing. Results ⑴ Inhibition of cell proliferation seemed more dependent on the increase of As 2O 3 concentration, ⑵Cell survival rate was lower in the combination of As 2O 3 an d r adiation than As 2O 3 alone, ⑶The apoptosis ratio was increased in 2?Gy and As 2O 3 with increase in As 2O 3 concentration, ⑷D q , D 2 value was decr eased in t he combined As 2O 3 and radiation than radiation only (D q: 1.44 vs 2.78, D 0: 0.85 vs 1.30, SF 2: 0.42 vs 0.87), with radiation enhancement ratio of 1.53 and 2.0 7 according to D 0 value and SF 2. Conclusions Arsenic trioxide is able to enhanc e radiation effect obviously ,especially at lower radiation dose.
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Objective: To investigate the effects and the mechanisms of c-raf-1 genes antisense oligodeoxynucleotides(ASODN) transfection in inhibiting the human ovarian carcinoma SKOV3 cell lines.Methods: There were 3 groups in our study: normal control group,c-raf-1 sense oligodeoxynucleotides(SODN) experimental group,and c-raf-1 antisense experimental group.at the different time points after liposome-mediated transfection,the cell proliferation,apoptosis,protein expressing level were observed by MTT assay,flow cytometry,fluorescent microscope and cloning test.Results: In the ASODN experimental group and SODN group,the OD-value were 0.272 and 1.307 respectively(P