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@#[摘 要] 目的：探讨卵巢癌组织中PD-L1与唾液酸结合性免疫球蛋白样凝集素15（Siglec-15）的关系及其临床意义以及两者对卵巢癌SKOV3细胞增殖、迁移及侵袭的影响。方法：收集2017年1月至2019年12月福建医科大学附属第二医院妇科50例手术切除的卵巢癌组织和配对输卵管组织的石蜡包埋标本，采用免疫组化染色Envision法检测癌组织和输卵管组织中PD-L1和Siglec-15的表达水平，Kaplan-Meier生存曲线和Logistic回归分析PD-L1和Siglec-15表达与患者预后的关系。利用瞬时转染技术在卵巢癌细胞SKOV3中分别转染si-PD-L1和si-NC，用qPCR和WB法检测SKOV3细胞中PD-L1的表达对Siglec-15的影响，用CCK-8及Transwell法验证PD-L1及Siglec-15表达对SKOV3细胞增殖、迁移及侵袭的影响。结果：50例卵巢癌组织中，PD-L1与Siglec-15均呈高表达（50.00%与42.00%）。PD-L1表达与肿瘤病理类型、有无腹水、淋巴结转移、FIGO分期及卵巢癌复发与否具有关联（均P<0.05）；Siglec-15表达与卵巢癌患者淋巴结转移及FIGO分期具有关联（均P<0.05）。成功构建PD-L1低表达SKOV3细胞株，降低PD-L1表达可使Siglec-15表达升高。结论：PD-L1和Siglec-15在卵巢癌组织中均有较高的阳性表达率，PD-L1是卵巢癌复发的独立风险因素。PD-L1和Siglec-15两者的表达呈负相关，降低PD-L1表达可使Siglec-15表达水平升高而抑制SKOV3细胞增殖、迁移和侵袭的能力。
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Objective:To investigate the effect of resveratrol on apoptosis of ovarian cancer cells and its molecular mechanism, and to find a potential new target for the treatment of ovarian cancer.Methods:Ovarian cancer SKOV-3 cells were divided into control group and resveratrol group.The survival rate of SKOV-3 cells treated with resveratrol was measured by MTT assay. 24 h after resveratrol intervention in SKOV-3 cells, Western blot was used to detect the expression of Bcl-2, Bax, Caspase-3, HMGB1, TLR4 and NF-?B. Ovarian cancer SKOV-3 cells were transfected with si-NC, si-HMGB1, Rb1+pcDNA3.1 or Rb1+pcDNA3.1-HMGB1, and the sensitivity of cells to resveratrol was detected by MTT assay. The transfected cells were treated with resveratrol and apoptosis was detected by flow cytometry.Results:Resveratrol could inhibit the growth of ovarian cancer SKOV-3 cells, and the higher the concentration of resveratrol, the more significant the inhibitory effect. The expression level of HMGB1 in control cells and resveratrol group was 1.24±0.15 and 0.86±0.11, respectively. 25μM resveratrol inhibited HMGB1 protein level ( P<0.01) . The sensitivity to resveratrol was increased after HMGB1 was downregulated, and the apoptosis of SKOV-3 cells was promoted. HMGB1 overexpression increased the resistance to resveratrol and inhibited the apoptosis of SKOV-3 cells. TLR4 expression quantity control and resveratrol cells were 0.98±0.12 and 0.63±0.08, amount of NF-?B expression were 1.21±0.14 and 0.45±0.07, 25 μM resveratrol could cut TLR4 and NF-?B protein levels. Conclusions:Resveratrol can promote apoptosis of ovarian cancer SKOV-3 cells. This mechanism may be related to the down-regulation of HMGB1/TLR4/NF-?B, which provides a basis for resveratrol treatment of ovarian cancer.
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@#[摘 要] 目的：探讨LINC01503在上皮性卵巢癌（EOC）中的表达水平和生物学功能及其可能的作用机制。方法：收集2015年5月至2016年5月间在河北医科大学第四医院妇瘤科手术切除并经病理学确诊的85例EOC患者的肿瘤组织和输卵管组织。常规培养人EOC细胞A2780、SKOV3、OVCAR3和OV90及正常人卵巢上皮细胞IOSE80，将si-LINC01503、si-NC及miR-342-3p mimic、miR mimic NC分别转染至SKOV3和A2780细胞，分别作为si-LINC01503组、si-NC组、miR-342-3p mimic组和miR mimic NC组。qPCR法检测EOC组织和细胞中LINC01503的表达水平，Kaplan-Meier法分析LINC01503表达水平与患者生存的关系。双荧光素酶报告基因实验验证LINC01503/miR-342-3p/IGF2R轴相关分子间的靶向关系。平板克隆、划痕愈合和Transwell实验分别检测敲低LINC01503及转染miR-342-3p mimic对A2780和SKOV3细胞增殖、迁移和侵袭能力的影响。WB法检测EOC细胞中LINC01503/miR-342-3p通路对IGF2R蛋白表达的影响。构建A2780细胞裸鼠移植瘤模型，观察敲低LINC01503对移植瘤生长的影响。结果：EOC组织和细胞中LINC01503表达水平分别显著高于输卵管组织和IOSE80细胞（均P<0.01），LINC01503高表达组患者术后PFS和OS均显著短于LINC01503低表达组患者（均P<0.01）。敲低LINC01503、转染miR-342-3p mimic均可抑制EOC细胞的增殖、迁移和侵袭能力（均P<0.01）。敲低LINC01503可下调IGF2R的表达（P<0.01），这一现象可通过转染miR-342-3p inhibitor挽救。敲低LINC01503可抑制A2780细胞裸鼠移植瘤的生长（P<0.01）。结论：在EOC组织和细胞中呈高表达的LINC01503与患者的不良预后密切相关，LINC01503可能通过吸附miR-342-3p影响IGF2R表达进而促进EOC的进展。
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@#[摘 要] 目的：探讨鱼藤素通过调控miR-520a-3p表达对卵巢癌SKOV3细胞增殖和凋亡的影响。方法：将SKOV3细胞分为对照组（鱼藤素0 μmol/L）、鱼藤素低剂量（5 μmol/L）、中剂量（10 μmol/L）、高剂量（20 μmol/L）组，miR-NC组、过表达miR-520a-3p组，鱼藤素+anti-miR-NC组、鱼藤素+anti-miR-520a-3p组。CCK-8法、细胞集落形成实验、FCM以及qPCR法分别检测SKOV3细胞的增殖抑制率、细胞克隆形成数、凋亡率以及miR-520a-3p表达水平。结果：与对照组比较，鱼藤素（低、中、高剂量）组SKOV3细胞增殖抑制率、凋亡率、miR-520a-3p表达水平均显著升高（均P<0.05），细胞克隆形成数显著减少（P<0.05）。与miR-NC组比较，过表达miR-520a-3p组SKOV3细胞的增殖抑制率、凋亡率均显著升高（均P<0.05），细胞克隆形成数显著减少（P<0.05）。与鱼藤素+anti-miR-NC组比较，鱼藤素+anti-miR-520a-3p组SKOV3细胞的增殖抑制率、凋亡率均显著降低（均P<0.05），细胞克隆形成数显著增多（P<0.05）。结论：鱼藤素通过增加miR-520a-3p表达抑制卵巢癌SKOV3细胞的增殖能力，并诱导其凋亡。
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Objective To explore the effect and mechanism of miR-581 on the autophagy of ovarian cancer SKOV3 cells. Methods miR-581 mimics and miR-581 NC were transfected into SKOV3 cells, and the transfection efficiency was detected by qRT-PCR. After successful transfection, Western blot was used to detect autophagy-related proteins expression in SKOV3 cells. TargetScanHuman database predicted miR-581 target genes, and Western blot verified the role of miR-581 and target genes. Results Overexpression of miR-581 significantly inhibited the expression of autophagy-related proteins LC3 Ⅱ and Beclin1 (P < 0.01); miR-581 negatively regulated FOXO1 expression (P < 0.01) and FOXO1 promoted autophagy of SKOV3 cells (P < 0.01). Conclusion miR-581 affects the autophagy of ovarian cancer SKOV3 cells by regulating the expression of FOXO1.
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Objective:To investigate the effects of anemoside B4 on proliferation, apoptosis and migration of ovarian cancer SKOV3 cells through a variety of biological methods, and further to explore its mechanism.Methods:Ovarian cancer SKOV3 cells were cultured in vitro. CCK-8 method was used to detect the proliferation of SKOV3 cells treated with different concentrations of anemoside B4, and IC50 value was calculated. Flow cytometry was employed to detect the apoptosis of cells treated with IC50 concentration of anemoside B4 for different time length; Transwell method was used to detect the migration and invasion of cells treated with IC50 concentration of anemoside B4 for different time length. Western blot was used to detect changes in the expression of related proteins.Results:Anemoside B4 can effectively inhibit the proliferation of SKOV3 cells, showing a concentration-dependent IC50 value of 6.08±0.56 μM, and the inhibitory effect is stronger than the positive control drug cisplatin, with statistically significant difference (P<0.05) . Flow cytometry showed that anemoside B4 could induce SKOV3 cells apoptosis, reduce Bcl-xl expression, and up-regulate the expression of Bax, cleaved-caspase-3 and PARP. Compared with the group of 0 h, the difference was statistically significant (P<0.05) . Crocetin could down-regulate the expression of N-cadherin and up-regulate the expression of E-cadherin, thereby inhibiting the migration of SKOV3 cells. Anemoside B4 could inhibit the expression of JAK/STAT3 signaling pathway proteins.Conclusion:Anemoside B4 can effectively inhibit the proliferation of cervical cancer cells, and induce SKOV3 cell apoptosis by regulating the JAK/STAT3 signaling pathway to inhibit their migration. Crocetin has great potential in the research and development of ovarian cancer therapy.
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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 observe the expression difference of lncRNA FAL1 in ovarian cancer cells and their drug-resistant cell lines, and to explore the effect and mechanism of lncRNA FAL1 down-regulation on cell chemotherapy resistance. Methods The expression levels of fal1 gene in SKOV3 and COC1 cells and their drug-resistant cell lines were detected by qRT-PCR. fal1 siRNA was transfected to downregulate fal1 gene expression. MTT was used to detect cell proliferation. Transwell method was used to detect cell invasion ability. Plate clone formation test was used to detect cell clone ability, and Western blot was used to detect MDR-1, mpr-1, ABCG2 and phosphorylation levels of p38 MAPK, ERK1/2 and JNK. SKOV3/DDP and COC1/DDP cells transfected with FAL1-siRNA were injected subcutaneously into BALB/c nude mice. The volume and mass of subcutaneous transplanted tumors were measured. Results Compared with SKOV3 and COC1 cells, SKOV3/DDP and COC1/DDP cells were less sensitive to DDP, and the expression levels of FAL1 gene increased (P < 0.01). After transfection with FAL1-siRNA, the sensitivity of SKOV3/DDP and COC1/DDP cells to DDP increased (P < 0.01), and the invasion (P < 0.05) and cloning ability (P < 0.01) decreased. The expression levels of MDR-1, MPR-1, ABCG2 (P < 0.01) and the phosphorylation levels of p38 MAPK, ERK1/2 and JNK (P < 0.05) decreased. The volume and mass of subcutaneous transplanted tumors were significantly reduced (P < 0.01). Conclusion Down-regulation of lncRNA FAL1 could significantly reduce the chemotherapy resistance of cisplatin-resistant ovarian cancer cell lines and inhibit the proliferation of drug-resistant cells in vivo. Its mechanism is related to inhibiting the activation of MAPK signaling pathway.
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Based on the PI3K/Akt signaling pathway, this study aimed to observe the proliferation and apoptosis of ovarian cancer SKOV3 cells at different concentrations of icaritin, in order to explore the possible molecular mechanisms. The research object was ovarian cancer SKOV3 cells. The cells were divided into the control group and icaritin groups(5, 10, 20 μmol·L~(-1)), and administrated with drugs for 48 hours. The cell counting kit-8(CCK-8)assay was used to detect the inhibitory effect of icaritin on the proliferation of ovarian cancer SKOV3 cells. The proliferation ability of the SKOV3 cells was detected by EdU assay. Hoechst 33342 fluorescence staining was used to observe the apoptotic morphology of SKOV3 cells in each group. The distribution of cell cycle and the apoptosis rate of each group were detected by flow cytometry. Quantitative Real-time PCR was used to detect mRNA expressions of PTEN, PI3K, Akt in each group of cells. Protein expressions of PTEN, PI3K, Akt and p-Akt were measured by Western blot. The results showed that the cell inhibition rates of icaritin groups were significantly increased compared with the control group(P<0.05). The rates of EdU-positive cells of icaritin groups were significantly decreased(P<0.05). SKOV3 cells in icaritin groups showed morphological changes of apoptosis. Apoptosis rates of icaritin groups were significantly increased(P<0.05). The proportions of cells in G_0/G_1 phase of icaritin groups were decreased(P<0.05), while the proportions of S phase cells were increased(P<0.05). The gene and protein expressions of PTEN in icaritin groups were elevated(P<0.05). The gene expressions of PI3K and Akt in icaritin groups were down-regulated(P<0.05). The protein expression of PI3K and p-Akt in icaritin groups were reduced(P<0.05). These results indicated that icarin may inhibit the proliferation of ovarian cancer SKOV3 cells in vitro, induce cell apoptosis and affect the cycle distribution of cells by inhibiting the PI3K/Akt signaling pathway.
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Female , Humans , Apoptosis , Cell Line, Tumor , Cell Proliferation , Flavonoids , Ovarian Neoplasms/genetics , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/geneticsABSTRACT
@#[摘 要] 目的：探讨ERBB2.1转导蛋白反义RNA1（transducer of ERBB2.1 antisense RNA 1，TOB1-AS1）在上皮性卵巢癌（epithelial ovarian cancer，EOC）组织中的表达情况及其临床意义，初步探讨TOB1-AS1对EOC细胞体外增殖、迁移和侵袭的影响。方法：使用TCGA数据库对EOC组织中TOB1-AS1表达情况进行分析；收集2017年7月至2018年1月在河北医科大学第四医院妇科行肿瘤切除并经病理检查证实为EOC的67例患者的肿瘤组织，收集同期因其他妇科疾病接受手术的30例患者的非肿瘤卵巢组织作为对照。采用qPCR法检测EOC组织和非肿瘤卵巢组织中TOB1-AS1的表达水平，χ2检验分析TOB1-AS1的表达与不同临床病理特征之间的相关性，Kaplan-Meier和Cox比例风险回归模型分析患者生存及预后的潜在影响因素。CCK-8实验、划痕实验和Transwell实验分别检测敲低TOB1-AS1表达对EOC细胞SKOV3和A2780增殖、迁移和侵袭的影响。结果：TCGA数据库中资料和qPCR检测结果均显示，在EOC组织中TOB1-AS1的表达水平显著高于非肿瘤卵巢组织（均P<0. 01）。TOB1-AS1的高表达与EOC患者较晚的FIGO分期、较差的组织分级、淋巴结转移及腹膜转移有关（均P<0.05）。Kaplan-Meier生存分析结果显示，TOB1-AS1高表达组患者术后DFS和OS均短于TOB1-AS1低表达组（均P<0.05）。Cox比例风险回归模型分析结果显示，FIGO分期、淋巴结转移、腹膜转移及TOB1-AS1表达是EOC患者预后的独立影响因素（均P<0.05）。TOB1-AS1在EOC细胞系SKOV3、A2780中的表达水平也显著高于正常卵巢上皮细胞系IOSE80（均P<0.01）。细胞功能实验结果显示，敲低TOB1-AS1可抑制SKOV3和A2780细胞的增殖、迁移和侵袭（均P<0.05）。结论：TOB1-AS1在EOC组织中高表达，与患者的不良预后显著相关。TOB1-AS1可能通过促进EOC细胞SKOV3、A2780的增殖、迁移和侵袭来影响EOC的恶性进展。
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Aim To explore the effects of EGCG on xenografts of ovarian cancer in nude mice and its possible mechanism. Methods Nude mice xenografts of ovarian cancer SK0V3 cells were established and divided into five groups after tumor formation, in which three groups were given EGCG (10, 30, 50 mg • k g-
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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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Aim To investigate the effect of Rhein derivative 4a containing amide structure on migration and invasion in ovarian cancer SKOV3 cells and its possible mechanism. Methods Ovarian cancer SK0V3 cells were used as target cells. Molecular docking and West-em blot were used to detect the regulatory effect of derivative 4a on Racl protein. CCK8, HE staining, Scratch and Transwell assay were used to detect the effects of derivative 4a on the proliferation, morphology , migration and invasion of SK0V3 cells, respectively. Western blot was employed to determine the expression of matrix metalloproteinases and EMT-related proteins. Results Derivative 4a could effectively bind to Racl protein, and the binding energy was-29. 10 kcal • mol"1, which was significantly lower than that of Rhein; it also could down-regulate the expression of Racl protein in SK0V3 cells. Derivative 4a could significantly inhibit the proliferation, invasion and migra tion of SKOV3 cells, and induce a large amount of cellular vacuolation; derivative 4a could also down-regu-\ late the expression of MMP-2 and MMP-9, up-regulate the expression of EMT epithelial marker protein E-ca-derin but down-regulate the expression of vimentin and j3-cantenin. Conclusions Derivative 4 a can inhibit the proliferation, migration and invasion of ovarian cancer SK0V3 cells. The mechanism may relate to its targeted regulation of Racl, thereby inhibiting the secretion of matrix metalloproteinases, up-regulating the expression of key molecule E-caderin and down-regula-ting the expression of Vimentin and (3-cantenin in EMT i process.•.
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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 effect of silencing sirtuin 3 (Sirt3) on the apoptosis of human ovarian cancer SKOV3 cells induced by resveratrol (Res), and to explore its mechanism of promoting apoptosis. Methods: The human ovarian cancer SKOC3 cells were cultured with different concentrations 0, 2. 5, 5. 0, 10.0, 20.0, 40.0 and 80.0 mg · L-1) of Res for 24 h. The survival rate of cells was measured by MTT assay. The SKOV3 cells were randomly divided into control group, Sirt3 inhibitory 3-1H-1, 2, 3-triazol-4-yl) pyridine 3-TYP group, Res group and 3-TYP+Res group. After 24 h of culture, the inhibitory rates of proliferation of the cells in various groups were detected by MTT assay; the nuclei were stained with Hoechst 33342, and the morphorgy nucleus was observed by laser confocal microscope; reactive oxygen species (ROS) probe was used to detect the intracellular ROS levels; Western blotting method was used to detect the expression levels of Sirt3, Bax, Bcl-2 and cleaved caspase-3 proteins in the cells in various groups. Results: The results of MTT assay showed that the survival rates of SKOV3 cells were significantly decreased with the increase of concentration of Res, and the median inhibitory concentration (IC50) was 42. 73 mg · L-1. Compared with control group, the inhibitory rates of proliferation of cells in Res group and 3-TYP+Res group were significantly decreased (P0.05); the protein expression levels of Sirt3 and Bcl-2 proteins in Res group were significantly decreased (P< 0.05), and the expression levels of Bax and cleaved caspase-3 proteins were significantly increased (P<0.05). Compared with Res group, the expression levels of Bax and cleaved caspase-3 proteins in 3-TYP + Res group were significantly increased (P<0.05), and the expression levels of Bcl-2 and Sirt3 proteins in 3-TYP+Res group were significantly decreased (P<0.05). Conclusion: Res can induce the apoptosis of SKOV3 cells, and the inhibition of Sirt3 expression by 3-TYP can enhance the effect of Res.
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Background: To investigate the potential of the phage display-identified tumor cellbinding peptide as a biomarker of epithelial ovarian cancer using phage display technology. Method: The Ph.D.-7 Phage Display Peptide Library was used to identify the specific conjugated phages with SKOV3 epithelial ovarian cancer cells, while Chinese hamster ovary cells formed the basis. After employing the rapid differential screening method invitro, the enzyme-linked immunosorbent assay (ELISA), DNA sequencing, immunohistochemistry, immunofluorescence, and the competitive inhibition test of synthetic peptides were used to determine the affinity and specificity of the phages with SKOV3 cells. Results: Using bio panning, we screened the phages, showing a 3590-fold increase after the third round. A total of 61 titers of the phage were randomly selected for ELISA and 10 kinds of the phages with an optical density >0.5 were used for DNA sequencing. Clones of the phage TRRNIPN were derived from DNA sequencing based on ELISA, exhibiting both the brown granular phenomenon and green fluorescence. The specific targeted peptide TRRNIPN was incorporated in tumor cells through the competitive inhibition test. Conclusion: The results of our study indicate that the phage display identified polypeptide TRRNIPN may be an effective biomarker for the early diagnosis and targeted therapy of ovarian cancer
Subject(s)
Humans , Female , Bacteriophages , DNA/analysis , Enzyme-Linked Immunosorbent Assay , Biomarkers, Tumor , Mass Screening/methods , Peptide Library , Early Diagnosis , Research Report , /therapyABSTRACT
Objective:To observe the effect of CoCl2on the cisplatin sensitivity of human ovarian cancer SKOV3cells, and to clarify the possible mechanism.Methods:The SKOV3cells were cultured in vitro and randomly divided into control group, CoCl2 group, cisplatin (DDP) group and CoCl2 combined with DDP (combination) group.The cells in CoCl2group were cultured in normal cell medium for 20hafter cultured in 200μmol·L-1 CoCl2for 4h, the cells in DDP group were cultured in normal cell medium containing 10mg·L-1 DDP for 24h, and the cells in combination group were cultured in 10mg·L-1 DDP for 20hafter cultured in 200μmol·L-1 CoCl2for 4h.The survival rates of SKOV3cells in various groups were detected by MTT method, and the positive expression intensities of hypoxia-inducible factor-1α (HIF-1α) and inducible nitric oxide synthase (iNOS) in the cells in various groups were detected by immunofluorescence method.Rhod 2-AM fluorescence probe was used to observe the levels of Ca2+in mitochondria in the cells in various groups.Western blotting method was used to observe the expression levels of cytochrome C (cyto C) , cysteinyl aspartase 3 (caspase 3) and cleaved cysteinyl aspartase 3 (cleaved caspase 3) .Muse○R apoptosis assay kit was used to detect the apoptotic rates of cells in various groups.Results:Compared with control group, the survival rate of the cells in CoCl2group had no significant change (P＞0.05) , and the survival rates of the cells in DDP and combination groups were decreased (P＜0.05) ;the survival rate in combination group was higher than that in DDP group (P＜0.05) .Compared with control group, the positive expression intensities of HIF-1αin CoCl2and combination groups were increased (P＜0.05) .Compared with control group, the positive expressions of iNOS in DDP and combination groups were increased (P＜0.05) .The Ca2+levels in the cells in DDP group and combination groups were higher than that in control group (P＜0.05) and the Ca2+level in DDP group was higher than that in combination group (P＜0.05) .Compared with control group, the expression levels of cyto C, caspase 3and cleaved caspase 3proteins in the SKOV3cells in CoCl2group had no significant changes (P＞0.05) , and the expression levels of cyto C, caspase 3and cleaved caspase 3in DDP group were increased significantly (P＜0.05) ;compared with DDP group, they were lower than those in combination group (P＜0.05) .Compared with control group, the apoptotic rate of SKOV3cells in DDP group was increased significantly (P＜0.05) ;the apoptotic rate of SKOV3cells in combination group was lower than that in DDP group (P＜0.05) .Conclusion:CoCl2can redece the mitochondrial apoptosis of human ovarian cancer SKOV3cells by inhibiting the DDP-induced enhancement of iNOS expression and decrease the sensitivity of SKOV3cells to cisplatin.
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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.