高低转移人卵巢癌细胞系基因表达谱差异

目的 用基因芯片技术研究高低转移人卵巢癌细胞系（HO－8910PM和HO－8910）基因表达谱差异,筛选与转移相关的基因。方法 分别抽提高低转移人卵巢癌细胞和对照正常卵巢上皮的总RNA并纯化mRNA;分别将等量的mRNA逆转录合成以图像,用软件对扫描图像进行数字化处理和分析。结果 HO－8910细胞与正常卵巢上皮比较差异3倍以上共有355个基因;HO－8910PM细胞与正常卵巢上皮比较差异3倍以上共有323个基因。HO－8910PM与母系HO－8910比较差异2倍以上共有163个基因,差异3倍以上共有21个基因。结论 两株人卵巢癌细胞与正常卵巢上皮细胞基因表达谱存在差异,提示这些基因与卵巢癌的发生和发展有关;HO－8910PM与HO－8910比较存在差异的基因可能与高转移特性相关。     

P16 GENE EXPRESSION IN OVARIAN EPITHELIAL CYSTADENOCARCINOMA

Itiswellknownthatc-oncogeneovereXPressionaswellasantioncogeneabsenceorinactivationplayaveryimportantroleincarcinogenesis.P16gene(orMTSI,Multipletumorsuppresser1)hasbeenwidelyanalyzedinvariousprimarytumors,suchasmelanoma,neuroblastomaandtumorsinkidney,esophagus,stomach,bladder,breastandpancreas.Theresultsfromdifferentresearchshowedahigh-frequentP16genemutationandabsence.'-ToexplaintherelationshipbetweenP16geneandovarian'cancer,wehadanalyzedP16expressioninhumanovarianpoorlydifferentiatedcyst…     

A STUDY OF MULTI-GENE EXPRESSION IN THE HIGHLY METASTASIZING HUMAN OVARIAN CANCER CELL LINE HO-8910PM AND ITS MOTHER CELL LINE HO-8910

Ｒｅｓｅａｒｃｈｅｓｏｆｔｕｍｏｒｍｏｌｅｃｕｌａｒｂｉｏｌｏｇｙｉｎｒｅｃｅｎｔｙｅａｒｓｈａｖｅｐｒｏｖｅｄｔｈａｔｐｒｏｔｏｏｎｃｏｇｅｎｅａｎｄｔｕｍｏｒｓｕｐｒｅｓｏｒｇｅｎｅｐｌａｙｖｅｒｙｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｔｈｅｐｒｏｃｅ...     

The gene expression profile of highly metastatic human ovarian cancer cell line by gene chip

In this paper, gene chip technique was used to analyze the difference of gene expression patterns in highly metastatic human ovarian tumor cell line HO-8910PM and in normal ovarian epithelial cells to explore the tumor-associated gene-cluster and its function in the process of occurrence an development of ovarian carcinoma. It will be helpful to comprehensively understand the molecular mechanism of cell transformation, to provide the molecular markers and target genes for clinical diagnosis a…     

斑蝥素抑制人高转移卵巢癌细胞HO-8910PM侵袭转移的体外实验研究

背景与目的:斑蝥素在治疗癌症方面显示出其独特的疗效,已有较多文献证实核因子鄄κB(nuclearfactor鄄kappaB,NF鄄资B)与肿瘤侵袭转移关系密切。本研究旨在观察斑蝥素对人高转移卵巢癌细胞HO鄄8910PM转移相关能力的影响,并探讨其作用机理。方法:MTT法及细胞粘附人工重组基底膜实验检测斑蝥素对HO鄄8910PM细胞的细胞毒作用及粘附能力的影响;用Transwell小室法检测斑蝥素对HO鄄8910PM细胞侵袭能力和趋化运动能力的影响;Westernblot法分析斑蝥素对HO鄄8910PM细胞中NF鄄κB和血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)的影响。结果:20μmol/L的斑蝥素作用6h对HO鄄8910PM细胞抑制率及体外侵袭、趋化运动和粘附的抑制率分别为(8.4±2.2)%及(38.8±1.7)%、(40.3±5.6)%和(55.1±6.7)%。20μmol/L斑蝥素能明显下调HO鄄8910PM细胞中NF鄄κB和VEGF的表达。结论:斑蝥素能抑制HO鄄8910PM细胞的侵袭、运动和粘附能力。斑蝥素抗肿瘤侵袭转移的作用机制与NF鄄κB和VEGF蛋白的表达下调有关。     

成纤维细胞激活蛋白对卵巢癌细胞增殖、迁徙和侵袭的影响

背景与目的:肿瘤间质活化的成纤维细胞能表达成纤维细胞激活蛋白(fibroblast activation protein,FAP),本研究通过体外实验研究FAP在卵巢癌细胞增殖、侵袭和迁徙过程中的作用。方法:人类卵巢癌细胞系HO-8910PM体外培养,加入不同浓度的FAP(30、100和300 pmol/L)处理,用MTT法检测FAP对HO-8910PM增殖的影响;细胞划痕实验检测FAP对HO-8910PM迁徙能力的影响;Transwell侵袭实验来研究FAP对HO-8910PM侵袭能力的影响。结果:MTT法及细胞生长曲线显示FAP对卵巢癌细胞有促增殖作用,并呈时间、剂量依赖性;细胞划痕试验结果显示,不同浓度的FAP对卵巢癌细胞系的迁徙均有促进作用,以100 pmol/L组最为明显(P<0.01);Transwell侵袭实验显示FAP对卵巢癌细胞有促侵袭作用,以100 pmol/L组最为明显(P<0.01)。结论:FAP具有促进卵巢癌细胞的增殖、迁徙和侵袭的作用。     

p16基因在人卵巢上皮癌细胞系中的缺失分析研究

目的：了解新型抑癌基因ｐ１６在人卵巢上皮癌细胞系中的变异情况,为认识卵巢上皮癌的形成和发展依据。方法：应用ＰＣＲ扩增、ｍＲＮＡ原位杂交和免疫细胞化学方法,对５种人卵巢上皮癌细胞系ＣＡＯＶ３、ＯＶＣＡＲ３、ＡＯ、ＨＯ－８９１０及ＨＯ－８９１０ＰＭ进行分析。结果：５种人卵巢上皮癌细胞系中只有ＣＡＯＶ３（２０％）显示ｐ１６基因纯合性缺失,ＣＡＯＶ３细胞亦无ｐ１６基因ｍＲＮＡ及蛋白表达。ＯＶＣＡＲ３、Ｈ０     

三氧化二砷联合抗坏血酸对高转移性卵巢癌细胞系HO-8910PM的抗肿瘤实验研究

[目的]在高转移性上皮性卵巢癌细胞株HO-8910PM中观察AS2O3联合抗坏血酸（VitaminC）的抗肿瘤效应。[方法]本研究以高转移性上皮性卵巢癌细胞株HO-8910PM为研究对象,以MTT药敏实验、流式细胞仪以及Western-blot方法检测AS2O3和VitaminC单药及联合的抗肿瘤效应及其机制。[结果]AS2O3和VitaminC单药对HO-8910PM细胞株均具有剂量依赖性的生长抑制作用。AS2O3联合VitaminC后具有明显的协同抗肿瘤效应,并且出现S期阻滞效应。Western-blot结果显示。AS2O3和VitaminC联合作用后,凋亡抑制蛋白bcl-2和凋亡促进蛋白bax分别进一步表达下调和上调。[结论]对高转移性的上皮性卵巢癌细胞株,VitaminC和AS2O3单药均具较好的抗肿瘤效应,VitaminC对AS2O3有较好的化疗增敏效应,两者的联合应用值得在卵巢癌方面进一步研究探讨。     

HLA-G expression is associated with metastasis and poor survival in the Balb/c nu/nu murine tumor model with ovarian cancer

Aberrant HLA-G expression is associated with tumor invasiveness and poor clinical prognosis; however, there is a lack of preclinical animal model to address whether HLA-G plays a causal role in the unfavorable prognosis of malignancies. In the current study, ovarian carcinoma cell lines (HO-8910 and Ovcar-3) were transfected with HLA-G gene. HLA-G expression was analyzed with western blot and flow cytometry. Transwell experiment was performed to analyze the cell migration and invasion capability and/or multicellular spheroid formation was investigated with the 3D culture assay in vitro. The effects of HLA-G expression for tumor cell organ metastasis and for mouse survival was analyzed with the Balb/c nu/nu mouse model. Our data showed that HO-8910-G and Ovcar-3-G cells are of higher invasion potential compared with the parental HO-8910 and Ovcar-3 cells. Multicellular spheroid formation exists only in HO-8910-G cells in a 3D culture assay. In Balb/c nu/nu mouse model, widespread metastasis was observed in mice xenografted with HO-8910-G cells, but not in the group with parental cells. Mouse survival was dramatically decreased in HO-8910-G and Ovcar-3-G xenografted mice than that with HO-8910 and Ovcar-3 cells, respectively. In summary, our study provided the first evidence that HLA-G expression is associated with tumor metastasis and with poor survival in an animal model with ovarian cancer.     

Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro

Background

IQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells.

Methods

We used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay.

Results

IQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAi-mediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration.

Conclusion

Our findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.     

Knockdown of UHRF1 by Lentivirus-mediated shRNA Inhibits Ovarian Cancer Cell Growth

Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has been reported to beover-expressed in many cancers, but its role in ovarian cancer remains elusive. Here, we determined whetherknockdown of UHRF1 by lentivirus-mediated shRNA could inhibit ovarian cancer cell growth. Lentivirusmediatedshort hairpin RNAs (lv-shRNAs-UHRF1) were designed to trigger the gene silencing RNA interference(RNAi) pathway. The efficiency of lentivirus-mediated shRNA infection into HO-8910 and HO-8910 PM cells wasdetermined using fluorescence microscopy to observe lentivirus-mediated GFP expressionand was confirmed to beover 80 percent. UHRF1 expression in infected HO-8910 and HO-8910 PM was evaluated by real-time PCR andWestern blot analysis. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability; flow cytometryand Hoechst 33342 assay was applied to measure cell cycle arrest and apoptosis. Cell invasion was assessedusing transwell chambers. Our results demonstrated that the loss of UHRF1 promoted HO-8910 and HO-8910PM cell apoptosis, while inhibiting cell proliferation. In addition, UHRF1 knockdown significantly inhibitedthe invasion of human ovarian cancer cells. In the present study, we also showed that depleting HO-8910 cellsof UHRF1 caused activation of the DNA damage response pathway, with the cell cycle arrested in G2/M-phase.The DNA damage response in cells depleted of UHRF1 was illustrated by phosphorylation of CHK (checkpointkinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser 216 and phosphorylation ofCDK1 (cyclin-dependent kinase 1) on Tyr 15.     

卵巢上皮恶性肿瘤侵袭转移相关miRNA的筛选与鉴定

目的 探索与卵巢上皮恶性肿瘤侵袭转移可能相关的miRNA.方法 采用miRNA芯片,筛选SKOV-3ip和SKOV-3细胞差异表达miRNA.利用生物信息学软件TargetScan、MicroCosm、PicTar和GO,预测差异表达miRNA的靶基因及其功能.采用实时逆转录聚合酶链反应(real-time RT-PCR)技术,验证与卵巢癌侵袭转移可能相关的5种miRNA(let-7a、let-7e、let-7f、miR-22和miR-886-5p)在SKOV-3ip和SKOV-3细胞的表达.同时,检测这5种miRNA在另外一组侵袭转移能力不同的卵巢癌细胞株HO8910和HO-8910PM中的表达,并进行统计学分析.结果 基因芯片筛选显示,42种miRNA在SKOV-3ip和SKOV-3细胞株表达差异明显.进一步分析显示,let-7a、let-7e、let-7f、miR-22和miR-886-5p等5种miRNA可能与卵巢癌的侵袭转移密切相关.real-time RT-PCR结果证实,let-7f和miR-22在两组侵袭转移能力不同的卵巢癌细胞(SKOV-3和SKOV-3ip细胞、HO-8910和HO-8910PM细胞)表达差异有统计学意义(均P＜0.05).结论 let-7f和miR-22在侵袭转移能力强的卵巢癌细胞中低表达,可能具有抑癌基因的作用.     

吉西他滨联合放射对人高转移卵巢癌细胞的体外实验研究

Objective  

The aim of the study was to investigate the mechanism of gemcitabine (GEM) combination with radiation on the high metastasis human ovarian cancer cell line (HO-8910PM).     

卵巢癌细胞及组织中P38MAPK信号通路调控uPA蛋白的表达及临床意义

目的 探讨P38MAPK信号通路与uPA在卵巢癌细胞及组织中的表达及临床意义。方法 应用免疫组织化学法检测uPA、P38MAPK、ERK、AKT蛋白在49例卵巢癌组织中的表达,Westernblot检测HO8910及HO-8910PM细胞系中uPA、P38MAPK蛋白的表达,特异性抑制剂SB203580阻断P38MAPK信号通路后uPA蛋白表达水平的变化。结果 uPA、P38MAPK、ERK、AKT蛋白在卵巢癌组织中表达阳性率分别为61.22%、57.14%、53.06%、55.10%。uPA与p38MAPK蛋白表达呈正相关（r=0.8645,P=0.001）,且与卵巢癌组织的临床病理分期、分化、转移程度有关（P均<0.05）,而与患者的年龄、组织学类型无明显相关（P>0.05）。ERK、AKT蛋白表达与卵巢癌淋巴结转移、大网膜转移有关（P均<0.05）,而与患者的年龄、组织类型、病理分期无明显关系（P>0.05）。HO-8910PM细胞系中uPA蛋白的表达水平明显高于HO8910,SB203580阻断P38MAPK信号通路后可降低uPA蛋白的表达,且随着SB203580 浓度升高,uPA蛋白表达逐渐降低。P38MAPK及uPA蛋白的表达与卵巢癌的预后显著相关（r=3.897, 11.044, P=0.048,0.001）。结论 卵巢癌组织中P38MAPK信号通路处于激活状态;该通路的激活可上调uPA的表达,促进卵巢癌的恶性进展;P38MAPK信号通路和uPA可能在卵巢癌侵袭和转移的过程中发挥重要作用。P38MAPK和uPA蛋白有望成为卵巢癌预后评估的重要指标之一。     

高转移人卵巢癌细胞系及其母系多基因表达研究

为研究高转移人卵巢癌细胞系ＨＯ８９１０ＰＭ及其母系ＨＯ８９１０多基因表达情况及其彼此关系,采用ＳＰ免疫组化染色方法,观察了９种基因产物的表达。结果显示,除ｂａｘ外,其余８种基因产物在２株细胞均呈不同程度的阳性表达。在ＨＯ８９１０ＰＭ细胞系中,以ｐ５３、ＣｙｃｌｉｎＤ１、ＣＤ４４Ｖ６、ＥＧＦＲ的表达强度强于母系;而ｐ１６、ｎｍ２３表达强度则较母系弱。２株细胞ｃｅｒｂＢ２、ｂｃｌ２表达无明显不同。结果表明,ＨＯ８９１０ＰＭ是一株较母系ＨＯ８９１０更具侵袭和转移生长潜能的细胞株。同时也证实肿瘤的发生、浸润和转移是多基因参于、多阶段协同作用的结果     

MTA1在卵巢癌中的表达及其对卵巢癌细胞侵袭转移影响的研究

  目的  研究转移相关基因1(metastasis-associated-gene1, MTA1)表达与卵巢癌发生发展转移的关系, 研究MTA1对卵巢癌侵袭转移能力的影响, 并探讨抑制卵巢癌侵袭转移的潜在靶点。  方法  免疫组织化学法检测110例卵巢癌组织中MTA1的蛋白表达水平, 分析MTA1蛋白表达与卵巢癌分化程度、临床分期及与腹腔转移的关系。并通过脂质体介导方法, 将特异性siRNA表达载体psilenter2.0-MTA1-siRNA转染入人卵巢癌细胞系HO-8910PM, 采用RT-PCR以及Western blot检测特异性siRNA对MTA1mRNA及蛋白表达的抑制效果。应用划痕损伤实验及Transwell实验检测MTA1对卵巢癌细胞侵袭转移能力的影响。  结果  MTA1随卵巢癌组织学分化程度的升高而降低, 呈负相关, MTA1的表达随着FIGO分期期别的增加而增加, 呈正相关, MTA1的表达随卵巢癌腹腔转移而增加, 呈正相关。RT-PCR及Western blot结果显示, siRNA成功抑制卵巢癌细胞系HO-8910PM中MTA1的表达。划痕损伤实验显示转染后划痕损伤愈合明显减慢, 迁移率明显降低, Transwell体外侵袭实验结果显示, 转染后穿膜细胞百分率显著降低(P < 0.05)。  结论  MTA1表达水平的增高与卵巢癌的分化程度、临床分期及远处转移密切相关, 体外研究显示抑制MTA1在卵巢癌细胞中的表达, 使细胞生长、侵袭及转移能力均受到抑制, 提示MTA1在卵巢癌的远处侵袭转移过程中发挥重要作用, 可能成为卵巢癌基因治疗的潜在靶点。      

Snail is critical for tumor growth and metastasis of ovarian carcinoma

Snail, a key inducer of epithelial‐mesenchymal transition (EMT), plays an important role in cancer metastasis. To better understand the role of Snail in the metastasis of ovarian carcinoma, expression of Snail was knocked down by antisense‐Snail in the highly metastatic ovarian cancer cell line HO8910PM. Gene array analysis revealed that blocking Snail expression suppressed the activity of matrix metalloproteinases (MMPs) and upregulated TIMP3, an MMP inhibitor. These findings suggest that Snail interacts with MMP during tumor invasion and metastasis. In addition, we examined the role of Snail in an ovarian cancer orthotopic model by using the antisense‐Snail HO8910PM cell line. We found that the size of primary ovarian cancer tumor and the number of metastatic lesions were significantly reduced when Snail was knocked down. Confirming our initial findings, the activity of MMP2 was greatly inhibited in tumors from antisense‐Snail cells. Furthermore, immunohistochemical analysis on ovarian cancer progression tissue array demonstrated that the expression of Snail was significantly higher in metastatic lesions, and Snail expression correlated with the stage of ovarian cancer. Interestingly, in early‐stage tumors, Snail was localized in both the cytoplasm and nucleus. In late stage and metastatic lesions, the level of Snail was elevated, and Snail was localized to the nucleus. The expression level and nuclear localization of Snail were also inversely correlated with E‐cadherin expression. Overall, our study indicates that Snail plays a critical role in tumor growth and metastasis of ovarian carcinoma through regulation of MMP activity.     

Inhibition of CD147 gene expression via RNA interference reduces tumor cell invasion, tumorigenicity and increases chemosensitivity to paclitaxel in HO-8910pm cells

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN or CD147), a member of the immunoglobulin family and a glycoprotein enriched on the surface of tumor cells, promotes invasion, metastasis, growth and survival of malignant cells, and confers resistance to some chemotherapeutic drugs. Here, we used a human U6 promoter-driven DNA template approach to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block CD147 gene expression in the human ovarian cancer cell line HO-8910pm. Knockdown of CD147 by shRNA resulted in decrease of the HO-8910pm invasion activity in vitro and tumorigenicity in nude mice. The suppression of CD147 expression also sensitized cells to be more sensitive to paclitaxel. These results suggested that CD147 was an ovarian cancer-related gene and CD147 might be a potential target for therapeutic anti-cancer drugs.