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.     

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.     

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。     

亚毒性浓度顺铂联合紫花牡荆素体外诱导人卵巢癌细胞调亡(英文)

Objective: The aim of the study was to investigate the effect of Casticin (CAS) combination with Cisplatin (DDP) in sub-toxic concentration on apoptosis of human ovarian cancer HO-8910 cells in vitro and unravel the associated mechanisms. Methods: Human ovarian cancer HO-8910 cells were cultured in vitro. The inhibitory effect of CAS combination with DDP in sub-toxic concentration on viability of human ovarian cancer HO-8910 cells was evaluated by the MTT assay. Morphological changes of cell apoptosis were detected by Hoechst 33258 staining assay. Cell apoptosis rate was analyzed by flow cytometry. The protein expression level was analyzed by Western blot. Results: CAS in sub-toxic concentration and DDP in sub-toxic concentration could slightly inhibit Human ovarian cancer HO-8910 cells, but CAS combination with DDP in sub-toxic concentration significantly inhibited the growth of HO-8910 cells, and growth inhibition rate was increased drastically compared with the control group (P﹤0.01), and the inhibiting effect showed synergistic action. Human ovarian cancer HO-8910 cells showed the typical morphological changes of apoptosis and apoptosis rate markedly increased when they were exposed to CAS combination with DDP in sub-toxic concentration for 48 h. Western blot showed that the expression of bcl-2 protein was down-regulated and protein level of caspase-3 was activated by CAS combination with DDP in sub-toxic concentration. Conclusion: CAS combination with DDP in sub-toxic concentration could inhibit the cells growth and lead to cell apoptosis in human ovarian cancer HO-8910 cells. And the down-regulation of bcl-2 protein expression and activation of caspase-3 protein might contribute to CAS combination with DDP in sub-toxic concentration in human cancer HO-8910 cells.     

Tiam1, negatively regulated by miR-22, miR-183 and miR-31, is involved in migration, invasion and viability of ovarian cancer cells

Tiam1 has been implicated in the invasive phenotype of various carcinomas. However, its role in ovarian cancer remains to be elucidated, including its upstream regulatory mechanisms. In the present study, we examined the differential expression of Tiam1 in 10 normal ovarian tissues and 17 paired primary and corresponding metastatic ovarian cancer tissues by semi-quantitative immunohistochemistry. It was found that Tiam1 expression was remarkably increased in both primary and metastatic ovarian cancer tissues relative to normal ovarian tissues. Loss-of-function study revealed that downregulation of Tiam1 in SKOV-3ip and HO-8910PM cells lead to reduced cell migration and invasion, and growth inhibition without significantly affecting cell apoptosis. Subsequent regulatory study further confirmed the negative regulatory effects of miR-22, miR-183 and miR-31 on Tiam1 expression. Taken together, our data suggested that Tiam1 may be involved in the aggressive behavior of ovarian cancer, and differential expression profiles of microRNA (miRNA) may contribute to the dysregulation of Tiam1 abundance, which contributes to the invasive, migratory and viability properties of ovarian cancer cells.     

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

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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.     

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.     

miRNA-27a靶向SPRY2调控Wnt/β-catenin信号通路对人卵巢癌细胞增殖和侵袭能力的影响

目的 探讨miRNA-27a靶向SPRY2调控Wnt/β-catenin信号通路对人卵巢癌细胞增殖和侵袭能力的影响.方法 采用荧光定量RT-PCR和Western blot法检测卵巢癌组织、癌旁正常上皮结缔组织和HO-8910细胞、正常卵巢上皮细胞的miRNA-27a和SPRY2表达水平;MTT法检测转染细胞的增殖情况;Transwell实验检测细胞侵袭情况;荧光素酶分析验证miRNA-27a对SPRY2的靶向调控能力;免疫荧光染色检测miRNA-27a与SPRY2对Wnt/β-catenin信号通路的调控能力.结果 卵巢癌组织、HO-8910细胞中miRNA-27a的表达水平分别高于癌旁正常上皮结缔组织、卵巢正常上皮细胞(P﹤0.05),而SPRY2的mRNA和蛋白表达水平均低于癌旁正常组织及卵巢正常上皮细胞(P﹤0.05).与B组相比,C组和E组HO-8910细胞增殖及侵袭能力均下调(P﹤0.05).F组的荧光素酶活性明显高于G组(P﹤0.01).C组的HO-8910细胞中,β-catenin表达上升,且进入细胞核.结论 miR-NA-27a通过靶向抑制SPRY2激活Wnt/β-catenin信号通路,从而促进人卵巢癌细胞的增殖和侵袭能力.     

Mechanism of induction apoptosis of Onychin in ovarian cells in vitro

Objective

The aim of the study was to investigate the possible mechanism of induction apoptosis of Onychin (ONY) in ovarian cancer HO-8910 cells in vitro.

Methods

Human ovarian cancer HO-8910 cells were cultured in vitro. Inhibitory effect of ONY on the viability of HO-8910 cells was evaluated by the MTT assay. Apoptosis of HO-8910 cells treated with different concentrations of ONY for 48 h was detected by FCM. Expression of proteins related to apoptosis was analyzed by Western blot.

Results

ONY significantly inhibited the viability of human ovarian cancer HO-8910 cells in a dose-dependent and time-dependent manner, and the IC₅₀ was 10.48 μg/mL for 48 h. The cells treated with ONY showed typical morphological change of apoptosis and increased cells of sub-G1 population by FCM in a dose-dependent. Western blot showed that expression of Bax, cytochrome C, caspase-9 and caspase-3 proteins were upregulated and protein level of Bcl-2 was depressed after treatment with ONY in a concentration dependent.

Conclusion

Apoptosis of ovarian cancer HO-8910 cells was induced by ONY through mitochondrial apoptosis pathway in vitro.     

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…     

长链非编码RNA FOXD2-AS1对卵巢癌细胞增殖和迁移的影响

  目的  观察长链非编码RNA（lncRNA）FOXD2-AS1对卵巢癌细胞增殖和迁移的影响。  方法  收集2017年2月至2017年9月华中科技大学同济医学院附属武汉儿童医院（武汉市妇幼保健院）手术切除的16例卵巢癌患者的组织标本,采用荧光实时定量聚合酶链式反应（quantitative PCR,qPCR）检测组织标本、人卵巢癌细胞系及人正常卵巢上皮细胞系中的FOXD2-AS1表达水平。将HO-8910细胞分为实验组和对照组,分别转染siRNA-FOXD2-AS1和阴性对照RNA。细胞计数CCK-8法、Transwell迁移实验分别检测沉默FOXD2-AS1表达对卵巢癌HO-8910细胞增殖活性和迁移能力的影响。生物信息学方法预测FOXD2-AS1的下游靶基因,qPCR检测下游靶基因表达,Western blot法检测葡萄糖调节蛋白94（glucose regulated protein94,GRP94）和信号通路Wnt/β-catenin的蛋白表达。  结果  卵巢癌组织中的FOXD2-AS1相对表达量8.56±0.51明显高于正常癌旁组织的2.38±0.21（P < 0.01）,FOXD2-AS1在卵巢癌细胞系OC3、HO-8910、A2780、SKOV-3中表达明显增加（P < 0.05）。下调FOXD2-AS1表达明显抑制卵巢癌HO-8910细胞的增殖活性和迁移能力（均P < 0.01）。生物信息学方法显示,FOXD2-AS1靶向结合miR-150-5p后,可靶向结合GRP94基因。实验组和对照组miR-150-5p相对表达量分别为5.45±0.91和1.01±0.08（P < 0.01）,GRP94 mRNA相对表达量分别为0.32±0.05和1.02±0.11（P < 0.01）。GRP94和Wnt/β-catenin信号通路蛋白表达减少。  结论  FOXD2-AS1在卵巢癌组织和细胞系呈高表达,下调FOXD2-AS1可调控miR-150-5p表达,抑制卵巢癌HO-8910细胞的增殖和迁移,可发挥抑癌基因的功能。      

抑癌基因DOC-2对卵巢癌细胞增殖的影响

目的:探讨抑癌基因DOC2对卵巢癌细胞系HO8910在生长代谢、超微结构及细胞周期等方面的影响及其作用机制。方法:实验分3组:卵巢癌细胞系HO8910、8910P93(转染并表达DOC2基因)、8910pcDNA3.1(转染空载体pcDNA3.1),通过细胞消化计数法和3HTdR掺入法分别测定3组细胞的生长曲线及DNA合成代谢情况;利用透射电镜对细胞的超微结构进行观察比较;最后利用流式细胞仪观察卵巢癌细胞转染前后细胞周期的变化。结果:8910P93在生长增殖及DNA合成方面均明显低于HO8910和8910pcDNA3.1(P<0.05),而后两者之间无明显差异;电镜结果显示,8910P93存在内质网扩张,线粒体空泡化,溶酶体增生,部分细胞可见染色质边集等凋亡前期改变;细胞周期检测结果显示,处于G1和G2期的8910P93细胞明显增多而S期细胞明显减少。结论:抑癌基因DOC2可通过改变卵巢癌细胞的形态及细胞周期而明显抑制细胞的增殖。     

Establishment of a highly metastatic human ovarian cancer cell line (HO-8910PM) and its characterization.

To establish a highly metastatic human ovarian cancer cell line and to study its characteristics tissue from the tumor mass of a nude mouse of 7th subtransplantation of the highly metastasizing human ovarian cancer model (NSMO) was cultured in vitro and the cell line HO-8910PM was established. The cell line grew well through 87 passages and the mitotic index, chromosome analysis, morphology under electron microscope and some oncoprotein expression were studied. The cell doubling time was 34.5 h and the mitotic index was 44.3%. The cells were all of epithelial type and most of them were of polygonal shape. Electron microscopic examination showed malignant nuclei with enlarged nucleoli and abundant microvilli. The plating efficiency in soft agar was 31.2%. The cell agglutination appeared in 4 ug/ml PHA. Chromosomal analysis revealed a mode of 54 per cell. The DNA index was 1.57 measured by FCM. Both of them showed hyperdiploid. Positive ER and PR granules were found in the cells. After hetero-transplantation of the cells into three nude mice all of the latter showed tumor growth with metastasis in lungs or lymph nodes. Eight of the nine kinds of oncoprotein detected by immnohistochemical method were found in the cells. The detection for mycoplasma showed negative. After storage in liquid nitrogen cell growth was stable. The cell line HO-8910PM can meet the criteria for the establishment cell lines. This cell line and NSMO model would be very useful in study of the mechanism of cancer metastasis in identifying various cellular factors regulating local and distant metastasis and also in establishing a rational approach for searching after anti-metastatic agents.     

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。     

人反义VEGF基因表达载体的构建及其对卵巢癌细胞增殖的影响

目的：构建人反义VEGF基因真核表达载体,进行抗血管生成治疗卵巢癌实验。方法：RT－PCR法获得人VEGF基因,将VEGF基因反向克隆入真核表达载体pcDNA3中,酶切鉴定结果。用此真核表达载体转染人卵巢癌细胞HO－8910,G418筛选获得阳性克隆,RNA斑点杂交鉴定HO－8910细胞中VEGF表达。Western blot及间接免疫荧光法检测转染前后HO－8910 细胞中VEGF蛋白表达。检测转染前后瘤细胞的生物学性状及裸鼠体内致瘤性。结果：RT－PCR法得到VEGF基因,并获得反向构建的VEGF真核表达载体。VEGF反义RNA部分阻断了HO－8910细胞中VEGF表达,转染后单个细胞的克隆形成能力明显减弱;细胞周期中,G1期细胞增多,S期细胞减少,细胞增殖能力降低;形态学超微结构显示细胞器扩张和肿胀,核染色质边集,凝集成块,可见明显的凋亡改变。裸鼠体内致瘤性降低。结论：成功构建了反义VEGF基因真核表达载体,该载体可明显抑制卵巢癌细胞增殖,为卵巢癌的基因治疗提供了一定的实验依据。     

DOC-2对卵巢癌细胞HO-8910致瘤力的影响

背景与目的：作为近年发现的新的肿瘤抑制基因，DOC-2的作用机制还不完全清楚。本实验的目的在于观察DOC-2转染后对卵巢癌细胞系HO-8910在克隆形成率、细胞周期、裸鼠致瘤能力等方面的影响，并对其作用机制进行初步探讨。方法：实验分3组：卵巢癌细胞系HO-8910（无DOC-2基因的表达）、8910-P93（经转染并表达DOC-2基因）、8910-pcDNA3．1（转染空载体pcDNA3．1），首先通过软琼脂克隆形成实验比较3组细胞克隆形成率的差异，之后利用流式细胞仪观察其细胞周期的变化，最后用裸鼠荷瘤实验进一步验证DOC-2对肿瘤细胞致瘤能力的影响。结果：卵巢癌细胞系HO-8910在转染DOC-2后其克隆形成率明显降低，与转染前差异明显（P〈0．05），同时G．和G，期细胞明显增多而S期细胞明显减少，移植瘤裸鼠荷瘤实验显示HO-8910-P93细胞在裸鼠体内生长缓慢，肿瘤体积及重量均明显低于对照组（P〈0．05）。结论：DOC-2基因能明显抑制卵巢癌细胞系HO-8910的致瘤力。