黏附诱导卵巢癌细胞基质金属

目的 观察黏附纤黏连蛋白后肿瘤细胞基质金属蛋白酶（MMP）基因的表达,揭示细胞侵袭发生的机理。方法 纤黏连蛋白与A2780人卵巢癌细胞相互作用后,用反转录PCR观察MMP mRNA的表达;从HT1080细胞基因组DNA克隆MMP9启动子区基因,构建MMP－9－gGL2报告基因载体,瞬时转染A2780细胞,通过测定萤火虫酶活性,观察纤黏连蛋白对MMP9启动子活性的影响。结果 A2780细胞不表达MMP基因,,黏附后细胞内MMP－9mRNA含量明显增加,这一作用与黏附时间有关。克隆MMP－9基因启动子并转染细胞,黏附后转染细胞内MMP－9基因启动子活性明显增强。结论 细胞－细胞外基质黏附可刺激MMP基因转录活性,诱导MMP基因表达,进而促进细胞侵袭。     

畸胎瘤细胞源性生长因子反义核酸载体对高度恶性人卵巢癌细胞增殖和侵袭的抑制效应及相关机制

目的 观察畸胎瘤细胞源性生长因子(PCDGF)反义核酸载体对高度恶性人卵巢癌细胞株Sw626和A2780增殖和侵袭的抑制效应,并初步探讨其相关机制.方法 采用二苯基溴化四氮唑蓝(MTT)法和Boyden小窒体外侵袭实验,检测PCDGF反义RNA真核表达载体对Sw626和A2780细胞增殖和侵袭能力的影响.采用Western blot技术,检测转染PCDGF反义RNA真核表达载体前后Sw626细胞cyclin D1和CDK4蛋自表达的变化.采用逆转录聚合酶链反应(RT-PCR)和明胶酶谱法,分析PCDGF反义RNA真核表达载体对Sw626细胞基质金属蛋白酶2(MMP-2)表达和活性的影响.结果 与空白对照组相比,PCDGF反义核酸载体转染组Sw626和A2780细胞的增殖抑制率分别为72.9%和70.9%,侵袭能力分别被抑制了62.9%和59.0%.转染组Sw626细胞cyclin D1和CDK4蛋白的表达水平分别为0.38±0.08和0.37±0.13,明显低于空白对照组(0.84±0.11和0.64±0.11,P<0.01).与空白对照组(0.89±0.09)相比,转染组Sw626细胞MMP-2 mRNA的表达水平(0.66±0.11)虽未见降低(P>0.05),但MMP-2酶原的活性被叨显抑制.结论 PCDGF反义核酸可显著抑制高度恶性人卵巢癌细胞株Sw626和A2780的增殖和侵袭能力,并逆转其部分恶性表型,这可能与其能下调cyclin D1和CDK4蛋白的表达并抑制MMP-2酶原的活性有关;PCDGF可以作为卵巢癌治疗的新靶点.     

基质金属蛋白酶-9反义寡核苷酸对卵巢癌细胞侵袭黏附能力的影响

目的 观察基质金属蛋白酶-9（MMP-9）反义寡核苷酸（ASODN）转染对卵巢癌细胞体外侵袭黏附行为的影响,并探讨其作用机制。方法 以Lipofectinmin介导的MMP-9反义寡核苷酸转染至经纤黏连蛋白诱导MMP-9表达的卵巢癌细胞株HO-8910PM,利用RT—PCR、Western blot及明胶酶谱法检测转染寡核苷酸后HO-8910PM细胞MMPO的mRNA、蛋白表达及酶活性的变化;通过细胞体外侵袭、迁移实验和黏附实验,检测细胞侵袭黏附能力的变化。结果 卵巢癌细胞HO-8910PM转染MMP-9反义寡核苷酸后,MMP-9的mRNA及蛋白的表达受到抑制,抑制率分别为34．8％和42．5％,与对照组比较,差异有统计学意义（P〈0．05）;明胶酶活性也受到了抑制。反义寡核苷酸的转染降低了肿瘤细胞体外侵袭、迁移和黏附能力,侵袭和迁移抑制率分别为22．4％和24．8％,在60min和90min黏附抑制率分别为49．8％和38．3％。结论 MMP-9反义寡核苷酸可抑制卵巢癌细胞的侵袭黏附能力,MMP-9有可能成为抗卵巢癌侵袭转移的分子靶点。     

纤粘连蛋白诱导卵巢癌细胞基质金属蛋白酶-2基因表达

目的　研究细胞外基质蛋白对肿瘤细胞基质金属蛋白酶 (MMP)基因表达的影响及机理。方法　以纤粘连蛋白处理SKOV3卵巢癌细胞后 ,用明胶 酶谱法和反转录PCR观察MMP分泌及基因表达。以MMP 2启动子转染细胞 ,通过测定萤火虫酶活性 ,观察纤粘连蛋白对MMP 2启动子活性的影响。细胞p5 3含量采用Westernblot分析。结果　 5 μg/mL纤粘连蛋白刺激MMP 2分泌 ,但不影响MMP 9分泌 ,这一作用随纤粘连蛋白浓度增加而增强。经 10 μg/mL纤粘连蛋白刺激 1h后 ,细胞内MMP 2mRNA量显著增加 ;作用时间延长 ,诱导作用减弱。纤粘连蛋白可诱导转染细胞中萤火虫酶活性增加 ,AP 1功能抑制剂姜黄素 (5 0 μmol/L)不能阻断这一作用。经纤粘连蛋白处理后 ,细胞内p5 3含量明显增加。结论　纤粘连蛋白可刺激卵巢癌细胞中MMP 2分泌 ,诱导MMP 2启动子活性增强及mRNA含量增加 ,这一作用可能与p5 3蛋白转录因子活性有关 ,而与AP 1通路无关。     

siRNA沉默FAM83A基因表达对非小细胞肺癌细胞增殖、迁移和侵袭的影响

目的:研究FAM83A基因对非小细胞肺癌细胞增殖、迁移、侵袭及肿瘤生长的影响。方法:运用qRT-PCR法检测非小细胞肺癌细胞H1299中FAM83A的表达;将blank组（未作任何处理）、si-control组（转染si-control）、si-FAM83A组（转染si-FAM83A）用脂质体法转染至H1299细胞;Western blot检测细胞中FAM83A、p16、p21、MMP-2、MMP-9的蛋白表达;MTT法检测细胞的增殖;Transwell小室检测细胞的迁移和侵袭;裸鼠成瘤实验检测肿瘤的生长。结果:沉默FAM83A后,H1299细胞的增殖、迁移和侵袭能力均得到显著下调,并且p16、p21的蛋白表达明显上调,MMP-2、MMP-9的蛋白表达明显下调。最重要的是,沉默FAM83A后的异种移植裸鼠体内的肿瘤生长能力得到明显抑制。结论:沉默FAM83A可抑制非小细胞肺癌细胞的增殖、迁移和侵袭,并抑制裸鼠体内肿瘤的生长。     

肺癌特异性脑转移细胞的生物学特性及VEGF、MMP-9的表达和意义

目的：分析特异性肺癌脑转移细胞株的生物学特性及细胞因子表达，揭示肺癌脑转移的部分机制。方法：通过裸鼠体内多次接种部分脑转移肺癌细株PC14，筛选出特异性肺癌脑转移细胞株PC14／B，与原株、非脑转移肺癌细胞株A549比较，测定它们在细胞外基质上的黏附、迁移、侵袭活性，并应用免疫组织化学方法分析，比较其VEGF、MMP-9的表达情况。结果：所筛选的PC14／B在细胞外基质上的黏附、迁移、侵袭活性都较PC14及A549增强（P〈0．05），PC14／B表达的VEGF和MMP-9也比PC14明显增高。但PC14及A549之间并无显著区别。结论：VEGF和MMP-9提高了肺癌细胞的侵袭活性，促进脑转移的发生，但仅VEGF和MMP-9表达增高尚不足构成肺癌脑转移的全部条件。     

Silencing of Twist Expression by RNA Interference Suppresses Epithelial-mesenchymal Transition,Invasion, and Metastasis of Ovarian Cancer

Purpose: This study aimed to explore the role of the Twist gene in the epithelial-mesenchymal transition ofovarian cancer. Methods: An RNA interference plasmid expressing a small interfering RNA (siRNA)-targetingTwist (Twist siRNA vector) was designed, constructed, and transfected into the human ovarian cancer cell lineA2780. Transfection efficiency was assessed under a fluorescence microscope. Changes in the expression of TwistmRNA in A2780 after transfection with the pGenesil Twist shRNA plasmid were analyzed through RT-PCR.MTT assays and adhesion experiments were applied to determine changes in proliferation and adhesion abilityof A2870 after transfection with the Twist shRNA plasmid. Changes in the expression of the E-cadherin andN-cadherin proteins in A2780 after transfection with the Twist shRNA plasmid were analyzed using Westernblotting. Result: The restructuring plasmid pGenesil-Twist shRNA was constructed successfully. After 48 h ofculture, 80% of the cells expressed high-intensity GFP fluorescence and stability. The expression of Twist decreasedsignificantly after the transfection of the Twist shRNA plasmid (P<0.05). Proliferation of the transfected TwistshRNA cells showed no difference with that of the A2780-nontransfection or A2780-si-control groups (P>0.05) butthe adhesion ability of A2780 decreased dramatically (P<0.05). Expression of the E-cadherin protein increased,whereas that of the N-cadherin protein decreased compared with that in the A2780-nontransfection or A2780-si-control groups (P<0.05). Conclusion: Twist is essential for epithelial-mesenchymal transition, invasion, andmetastasis of ovarian cancer.     

XIAP基因对食管癌细胞黏附、侵袭和迁移能力的影响

目的:探讨沉默X连锁凋亡抑制蛋白（XIAP）基因表达对食管癌细胞黏附、侵袭和迁移能力的影响及机制。方法:参照LipofectamineTM2000说明将设计合成的XIAP特异性siRNA（si-XIAP）转染人食管癌EC9706细胞,将转染阴性对照siRNA(NC组）和仅加入脂质体的空白细胞作为两个对照组,siRNA转染48 h,通过Western blotting检测XIAP、AKT、p-AKT、E-cadherin和MMP-2蛋白表达;通过MTT法、Transwell法分别检测细胞黏附、侵袭和迁移能力;MTT法检测转染siRNA后24 h、48 h和72 h的细胞增殖。结果:XIAP特异性siRNA转染后,EC9706细胞XIAP表达明显降低,与空白组比较差异有统计学意义（P<0.05）。与空白组比较,si-XIAP组细胞增殖明显降低,黏附率明显升高,侵袭和迁移能力明显降低,p-AKT和MMP-2表达明显降低,E-cadherin表达明显升高（P<0.05）。结论:沉默XIAP基因可增强食管癌细胞黏附率,抑制侵袭和迁移能力,机制与抑制PI3K/AKT信号通路有关。     

HPV-16癌蛋白对肺癌细胞A549迁移和侵袭的影响及其机制

目的:探讨HPV-16 E6和E7癌蛋白对肺癌细胞A549迁移和侵袭能力的影响及其机制。方法:A549细胞分别转染含HPV-16 E6、E7的EGFP质粒,采用划痕实验观察转染后不同时间的迁移能力,Transwell侵袭实验检测侵袭能力,Western blotting和实时定量PCR分别分析MMP-2和MMP-9的蛋白、mRNA表达。结果:同对照细胞相比,转染含HPV-16 E6、E7质粒的细胞迁移能力增强,在36h 和48h时趋势较为明显;转染HPV-16 E6、E7组的侵袭细胞数分别为每高倍视野121.3±13.3、129.7±5.5,其显著高于对照组（P＜0.01）。而且,转染HPV-16 E6、E7的细胞中MMP-2和MMP-9的蛋白和mRNA表达显著增加（P＜0.01）。结论:HPV-16 E6和E7癌蛋白可增强A549细胞的迁移和侵袭能力,其机制与上调MMP-2和MMP-9表达有关。     

PTEN磷酸酶活性对乳腺癌细胞ZR-75-1迁移能力的影响

目的:探求PTEN蛋白的磷酸酶活性对乳腺癌细胞ZR-75-1转移能力的影响。方法:采用脂质体介导法分别将野生型PTEN质粒(wt-PTEN)、磷酸酶失活的PTEN质粒(G129R-PTEN)和只具有蛋白磷酸酶活性的PTEN质粒(G129E-PTEN)转染PTEN基因缺失的人乳腺癌细胞株ZR-75-1,Western印迹法检测PTEN蛋白及P397-FAK的表达水平,体外细胞划痕实验观察PTEN磷酸酶活性对ZR-75-1细胞迁移能力的影响,细胞基质黏附试验和人工重组基底膜侵袭试验测定PTEN质粒转染和未转染的ZR-75-1细胞的黏附抑制率和侵袭抑制率,免疫组化法检测MMP-2的水平。结果:wt-PTEN、G129R-PTEN及G129E-PTEN3种质粒均成功转染ZR-75-1细胞并有PTEN蛋白的表达,其中wt-PTEN、G129E-PTEN均能抑制ZR-75-1细胞迁移;wt-PTEN和G129E-PTEN转染细胞之间的黏附抑制率和侵袭抑制率或侵袭细胞相对数均无显著性差异,但与G129R-PTEN转染的和未经转染的ZR-75-1细胞相比有显著性差异(P<0.01)。wt-PTEN和G129E-PTEN质粒转染的ZR-75-1细胞其P397-FAK水平均显著低于G129R-PTEN质粒转染的ZR-75-1细胞;wt-PTEN与G129E-PTEN质粒转染的ZR-75-1细胞MMP-2水平对比于G129R-PTEN质粒转染的和未经质粒转染的ZR-75-1细胞有显著性差异(P<0.01)。结论:具有双特异磷酸酶活性的野生型PTEN基因和只具蛋白磷酸酶活性的PTEN基因均能抑制乳腺癌细胞ZR-75-1的迁移,而磷酸酶失活的PTEN基因则无此作用。     

Culture of human breast cancer cell line (MDA-MB-231) on fibronectin-coated surface induces pro-matrix metalloproteinase-9 expression and activity

Interaction between cell surface integrin receptors with extracellular matrix (ECM) plays an important role in cell survival, proliferation, and migration including tumor development and invasion. Binding of ECM to integrins initiates intracellular signaling cascades, modulating expression and activity of different matrix metalloproteinases (MMPs) which is important in ECM degradation. The present study investigates fibronectin–integrin-mediated signaling and thereby modulation of MMPs expression and activity in human breast cancer cell line, MDA-MB-231. Culture of MDA-MB-231 cells on fibronectin (FN) induced expression and activity of pro-matrixmetalloproteinase-9 (MMP-9). Appreciable reduction of FN-induced pro-MMP-9 activity was observed in anti-α5 antibody treated cells. Inhibitor studies revealed that inhibitors of phosphatidyl inositiol-3-kinase (PI-3K), and nuclear factor kappa B (NF-κB) inhibited FN-induced pro-MMP-9 activity. FN increased tyrosine phosphorylation of focal adhesion kinase (FAK), integrin linked kinase (ILK), and PI-3K in MDA-MB-231 cells. FN-induced the transactivation of MMP-9 promoter by enhancing DNA binding activity of NF-κB and Sp1. Wound healing assay showed faster migration of MDA-MB-231cells grown on fibronectin-coated as surface as compared to control. Our findings indicated that culture of MDA-MB-231 on fibronectin perhaps send signals via fibronectin–integrin-mediated signaling pathways recruiting FAK, PI-3K, ILK, NF-κB, and modulate expression and activation of pro-MMP-9. These observations may enrich fundamental aspects of cancer biology especially role of α5β1 integrin in regulation of MMPs expression and activity.     

Effects of Tissue Factor,PAR-2 and MMP-9 Expression on Human Breast Cancer Cell Line MCF-7 Invasion

Objective: This study aimed to explore the expression of tissue factor (TF), protease activated receptor-2(PAR-2), and matrix metalloproteinase-9 (MMP-9) in the MCF-7 breast cancer cell line and influence oninvasiveness. Methods: Stable MCF-7 cells transfected with TF cDNA and with TF ShRNA were established.TF, PAR-2, and MMP-9 protein expression was analyzed using indirect immunofluorescence and invasivenesswas evaluated using a cell invasion test. Effects of an exogenous PAR-2 agonist were also examined. Results: TFprotein expression significantly differed between the TF cDNA and TF ShRNA groups. MMP-9 protein expressionwas significantly correlated with TF protein expression, but PAR-2 protein expression was unaffected. The PAR-2 agonist significantly enhanced MMP-9 expression and slightly increased TF and PAR-2 expression in the TFShRNA group, but did not significantly affect protein expression in MCF-7 cells transfected with TF cDNA. TFand MMP-9 expression was positively correlated with the invasiveness of tumor cells. Conclusion: TF, PAR-2,and MMP-9 affect invasiveness of MCF-7 cells. TF may increase MMP-9 expression by activating PAR-2.     

Inhibition of Metastasis and Invasion of Ovarian Cancer Cells by Crude Polysaccharides from Rosa Roxburghii Tratt in Vitro

Background: Rosa Roxburghii Tratt is a promising wild fruit crop in Southwest China. Its extracts havebeen used as traditional Chinese medicine, which benefit immune responses and cure various health disorders.However, whether Rosa Roxburghii Tratt polysaccharides could inhibit metastasis and invasion of ovarian cancercells remains unknown. Materials and Methods: Effects of crude polysaccharides from Rosa Roxburghii Tratton the viability of ovarian cancer A2780 cells were detected by MTT assay. Ovarian carcinoma cell migrationand invasion after exposure to Rosa Roxburghii Tratt polysaccharides were quantified by wound healing andTranswell assays, respectively. Western blotting was applied to assess protein levels of MMP-9. Results: Theresults indicated that Rosa Roxburghii Tratt polysaccharides significantly reduced wound closure rate of A2780cells, inhibited their migration and invasion, and suppressed the expression of MMP-9. Conclusions: Our findingsindicated that Rosa Roxburghii Tratt polysaccharides have potential for develop as anti-metastatic cancer drugpreparations for ovarian cancer patients.     

沉默MTA1抑制肝癌细胞黏附、迁移和侵袭能力的实验研究

目的:研究沉默转移相关基因1(MTA1)对肝癌细胞黏附、迁移和侵袭能力的影响。方法:在肝癌细胞中转染MTA1 siRNA、siRNA control,同时以不做转染的细胞作为对照,qRT-PCR和Western blot分别检测细胞中MTA1的表达水平,MTT检测细胞增殖,细胞黏附试验检测细胞黏附能力,Transwell小室检测细胞侵袭和迁移能力,Western blot检测细胞中基质金属蛋白酶-2（MMP-2）、上皮钙黏附素(E-cadherin)、基质金属蛋白酶-9（MMP-9）、神经型钙黏素(N-cadherin)蛋白水平,明胶酶谱实验检测MMP-2和MMP-9活性。结果:细胞中转染MTA1 siRNA能够下调肝癌细胞中MTA1 mRNA和蛋白水平,转染siRNA control对细胞中MTA1 mRNA和蛋白水平没有影响。沉默MTA1后的肝癌细胞存活率和黏附率降低,侵袭细胞数目减少,与没有转染的细胞比较,差异有统计学意义（P＜0.05）。沉默MTA1后的肝癌细胞中MMP-2、MMP-9蛋白水平及活性降低,细胞中E-cadherin蛋白水平升高,N-cadherin蛋白水平降低,与没有转染的细胞比较,差异有统计学意义（P＜0.05）。结论:沉默MTA1抑制肝癌细胞黏附、迁移和侵袭能力,作用机制与抑制细胞分泌MMP-2、MMP-9及抑制细胞EMT有关。     

Steroidal Saponins from Paris polyphylla Suppress Adhesion,Migration and Invasion of Human Lung Cancer A549 Cells Via Down-Regulating MMP-2 and MMP-9

Background: Tumor metastases are the main reasons for oncotherapy failure. Paris polyphylla (Chinese name:Chonglou) has traditionally been used for its anti-cancer actions. In this article, we focus on the regulation ofhuman lung cancer A549 cell metastases and invasion by Paris polyphylla steroidal saponins (PPSS). Materialsand Methods: Cell viability was evaluated in A549 cells by MTT assay. Effects of PPSS on invasion and migrationwere investigated by wound-healing and matrigel invasion chamber assays. Adhesion to type IV collagen andlaminin was evaluated by MTT assay. Expression and protease activity of two matrix metalloproteinases (MMPs),MMP-2 and MMP-9, were analyzed by Western blotting and gelatin zymography, respectively. Results: PPSSexerted growth inhibitory effects on A549 cells, and effectively inhibited A549 cell adhesion, migration andinvasion in a concentration-dependent manner. Western blotting and gelatin zymography analysis revealed thatPPSS inhibited the expression and secretion of MMP-2 and MMP-9 in A549 cells. Conclusions: PPSS has thepotential to suppress the migration, adhesion and invasion of A549 cells. PPSS could be a potential candidatefor interventions against lung cancer metastases.     

miR-126下调MMP-2抑制人脑胶质瘤细胞侵袭

目的初步探讨miR-126抑制人胶质瘤细胞侵袭的可能机制。方法化学合成miR-126,脂质体转染人脑胶质瘤U87细胞,应用RT-PCR、Western blot检测MMP-2基因和蛋白的表达情况,并应用Transwell小室检测转染前后细胞侵袭力的变化。结果miR-126上调后U87细胞的MMP-2基因和蛋白表达降低,并且细胞侵袭力明显降低。结论化学合成的miR-126在抑制人胶质瘤细胞侵袭过程中发挥重要作用,可能成为胶质瘤基因治疗的新靶点。     

DUSP10对人卵巢癌A2780细胞迁移及侵袭的影响

目的：探讨DUSP10在浆液性卵巢癌组织中的表达情况及其与卵巢癌细胞迁移及侵袭能力的关系。方法：实时荧光定量PCR方法检测浆液性卵巢腺癌和正常输卵管伞端组织中 DUSP10的表达情况;转染DUSP10过表达和干扰质粒至人卵巢癌A2780细胞,划痕实验和Transwell实验观察癌细胞迁移及侵袭能力的变化。结果：DUSP10在浆液性卵巢癌组织的表达低于正常输卵管伞端组织（ P﹤0.05）。与对照组相比,转染DUSP10过表达质粒后A2780细胞迁移及侵袭能力受到抑制,转染DUSP10干扰质粒后A2780细胞迁移及侵袭能力增强。结论：DUSP10在浆液性卵巢癌组织中低表达;DUSP10可抑制人卵巢癌A2780细胞的迁移及侵袭能力。