脂筏结构蛋白1基因在乳腺癌细胞中的表达及靶向抑制其表达对癌细胞凋亡的诱导

目的探讨脂筏结构蛋白(FLOT)1基因在乳腺癌细胞中的表达及靶向抑制其表达对癌细胞凋亡的影响及机制。方法 Western印迹法检测正常乳腺上皮细胞MCF-10A及MCF7、MDA-MB-231和HCC1569乳腺癌细胞FLOT1的蛋白表达。将设计合成的针对FLOT1的特异性siRNA序列(si-FLOT1组)及阴性对照siRNA序列(NC组)转染至MDA-MB-231细胞,仅仅加入脂质体的为空白对照组,AG490作为信号转导与转录因子(STAT)3信号通路抑制剂,转染48 h, Western印迹法检测FLOT1、磷酸化蛋白酪氨酸激酶(p-JAK)2、p-STAT3、细胞增殖核抗原(PCNA)和B细胞淋巴瘤(Bcl)-2的蛋白表达。流式细胞术检测细胞凋亡率。结果 FLOT1在MCF7、MDA-MB-231和HCC1569乳腺癌细胞中的蛋白表达均显著高于正常乳腺上皮细胞MCF-10A(P0.05)。与空白对照组比较,si-FLOT1组FLOT1表达受到抑制(P0.05);与NC组比较,si-FLOT1组细胞凋亡率显著升高(P0.05),p-JAK2、p-STAT3、PCNA和Bcl-2的蛋白表达显著下调(P0.05)。与si-FLOT1组比较,si-FLOT+AG490组细胞凋亡率显著升高(P0.05)。结论 FLOT1在乳腺癌细胞中呈高表达,通过RNA干扰抑制其表达可诱导癌细胞凋亡,机制与下调STAT3信号通路有关。     

沉默ATAD2增加乳腺癌细胞放疗敏感性

目的研究沉默三磷酸腺苷酶家族蛋白(ATAD)2对乳腺癌细胞放疗敏感性的影响。方法以乳腺癌细胞MDA-MB-231作为探讨对象,在乳腺癌细胞中转染ATAD2 shRNA载体,以Realtime PCR和Western印迹检测细胞中ATAD2表达水平。噻唑蓝(MTT)检测细胞增殖,碘化丙啶(PI)单染法检测细胞周期,膜联蛋白(Annexin) V-FITC/PI双染法检测细胞凋亡,Western印迹检测细胞中激活型含半胱氨酸的天冬氨酸蛋白水解酶(Cleaved Caspase)-3、细胞周期依赖性蛋白激酶(CDK)4、p21蛋白表达水平,克隆形成实验检测放射敏感性。结果转染ATAD2 shRNA载体后的乳腺癌细胞中ATAD2表达水平降低。沉默ATAD2或放射处理后的乳腺癌细胞增殖能力降低,G0/G1期细胞比例增多,细胞凋亡率升高,细胞中Cleaved Caspase-3、p21蛋白水平升高,CDK4蛋白水平降低。沉默ATAD2和放射共同处理后的乳腺癌细胞增殖能力下降,细胞凋亡率升高,细胞中Cleaved Caspase-3、p21蛋白水平升高,CDK4蛋白水平降低,与沉默ATAD2或放射处理后的乳腺癌细胞比较,差异有统计学意义(P0.05)。沉默ATAD2后的乳腺癌细胞放射增敏比为1.396。结论沉默ATAD2协同放射诱导乳腺癌细胞凋亡,抑制乳腺癌细胞增殖并阻滞细胞周期,沉默ATAD2具有提高乳腺癌细胞放射敏感性的作用。     

miR-509-3p靶向BCL2基因调控乳腺癌细胞增殖、侵袭、迁移及凋亡的分子机制

目的探讨微小RNA-509-3p(miR-509-3p)调控乳腺癌细胞增殖、侵袭、迁移及凋亡的分子机制。方法 qRT-PCR检测miR-509-3p、B淋巴细胞瘤(BCL)2在乳腺癌组织及不同乳腺癌细胞株中的表达情况;双荧光素酶报告基因实验检测miR-509-3p与BCL2之间的相互作用;噻唑蓝(MTT)实验与流式细胞术分别检测miR-509-3p对乳腺癌MDA-MB-231细胞增殖及凋亡能力的变化情况;Transwell迁移及侵袭实验检测miR-509-3p对MDA-MB-231细胞迁移及侵袭行为的变化情况;Western印迹检测miR-509-3p对BCL2、细胞周期蛋白(CyclinD)1、p21、基质金属蛋白酶(MMP)-2、MMP-9蛋白的调控情况。结果与癌旁正常组织相比,乳腺癌组织中miR-509-3p的表达水平显著下调(P<0.05),与其他乳腺癌细胞相比,乳腺癌MDA-MB-231细胞中miR-509-3p表达最低,而BCL2的表达水平相反;上调miR-509-3p表达MDA-MB-231细胞的增殖、迁移及侵袭能力明显降低(P<0.05),而细胞凋亡水平明显升高(P<0.05),CyclinD1、MMP-2、MMP-9表达水平明显降低(P<0.05),而p21表达水平明显升高(P<0.05);miR-509-3p可靶向调控BCL2表达;上调BCL2表达可逆转上调miR-509-3p表达对乳腺癌MDA-MB-231细胞增殖、凋亡、迁移及侵袭的作用。结论 miR-509-3p能够靶向抑制BCL2而抑制乳腺癌细胞增殖、迁移、侵袭并促进细胞凋亡。     

姜黄素对乳腺癌MCF-7、MDA-MB-231细胞增殖、侵袭的影响及其机制探讨

目的观察姜黄素对雌激素受体阳性（ER＋）乳腺癌细胞MCF-7和ER-乳腺癌细胞MDA-MB-231增殖、侵袭的影响,并探讨其机制。方法将MCF-7/MDA-MB-231随机分为四组,姜黄素高、中、低剂量组（姜黄素Ⅰ、Ⅱ、Ⅲ组）分别加入5、10、20μg/ml的姜黄素,对照组不加。MTT法检测细胞的增殖能力,流式细胞仪分析细胞周期,通过Boyden小室观察细胞侵袭力,RT-PCR法检测细胞中的MMP-9 mRNA。结果与对照组相比,姜黄素各剂量组细胞增殖下降,G0/G1期及S期细胞比例减少,而G2/M期细胞比例显著增加,细胞侵袭指数降低,细胞中的MMP-9 mRNA表达下降,P均〈0.05。结论姜黄素能抑制MCF-7/MDA-MB-231细胞的增殖、侵袭作用,与其降低MMP-9基因表达有关。     

siRNA干扰LOX基因对乳腺癌细胞株侵袭性影响的研究

目的检测赖氨酰氧化酶（LOX）基因在低侵袭性乳腺癌细胞MCF-7和高侵袭性乳腺癌细胞MDA-MB-231中的表达差异,并探讨LOX基因对乳腺癌细胞生物学行为的影响。方法 RT-PCR扩增MCF-7和MDA-MB-231LOX基因,采用SYBR G reen I实时荧光定量RT-RCR技术相对定量两种细胞的LOX基因表达。瞬转MDA-MB-231,并于转染后24、48、72 h行体外运动试验侵袭实验,观察干扰LOX基因表达后对MDA-MB-231侵袭的影响。结果 MDA-MB-231较MCF-7高表达LOX基因（P〈0.05）。MDA-MB-231细胞中LOX mRNA在转染48 h表达下降明显。干扰LOX表达后,体外运动实验结果显示RNA i组中MDA-MB-231细胞穿过微孔滤膜到达下室面的细胞数少于未转染组和阴性对照组（P均〈0.05）。结论 MDA-MB-231细胞高表达LOX基因,干扰其LOX基因表达后细胞的侵袭运动能力明显减弱。     

RNA干扰抑制cyclin E表达对食道癌细胞Eca-109增殖的影响

目的 探讨利用RNAi技术抑制cyclin E的表达,研究cyclin E在食道癌细胞Eca-109生长、增殖过程中的作用.方法 设计针对cyclin E基因的siRNA序列,合成并克隆入pGFU6/neo质粒中,用脂质体转染法将重组质粒转染Eca-109细胞.采用RT-PCR和免疫印迹试验检测稳定转染的Eca-109细胞中cyclin E的表达水平.台盼蓝排斥法、MTT法、3H-TdR掺人试验、平板克隆形成试验检测干扰cyclin E后对细胞活力以及增殖能力的影响,流式细胞仪测定细胞周期时相分布.Westem印迹检测细胞增殖有关蛋白PCNA、p21、bax和bcl-2表达变化.结果 台盼蓝排斥法、MTT法、3H-TdR掺入试验和平板克隆形成试验显示转染组细胞活力及增殖受到抑制;阻滞在G0/G1期细胞比例增加.Western印迹表明PCNA和bcl-2蛋白表达量下降;而p21蛋白和Bax蛋白表达量上升.结论 沉默cyclin E基因能明显抑制食道癌细胞Eca-109的活力及增殖能力,并且细胞周期被阻滞,机制可能与下调肿瘤细胞内PCNA以及上调p21的表达有关.     

siRNA沉默Cyclin E基因对肝癌HepG2、SMMC-7721和BEL-7402细胞增殖和侵袭能力的影响

目的:观察siRNA沉默Cyclin E基因表达对肝癌HepG2、SMMC-7721和BEL-7402细胞增殖和侵袭能力的影响.方法:构建2个靶向Cyclin E基因siRNA载体,转染人肝癌HepG2、SMMC-7721和BEL-7402细胞.RT-PCR、Western blot检测转染后HepG2、SMMC-7721和BEL-7402细胞Cyclin E基因mRNA和蛋白表达水平.CCK-8试验、软琼脂克隆形成实验检测HepG2、SMMC-7721和BEL-7402细胞增殖、克隆形成能力.流式细胞术、transwell试验分别检测HepG2、SMMC-7721和BEL-7402细胞周期和侵袭能力.结果:构建的2个Cyclin E基因siRNA载体插入序列与所设计序列均一致;转染HepG2、SMMC-7721和BEL-7402细胞后,干扰1组、干扰2组与空白对照组和阴性对照组比较,C y c l i n E m R N A和蛋白表达量均显著降低(P<0.05),细胞生长速度延缓,软琼脂细胞集落形成数、穿透细胞数均显著降低(P<0.05),S和G2/M期细胞比例减少,G0/G1期细胞比例增加.结论:沉默肝癌细胞Cyclin E表达水平,可有效抑制细胞生长、增殖和侵袭能力.     

中药扶正祛瘤对乳腺癌干细胞MDA-MB-231增殖、凋亡的影响及其机制的研究

目的探究中药扶正祛瘤对乳腺癌干细胞MDA-MB-231抑制增殖、凋亡的影响。方法体外富集培养乳腺癌干细胞MDA-MB-231,饲养小鼠并灌输中药获得含药血清;实验分为对照组、等药量组、1倍药量组、2倍药量组,MTT法检测每组乳腺癌干细胞MDA-MB-231细胞抑制率;在最适药物浓度条件下连续培养MDA-MB-23细胞5 d,MTT法测定每天细胞的抑制率,绘制乳腺癌干细胞MDA-MB-231抑制增长曲线;在最佳药物浓度及培养时间下培养细胞,用流式细胞仪检测乳腺癌干细胞MDA-MB-231的凋亡情况;q RT-PCR法检测中药扶正祛瘤处理后细胞MDA-MB-231中mi R-34a及其靶基因SIRT1的基因表达。结果含药血清对乳腺癌干细胞MDA-MB-231有不同程度的抑制,抑制率:2倍药量组1倍药量组等量药量组对照组;采用2倍药量的小鼠血清连续培养细胞,1～4 d内随处理时间增长细胞抑制率增加,第5天有下降趋势;含药血清处理后的细胞内mi R-34a基因表达量高于对照组,SIRT1基因表达量低于对照组;2倍药量的中药扶正祛瘤血清作用乳腺癌干细胞MDA-MB-231 4 d后与对照组相比出现大量的凋亡。结论中药扶正祛瘤能够效抑制乳腺癌干细胞MDA-MB-231的增殖,可能通过调控mi R-34a靶基因SIRT1的表达而抑制癌细胞的增殖凋亡。     

慢病毒介导SOX4表达下调对乳腺癌细胞生长及迁移能力的影响

目的研究慢病毒介导的性别决定区Y框蛋白(SOX)4表达下调对乳腺癌细胞生长及迁移能力影响。方法以乳腺癌MCF-7细胞为研究对象,用含有SOX4 siRNA的重组慢病毒和阴性对照慢病毒感染MCF-7细胞,qRT-PCR和Western印迹方法检测细胞中SOX4表达变化。噻唑蓝(MTT)方法测定增殖变化,碘化丙啶(PI)单染法测定细胞周期分布变化,Transwell小室检测侵袭和迁移能力变化,Western印迹检测细胞中细胞周期蛋白(Cyclin)D1、波形蛋白(Vimentin)、基质金属蛋白酶(MMP)-9、上皮性钙黏附素(E-cadherin)、MMP-2、p21蛋白表达水平。结果 SOX4 siRNA重组慢病毒感染可以抑制乳腺癌细胞中SOX4基因的表达和转录。敲减SOX4后的乳腺癌细胞的增殖能力显著降低,细胞G0/G1期比例显著升高,细胞侵袭和迁移能力显著下降,细胞中CyclinD1蛋白表达水平显著降低,p21蛋白表达水平显著升高,MMP-2、MMP-9、Vimentin蛋白表达水平显著下降,E-cadherin蛋白表达水平显著升高(均P0.05)。阴性对照慢病毒感染对MCF-7细胞中SOX4表达水平没有影响(均P0.05),并且对细胞增殖、周期分布、侵袭、迁移水平和细胞中CyclinD1、p21、MMP-2、MMP-9、Vimentin、E-cadherin蛋白表达水平均没有影响。结论敲减SOX4可以抑制乳腺癌细胞生长、侵袭和迁移,并将乳腺癌细胞周期阻滞在G0/G1期。     

睾丸蛋白聚糖1对胆囊癌细胞生长凋亡的影响及机制

目的研究睾丸蛋白聚糖(SPOCK)1对胆囊癌细胞生长、凋亡影响及机制。方法胆囊癌细胞GBC-SD中分别转染SPOCK1 siRNA和siRNA control,记为干扰组和阴性组,同时以不做处理的GBC-SD细胞作为对照组。Real time PCR和Western印迹法分别测定转染后的各组胆囊癌细胞中SPOCK1的表达水平,噻唑蓝(MTT)测定各组细胞增殖,克隆形成实验测定各组细胞克隆能力,流式细胞术测定各组细胞凋亡,Western印迹测定各组细胞中信号转导与转录因子(STAT)3、剪切的含半胱氨酸的天冬氨酸蛋白水解酶(Cleaved Caspase)-3、磷酸化的STAT(p-STAT)3蛋白水平。结果阴性组胆囊癌细胞中的SPOCK1 mRNA和蛋白水平、细胞A值、细胞克隆形成率、凋亡率及STAT3、Cleaved Caspase-3、p-STAT3蛋白水平与对照组相比差异均无统计学意义(P>0.05)。干扰组较对照组胆囊癌细胞中的SPOCK1 mRNA和蛋白水平明显降低,细胞A值和克隆形成率也明显下降,细胞凋亡率明显升高,细胞内的Cleaved Caspase-3水平明显升高,p-STAT3蛋白水平明显下降(P<0.05)。结论 SPOCK1下调可以抑制胆囊癌细胞生长,诱导Caspase-3介导的细胞凋亡,其作用机制可能与抑制STAT3信号通路的激活有关。     

Inhibitory effects of resistin-13-peptide on the proliferation, adhesion, and invasion of MDA-MB-231 in human breast carcinoma cells

To investigate the potential effects of resistin-13-peptide on the growth, adhesion, and invasion in human breast carcinoma cells, MDA-MB-231. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and colony-forming assay were used to assess the proliferation effects of resistin-13-peptide. The adhesive ability was investigated by cell adhesion assay, and the invasive potential was assessed using a transwell model. Activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by zymography analysis and western blotting. Tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2 were determined by western blotting. In this study, we performed in vivo experiments and determined the effect of resistin-13-peptide on tumor growth and other organs, especially ovaries in a xenograft model using the cell line studied. Resistin-13-peptide inhibited MDA-MB-231 cell growth and colony formation in a dose- and time-dependent manner. Meanwhile, the invasive and adhesive abilities of MDA-MB-231 cells were yet cut down by resistin-13-peptide in a dose-dependent manner. Resistin-13-peptide decreased the gelatinolytic activities of both MMP-2 and MMP-9 and enhanced the protein expression of TIMP-1 and TIMP-2, which were secreted from the MDA-MB-231 cells. The animal experiments found that the growth of tumors was repressed by resistin-13-peptide, which affected other organs in the same time. Especially, ovaries did not have pathological changes yet. Treatment with resistin-13-peptide is effective in suppressing tumor proliferation, adhesion, and invasion. The possible mechanism is downregulation of MMPs and upregulation of TIMPs.     

人参皂苷Rg3对乳腺癌细胞增殖的影响及其机制探讨

目的观察人参皂苷Rg3对乳腺癌细胞MDA-MB-231增殖的影响,并探讨其机制。方法取处于对数生长期的MDA-MB-231,加入不同终浓度（50、100、200、400、600μmol/L）人参皂苷Rg3,以不加人参皂苷Rg3为对照。MTT法检测细胞生长情况;Western blot法检测细胞中细胞周期蛋白E（Cyclin E）及细胞分裂周期蛋白25A（CDC25A）表达水平。结果低浓度（≤200μmol/L）的人参皂苷Rg3可促进MDA-MB-231增殖,高浓度（≥400μmol/L）的人参皂苷Rg3则抑制其增殖。与对照细胞比较,低浓度人参皂苷Rg3处理的细胞中Cyclin E及CDC25A的表达增高,高浓度处理者Cyclin E及CDC25A的表达下降（P均〈0.01）。结论高浓度人参皂苷Rg3在体外对乳腺癌细胞的生长具有抑制作用,其机制可能与抑制细胞周期关键蛋白Cyclin E及CDC25A的表达有关。     

全硫代反义寡核苷酸对乳腺癌MDA-MB-231细胞的影响

目的 本研究探讨全硫代反义寡核苷酸( PS-ASODN)对乳腺癌MDA-MB-231细胞端粒酶活性及细胞生长、迁移、侵袭的影响.方法 本实验将PS-ASODN经脂质体转染作用于乳腺癌MDA-MB-231细胞,将实验分为空白对照组、对照正义全硫代寡核苷酸(PS-SODN)组和不同剂量的PS-ASODN组;脂质体介导的PS-ASODN和PS-SODN作用于乳腺癌MDA-MB-231细胞后,分别采用四甲基偶氮唑蓝比色法(MTT)、酶联免疫吸附法(EUSA)、流式细胞术、Transwell小室迁移、侵袭实验检测细胞的体外增殖、端粒酶活性、细胞凋亡、细胞迁移、侵袭能力的影响.结果 终浓度为1、3及5 μmol/L的PS-ASODN对MDA-MB-231细胞端粒酶活性及细胞的增殖均有抑制作用,与空白对照组比较差异显著(P＜0.01),并呈一定剂量依赖性;ELISA法检测结果,1、3及5μmol/L的PS-ASODN对MDA-MB-231细胞作用72 h后端粒酶皆为阴性,表明端粒酶PS-ASODN能够抑制端粒酶活性,对照组端粒酶皆为阳性.1、3及5μmol/L的PS-ASODN作用24h可以明显抑制MDA-MB-231细胞的迁移和侵袭能力(P＜0.01).结论 PS-ASODN能有效抑制人乳腺癌MDA-MB -231细胞的生长、促进凋亡、抑制其侵袭和迁移能力,其机制可能是通过PS-ASODN降低端粒酶活性而实现.     

曲古抑菌素A对乳腺癌细胞株MDA-MB-231增殖的影响

目的探讨曲古抑菌素A（TSA）对乳腺癌细胞株增殖的影响。方法培养乳腺癌细胞株MDA-MB-231,用组不同浓度TSA分别作用于细胞,于24、48、72、96h用MTT比色法观察细胞生长情况,流式细胞术检测S期细胞的比例和周期素（Cyclin）D1、A2表达。结果72h内和4μmol／L的浓度范围内,乳腺癌细胞生长受到抑制,并有一定的时效关系;TSA作用后周期素Cyclin A2表达下降,Cyclin D1表达上升（P均〈0．05）。结论TSA能抑制乳腺癌细胞生长,周期素Cyclin D1、Cyclin A2参与了抑制剂对细胞增殖周期的调控。     

Expression of new prognostic markers, peripheral-type benzodiazepine receptor and carbonic anhydrase IX, in human breast and ovarian carcinoma cell lines

Peripheral benzodiazepine receptor (PBR), a mitochondrial protein involved in cell proliferation and differentiation, and carbonic anhydrase IX (CA IX), an intrinsic marker of hypoxia, have been studied in the panel of human breast (MCF-7, BT- 20, MDA-MB-453, MDA-MB-231) and ovarian (A2780, A2780/CP, A2780/ADR, CH1, SKOV-3) carcinoma cell lines that differ by malignant progression. The expression of both antigens was detected by staining with the PBR-specific 8D7 and CA IX-specific M75 monoclonal antibodies and quantitated by flow cytometry. PBR was related to mitochondrial mass and CA IX to the cell density. Breast carcinoma cell lines showed higher relative fluorescence intensity of PBR expression than ovarian cell lines, with the exception of A2780/CP cisplatin-resistant subline that was comparable to highly invasive MDA-MB-231 breast line. Among the breast cell lines, PBR expression increased with their invasive potential. The ovarian cell lines showed greater variability in fluorescence intensities and the expression of PBR did not correlate with the amount of mitochondria. Mitochondrial PBR density disclosed significant difference between cisplatin-sensitive (low PBR density) and -resistant (high PBR density) ovarian cell lines. MTT test showed higher sensitivity of 2 breast cell lines MCF-7 and MDA-MB-231 (IC50 < 75 microM) to PBR ligand PK 11195 than all examined ovarian cell lines (IC50 > 90 microM, in chemo- and radio- resistant lines IC50 > 110 microM). Growth inhibitory effect of PK 11195 did not correlate with the amount of PBR and was mediated probably by another, PBRindependent mechanisms. The expression of CA IX was only marginal in majority of tested cell lines in subconfluent conditions and was inducible by high cell density. More than 5% of positive cells in sparse culture have been found in MDA-MB-231 and MDA-MB-453 breast cell lines while more than 15% of A2780/ADR adriamycin-resistant ovarian cells were positive for CA IX expression under the same conditions. Our data indicate that PBR expression in breast and ovarian carcinoma cell lines is not proportional to the amount of mitochondria and should be expressed relatively to the cell mitochondrial mass. This assessment allows establishing high PBR density as a measure of aggressiveness (invasion in breast and resistance in ovarian cancer). Observation of relatively high CA IX expression in A2780/ADR cells evokes the assumption that multidrug resistance might be connected with selection advantage towards CA IX expressing cells.     

c—Src在RANKL诱导的乳腺癌MDA-MB-231细胞迁移中的作用

目的探讨非受体酪氨酸激酶c—Src在核因子-κB受体活化因子配体（RANKL）诱导的乳腺癌MDA-MB-231细胞迁移中的作用。方法流式细胞仪检测MDA—MB-231细胞表面受体核因子-κB受体活化因子（RANK）蛋白的表达及RANKL刺激后细胞p-Src及c-Src的表达;Transwell法测定细胞迁移能力。结果MDA—MB-231细胞表达RANK蛋白,RANKL诱导MDA-MB-231细胞迁移能力增强。应用RANKL的圈套受体OPG可阻断RANKL诱导的细胞迁移。RANKL刺激后MDA—MB-231细胞p-Src表达升高,应用src激酶抑制剂PP2可显著抑制RANKL诱导的细胞迁移。结论c-Src信号通路参与RANKL诱导的乳腺癌MDA-MB-231细胞迁移。     

17Beta-hydroxysteroid dehydrogenase Type 1 and Type 2: association between mRNA expression and activity in cell lines

17Beta-hydroxysteroid dehydrogenases (17beta-HSDs) are a family of enzymes that regulate steroid availability within a tissue by catalysing the interconversion of active and inactive forms. Type 1 is up-regulated in many breast tumours, and is responsible for the reduction of oestrone to active oestradiol which stimulates cell proliferation within the tumour. Type 2 oxidises many active steroids to their inactive forms, including oestradiol to oestrone. In this study, we have compared the mRNA expression and enzyme activities of Type 1 and Type 2 in MCF-7, MDA-MB-231, T47D, JEG3 and 293-EBNA cell lines. Also studied were two cell lines stably expressing transfected Type 1 cDNA. RT-PCR indicated that little Type 1 mRNA is expressed in two of the breast cancer cell lines, MCF-7 and MDA-MB-231, and in 293-EBNA cells, but that expression is much higher in the T47D breast cancer cell line, and in the choriocarcinoma cell line, JEG3. However, a higher level of expression of Type 1 is seen in the transfected cell lines MCF-7.8H and 293-EBNA[His617beta-HSD1]. Activity assays show that there is high association between mRNA expression and enzyme activity. Assays indicate that, with the exception of MDA-MB-231 cells, Type 2 activity is low in these lines. The study of the basal activities of these enzymes will be used in future studies investigating the regulation of the enzymes by endogenous and exogenous factors. An understanding of their regulation in both healthy and malignant tissues may lead to future therapeutic intervention at the regulatory level.     

Novel biological activities of the fish pro-IGF-I E-peptides: studies on effects of fish pro-IGF-I E-peptide on morphological change,anchorage-dependent cell division,and invasiveness in tumor cells

E-peptides of proinsulin-like growth factor-I (pro-IGF-I) are proteolytically cleaved from the prohormone after translation and have long been regarded as biologically inactive. Tian et al. [Endocrinology 140 (1999) 3387-3390] recently demonstrated that recombinant rainbow trout pro-IGF-I E-peptides (rtEa-2-, rtEa-3-, and rtEa-4-peptides), like hIGF-I, exhibited a dose-dependent mitogenic activity in several nontransformed mammalian cell lines. We show in this report that treatment of established human and fish cancer cells (MCF-7, HT-29, HepG2, ZR-75-1, SK-N-F1, and HC) and retroviral transformed human embryonic kidney cells (293GP) with recombinant rtEa-2- or rtEa-4-, but not rtEa-3-peptide, resulted in a dose-dependent induction of morphological change and enhanced cell attachment. The E-peptide-induced morphological changes are sensitive to treatment with alpha-amanitin or cycloheximide, known inhibitors of RNA and protein synthesis. The in vitro colony formation activity of established human tumor cells (HT-29 and MDA-MB-231) is greatly reduced or diminished by treatment with the rtEa-4-peptide. Both morphological change and reduction of colony formation activity in MDA-MB-231 cells were also observed following transfection with an Ea-4 transgene construct. Furthermore, the invasive activity of HT1080 cells, known invasive cancer cells, is reduced three to fourfold by treatment with the rtEa-4-peptide. These results suggest that E-peptides of rainbow trout pro-IGF-I possess novel biological activities controlling malignant properties of cancer cells in vitro.     

不同片段的NPM1-EGFP融合表达载体的构建和表达

目的构建不同片段的NPM1与绿色荧光蛋白(EGFP)的真核融合表达质粒,检测其在人乳腺癌MDA-MB-231细胞中的表达和定位。方法 RT-PCR法从人乳腺癌MDA-MB-231细胞cDNA中扩增不同片段的NPM1,产物经电泳分离、切胶纯化后,分别采用XhoⅠ和EcoRⅠ进行酶切,然后与同样酶切后的pEGFP-C3载体进行连接反应,将连接产物转化大肠杆菌感受态细胞,挑取克隆,提取质粒并酶切鉴定。将酶切及测序鉴定后的pEGFP/NPM1质粒采用脂质体介导后将其转入MDA-MB-231细胞中,通过Western blot技术检测其蛋白的表达。倒置荧光显微镜观察其亚细胞定位。结果酶切鉴定和基因测序结果显示不同片段的pEGFP-NPM1融合表达质粒构建成功,并可在MDA-MB-231细胞内表达,倒置荧光显微镜下可以清晰显见融合蛋白的亚细胞定位。结论 NPM1及其不同结构域的真核重组质粒构建成功,在细胞中成功表达,并可以用于分析其亚细胞定位,从而为进一步研究NPM1在乳腺癌发生发展中的作用奠定基础。