胰岛素通过Erk通路促进人乳腺癌细胞MCF-7增殖和迁移

目的观察胰岛素对人乳腺癌细胞MCF-7中Erk信号通路的影响及其对MCF-7细胞增殖、侵袭和迁移的影响。方法分别采用0、50、100和200n M不同剂量的胰岛素处理MCF7细胞0、24、48和72h,采用Western blot方法检测MCF-7细胞中Erk信号通路的磷酸化活化情况,采用MTT方法检测MCF-7细胞的增殖情况。通过伤口愈合实验和Boyden小室检测200n M胰岛素处理72h对MCF-7细胞侵袭和迁移能力的影响。采用Erk信号通路抑制剂预处理MCF-7细胞,观察Erk信号通路在胰岛素促MCF-7细胞增殖、迁移和侵袭能力中的作用。结果同0nM胰岛素处理组相比,不同剂量的胰岛素均可显著上调Erk的磷酸化水平,加强MCF-7细胞的增殖能力(P0.05),且这种作用具有计量依赖性。同对照组相比,200nM胰岛素处理72h后,MCF-7细胞侵袭和迁移能力均显著增强(t=188.000、95.640,P0.05)。采用Erk信号通路阻断剂U0126阻断MCF-7细胞中的Erk信号通路后,胰岛素促MCF-7细胞增殖、迁移和侵袭的能力显著降低(P0.05)。结论胰岛素可通过激活MCF-7细胞中的Erk信号通路,增强肿瘤细胞的增殖、迁移和侵袭能力。     

蟾毒灵抑制低氧条件下乳腺癌细胞干性机制研究

目的:探究蟾毒灵(bufalin, BUF)对低氧条件下乳腺癌细胞干性的影响以及可能的机制。方法:慢病毒转染构建稳转低氧乳腺癌细胞模型MDA-MB-231/HIF1α^OE细胞株，以CCK-8、平板克隆、Transwell、划痕实验、克隆球悬浮实验检测BUF对低氧模型组乳腺癌细胞增殖、迁移、侵袭能力的影响；Western blot检测BUF对低氧模型组干性指标和PI3K/AKT信号通路的影响。结果:成功构建乳腺癌低氧细胞模型；低氧模型组的增殖率、克隆形成率、侵袭迁移能力以及克隆球形成能力明显高于Ctrl组；BUF干预后，上述干性指标明显降低；Western blot实验结果显示，低氧刺激激活PI3K/AKT信号通路，BUF可抑制低氧模型组PI3K/AKT信号通路的激活。结论:BUF可抑制低氧条件下乳腺癌细胞的干性，其机制可能与调控PI3K/AKT信号通路相关。     

miR-96在人乳腺癌中表达及其对乳腺癌细胞侵袭和迁移活性的影响

目的研究miR-96在人乳腺上皮细胞株和乳腺病变组织中表达状况及其对人乳腺癌细胞增殖、侵袭和迁移的影响。方法抽提4株人乳腺上皮细胞系和28例人乳腺病变新鲜组织总miRNA,实时定量PCR方法检测它们miR-96的表达状况;脂质体介导的转染方法将miR-96抑制物转染人乳腺癌MCF-7细胞株;通过MTS试剂盒检测细胞增殖能力,Transwell侵袭和迁移实验检测细胞侵袭及迁移能力。结果miR-96在高侵袭乳腺癌细胞株中表达较低侵袭乳腺细胞明显下调(P<0.01);人乳腺癌组织中miR-96表达较乳腺良性病变组织明显下调(P<0.01);miR-96抑制物转染乳腺癌MCF-7后,乳腺癌细胞侵袭和迁移活力较阴性对照组细胞明显增强(P<0.05),而细胞增殖活力改变无统计学意义(P>0.05)。结论 miR-96在人乳腺癌细胞株和乳腺癌组织中存在表达下调,并对人乳腺癌细胞的侵袭及迁移能力可能存在一定负性调控作用。     

siRNA沉默Twist基因对鼻咽癌细胞CNE-2增殖的影响

目的探讨RNA干扰沉默Twist基因对人鼻咽癌细胞株CNE-2的迁移、侵袭和增殖等生物学活性的影响。方法构建Twist-shRNA表达载体,脂质体法将重组质粒分别转染入鼻咽癌细胞株CNE-2中,RT-PCR法检测Twist mRNA、Western blot法检测Twist蛋白表达量的变化。MTT法检测细胞的增殖,细胞创伤愈合试验观察细胞迁移能力,Tran-swell小室试验检测细胞侵袭能力的变化。结果沉默Twist基因可减慢CNE-2细胞生长速度(与对照组比较P<0.05),降低CNE-2细胞的迁移速度,抑制CNE-2的体外侵袭能力。结论靶向封闭Twist基因的表达,可明显抑制CNE-2细胞的增殖、迁移及侵袭能力,提示Twist可作为鼻咽癌防治的一个新的靶点。     

细胞外基质磷酸糖蛋白对细胞增殖能力的影响

目的:探讨细胞外基质磷酸糖蛋白对细胞增殖能力的影响。方法:构建含细胞外基质磷酸糖蛋白基因的质粒,稳定转染293T细胞,通过激光共聚焦显微镜与RT-PCR检测转染细胞株hMEPE蛋白表达结果。通过MTT实验检测细胞增殖与迁移能力。通过RT-PCR检测p53基因的表达水平的差异,进一步探讨肿瘤细胞增殖的分子机制。结果:与转染空载体细胞相比,稳定转染hmepe细胞株增殖能力有明显提高,RT-PCR结果显示p53基因在转染目的基因细胞中较转染空载体细胞其表达增高。结论:细胞外基质磷酸糖蛋白可以提高细胞增殖能力。     

siRNA 靶向沉默 Lipocalin-2基因下调 MMP-9表达对降低人肺癌 A549细胞的侵袭、迁移能力的影响

目的：利用针对 Lipocalin-2的小干扰 RNA（siRNA）,观察 Lipocalin-2基因沉默对人肺癌 A549细胞MMP-9表达及侵袭、迁移能力的影响。方法采用 Real-time PCR 和 Western blot 检测 Lipocalin-2在3种肺癌细胞株（A549、NCI-H661、NCI-H446）及正常人支气管上皮细胞（HBE）的表达情况。设计并构建靶向 Lipocalin-2的 siRNA 转染 A549细胞,采用 Real-time PCR 和 Western blot 检测转染效率及 Lipocalin-2基因沉默对 MMP-9 mRNA 和蛋白表达的影响。Transwell 小室检测细胞侵袭能力。细胞划痕实验检测细胞迁移能力。结果 Lipocalin-2 mRNA 和蛋白在肺癌A549、NCI-H661、NCI-H446细胞株中的表达水平均显著高于 HBE 细胞,差异有统计学意义（ P ＜0.05）。siRNA-Li-pocalin-2转染组 Lipocalin-2 mRNA 和蛋白表达水平与阴性对照组和空载体对照组比较,差异有统计学意义（ P ＜0.05）;siRNA-Lipocalin-2转染组 MMP-9 mRNA 和蛋白表达水平与阴性对照组和空载体对照组比较,差异有统计学意义（ P ＜0.05）;siRNA-Lipocalin-2转染组穿膜细胞数、平均迁移距离与阴性对照组和空载体对照组比较,差异有统计学意义（ P ＜0.05）。结论 Lipocalin-2基因沉默通过下调 MMP-9表达降低了人肺癌 A549细胞的侵袭和迁移能力。     

下调miR-19b表达对骨肉瘤MG63细胞侵袭、迁移及Wnt/β-Catenin通路的影响

摘要：目的:探讨下调miR-19b表达对骨肉瘤MG63细胞侵袭、迁移能力的影响,并探讨可能机制。方法:采用脂质体转染法转染人骨肉瘤MG63细胞,实验分为空白组、miR-19b inhibitor NC组、miR-19b inhibitor组,采用细胞计数试剂盒-8(CCK-8)法检测细胞增殖情况,通过Transwell小室法检测各组细胞侵袭、迁移情况,通过免疫印迹(WB)法检测侵袭、迁移以及Wnt/β-连环蛋白(β-Catenin)通路相关蛋白表达情况。结果:与空白组、miR-19b inhibitor NC组相比,miR-19b inhibitor组miR-19b相对表达量、转染后48 h、72 h吸光度(A)值、侵袭与迁移细胞数、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、Wnt、细胞核β-Catenin表达水平显著降低(P<0.05),细胞质β-Catenin表达水平显著升高(P<0.05)。结论:下调miR-19b表达可能通过抑制Wnt/β-Catenin信号通路激活,从而抑制骨肉瘤MG63细胞侵袭、迁移。     

沉默LIMK1促进DADS抑制SW480细胞迁移与侵袭

目的探讨LIMK1沉默对DADS抑制SW480细胞迁移与侵袭的影响。方法 RNA干扰技术建立稳定LIMK1-miRNA/SW480细胞株;免疫组化和Western blot检测DADS对沉默LIMK1结肠癌SW480细胞LIMK1和磷酸化LIMK1蛋白表达的影响。划痕实验和侵袭实验检测LIMK1 RNA沉默与DADS对SW480细胞迁移与侵袭的影响。结果 RT-PCR与Western blot显示,LIMK1-miR/SW480细胞LIMK1mNRA与蛋白表达明显下调,表明成功构建稳定沉默LIMK1基因的SW480细胞株。免疫组化和Western blot显示,45 mg.L-1DADS处理和沉默LIMK1组较未转染组与空载体组SW480细胞LIMK1蛋白和磷酸化LIMK1明显下调(P<0.05)。划痕实验和侵袭实验发现,沉默LIMK1或DADS处理SW480细胞的迁移与侵袭能力较未转染组与空载体组明显抑制(P<0.05)。而DADS处理沉默组抑制SW480细胞迁移和侵袭能力更为明显(P<0.05)。结论沉默LIMK1基因可抑制SW480细胞迁移与侵袭,增强DADS抑制SW480细胞迁移与侵袭作用。     

miR-106b对宫颈癌SiHa细胞运动侵袭功能的影响

目的 研究miR-106b对宫颈癌SiHa细胞转移侵袭功能的影响及作用机制.方法 SiHa细胞转染miR106b inhibitors和mimics,实时定量PCR检测转染效率,Transwell及划痕法检测细胞运动侵袭功能,分析E-cadherin 、PTEN、p-AKT(Ser473)蛋白表达情况.结果 miR-106b inhibitors和mimics改变SiHa细胞中miR-106b表达.转染miR-106b inhibitors后,细胞迁移数、移动距离减少(P＜0.05);PTEN及E-cadherin表达增强,p-AKT表达降低.转染miR-106b mimics后,细胞迁移数、移动距离增加(P＜0.05);PTEN及E-cadherin表达降低,p-AKT表达增强.结论 miR-106b可能通过E-cadherin/AKT、PTEN/AKT信号通路影响SiHa细胞运动侵袭功能.     

Six1通过PI3K通路抑制肺癌细胞A549的增殖和迁移

目的研究Six1基因对人肺癌细胞A549增殖和迁移能力的影响及其作用机制。方法将Six1的特异小干扰RNA序列转染入A549细胞中,应用Western印迹法检测Six1在A549细胞中的蛋白水平的表达情况,应用MTT法和细胞计数观察低表达Six1对A549细胞增殖的作用,应用划痕实验和transwell侵袭小室实验检测A549细胞迁移能力的变化情况,应用Western印迹法检测PI3K信号通路蛋白的表达变化。结果在A549细胞中,特异的小干扰RNA使Six1在蛋白水平表达降低,低表达Six1的A549细胞系增殖和迁移能力下降,p-AKT的蛋白表达降低。结论在A549细胞系中Six1通过PI3K通路抑制A549细胞的侵袭和转移能力。     

新的抗肿瘤药7-hydroxystaurosporine(UCN-01)抑制肿瘤浸润和转移

目的和方法研究7_hydroxystaurosporine(UCN_01)对转移性前列腺肿瘤DU_145细胞浸润和转移的影响。用体外转移和创伤法检查细胞浸润和转移 ,用Westernblotting 法检查蛋白质的表达。结果UCN_01在非毒性剂量下(100nmol·L -1)明显地抑制DU_145细胞浸润和转移。而且 ,UCN_01这种抗肿瘤浸润和转移功能与它增加细胞_细胞粘连分子E_cadherion的表达有关。结论这些实验首次证实UCN_01能抑制人前列腺肿瘤细胞的浸润和转移。UCN_01的临床应用可能更有效地控制前列腺肿瘤转移     

乳腺癌中神经细胞黏附分子的表达及临床意义

目的探讨神经细胞黏附分子（NCAM）在乳腺癌中的表达及临床意义。方法应用免疫组织化学方法检测NCAM在30例乳腺癌组织、癌旁组织及30例乳腺良性病变组织中的表达情况。应用酶联免疫吸附法（ELISA）测定NCAM在30例乳腺癌患者、30例乳腺良性病变患者及30例正常人群中的血清浓度并进行统计学分析。结果免疫组化结果：乳腺癌组织、癌旁组织及乳腺良性病变组织中NCAM阳性率分别为26.67%、3.33%、3.33%,NCAM在癌组织中表达明显高于癌旁组织及良性病变组织（P〈0.0125）,乳腺癌中有神经侵袭组的NCAM阳性率明显高于无神经侵袭组（P〈0.05）。ELISA结果：乳腺癌患者的NCAM血清浓度均显著高于良性病变组及正常人群组（P〈0.05）。结论神经细胞黏附分子在乳腺癌的发生发展中可能起到了重要作用。NCAM的高表达对于乳腺癌的神经侵袭和转移可能很重要。     

Modulation of peroxisome proliferator-activated receptor delta activity affects neural cell adhesion molecule and polysialyltransferase ST8SiaIV induction by teratogenic valproic acid analogs in F9 cell differentiation

It has been suggested that the teratogenic effects of the antiepileptic drug valproic acid (VPA) is reflected in vitro by the differentiation of F9 cells, activation of peroxisome proliferator-activated receptor delta (PPARdelta), and inhibition of histone deacetylases (HDACs). The aim of this study was to identify genes involved in the differentiation of F9 cells induced by VPA, teratogenic VPA derivatives, or the HDAC inhibitor trichostatin A (TSA) and to characterize the role of PPARdelta. Treatment of the cells with teratogenic VPA derivatives or TSA induced differentiation of F9 cells, mRNA, and protein expression of the neural cell adhesion molecule (NCAM) as well as activated the 5'-flanking region of the NCAM promoter, whereas nonteratogenic VPA derivatives had no effect at all. The polysialyltransferases [ST8SiaIV (PST1) and ST8SiaII] are responsible for the addition of polysialic acid (PSA) to NCAM. The mRNA expression of PST1 was highly induced by only teratogenic VPA derivatives and TSA. As shown by fluorescence-activated cell sorting analysis the level of PSA was higher after treatment of F9 cells with teratogenic VPA derivatives. It is interesting that overexpression of the PPARdelta but not PPARalpha or PPARgamma in F9 cells resulted in higher induction of NCAM mRNA and protein expression and of PST1 mRNA expression (and a higher PSA level) than in mock-transfected F9 cells. Furthermore, repression of PPARdelta activity in F9 cells inhibited these effects. We conclude that NCAM and PST1 are molecular markers in F9 cell differentiation caused by treatment with teratogenic VPA compounds or TSA and suggest that in addition to HDAC inhibition PPARdelta is involved in the signaling pathway.     

Valproic acid modulates NCAM polysialylation and polysialyltransferase mRNA expression in human tumor cells

Polysialic acid (PSA) is a dynamically regulated carbohydrate modification of the neural cell adhesion molecule NCAM, which has been linked to cancer development and dissemination. Two enzymes, the polysialyltransferases ST8SiaIV and ST8SiaII, are known to be involved in the polysialylation of NCAM. The antiepileptic drug valproic acid (VPA) is associated with anti-cancer activity. In this study, VPA blocked the adhesion of several neuroectodermal tumor cell lines to human umbilical vein endothelial cells. Furthermore, VPA induced intracellular PSA accumulation and enhanced expression of PSA-NCAM on the cell surface. Using a semiquantitative RT-PCR strategy, VPA was shown to up-regulate ST8SiaIV mRNA, whereas ST8SiaII mRNA was down-regulated by this compound. Our data indicate that increased expression of ST8SiaIV enables accelerated polysialylation of NCAM, which might be coupled to a loss of adhesive functions of tumor cells.     

Low-dose zoledronic acid reduces spinal cord metastasis in pulmonary adenocarcinoma with neuroendocrine differentiation

Zoledronic acid (ZOL), a nitrogen-containing compound, is effective in the treatment of skeletal disorders, but its long-term use in high doses gives rise to complications such as osteonecrosis. We aimed to investigate the effect of low-dose ZOL on the expression of the neural cell adhesion molecule (NCAM), which may be correlated with tumor growth and spinal cord metastasis in lung adenocarcinoma with neuroendocrine differentiation. First, we used the small hairpin RNA technique to directly knock down NCAM expression in cells of a murine lung adenocarcinoma line, line 1 cells, and found that the tumor cells generated showed lower invasive capacity, slower tumor growth, and lesser tendency for spinal cord metastasis than control cells. Further, ZOL decreased NCAM expression and invasiveness in line 1 tumor cells in vitro. Line 1/lacZ cells, a stable clone tagged with the lacZ gene, were introduced into mice, followed by ZOL treatment (1 μg/kg/weekly). Low-dose ZOL significantly reduced spinal cord metastasis probably through reduced NCAM expression in vivo. These findings indicated that NCAM is involved in tumor growth and spinal cord metastasis of lung adenocarcinoma with neuroendocrine differentiation. Treatment with low-dose ZOL can reduce NCAM expression that may contribute toward reduced spinal cord metastasis, suggesting that NCAM is an alternative therapeutic target and that the low-dose ZOL treatment protocol is a reasonable approach for its treatment.     

Effect of 7-hydroxystaurosporine on glioblastoma cell invasion and migration

AIM: To investigate the effect of 7-hydroxystaurosporine (UCN-01), a selective protein kinase C (PKC) inhibitor, on cell growth, migration, and invasion in invasive human glioblastoma U-87MG cells. METHODS: PKC activity was determined based on the PKC-catalyzed transfer of the (32)P-phosphate group from [g-(32)P]ATP into a PKC-specific peptide substrate. Cell viability was measured by MTT assay. Cell invasion and migration were evaluated by a Boyden chamber assay and scratch wound assay, respectively. Protein expression was analyzed using Western blot assay. The formation of 3-dimensional cellular aggregates was examined by a cell-cell aggregation assay. RESULTS: UCN-01 treatment resulted in concentration- and time-dependent inhibition of U-87MG cell growth at higher doses (>100 nmol/L), and reduced cell invasion and migration capability at less cytotoxic doses (<100 nmol/L). UCN-01 significantly repressed PKC activity. Consistent with this result, UCN-01 blocked cell invasion stimulated by phorbel 12-myristate-13-acetate (PMA) and ethanol (EtOH), 2 PKC activators. Enforced expression of the tumor suppressor genes BRCA1 and PTEN increased the anti-invasion potential of UCN-01. Exposure to UCN-01 caused a dose-dependent increase in cell adhesion molecule E-cadherin. The effect of UCN-01 on the formation of cell-cell aggregation was significantly reduced by the addition of an anti-E-cadherin antibody. CONCLUSION: UCN-01 inhibits the invasion and migration of human glioma cells. Accordingly, UCN-01 can have potential clinical applications for the treatment of human glioma metastasis.     

蝎毒多肽提取物体外抑制胰腺癌细胞侵袭转移及相关机制

目的研究蝎毒多肽提取物PESV对人高转移胰腺癌细胞MIA-PaCa2-3转移相关能力的影响及其作用的机制。为PESV在临床上治疗胰腺肿瘤提供理论根据。方法以高转移的人胰腺癌细胞系MIA-PaCa2-3为研究对象,通过细胞增殖实验(MTT法)观察在PESV影响下肿瘤细胞活性及增殖情况,通过细胞黏附实验、细胞划痕实验及Transwell体外侵袭实验检测PESV干预后细胞的黏附、侵袭及运动能力变化。免疫细胞化学实验及Western blot检测胰腺癌细胞的E-钙粘蛋白、纤粘蛋白和基质金属蛋白酶-9的表达。结果细胞增殖实验结果显示,PESV对胰腺癌细胞呈明显剂量及时间效应关系的增殖抑制作用,与对照组比较细胞增殖抑制作用明显(P<0.05);黏附实验表明PESV可降低胰腺癌细胞的黏附力,侵袭实验及划痕实验的结果表明PESV可以使细胞的侵袭力和运动能力下降,PESV(40mg.L-1)干预8h后,黏附力、侵袭力和运动能力的抑制率分别为(30.3±4.7)%、(42.1±3.7)%、(47.6±3.3)%,与对照组比较差异均有显著性(P<0.05);免疫细胞化学及Western blot实验检测均显示,PESV干预后,胰腺癌细胞E-钙粘蛋白阳性表达细胞数量上升,灰度值明显上调(P<0.05),FN及MMP-9蛋白阳性表达数量减少,灰度值明显下调(P<0.05)。结论PESV能够明显抑制胰腺癌细胞的增殖、黏附力、侵袭及运动能力,其机制可能是通过提高E-cad表达、降低FN和MMP-9表达而实现的。     

紫花牡荆素抑制小细胞肺癌NCI-H446细胞系肺癌干细胞样细胞侵袭和转移的研究

目的探讨紫花牡荆素(CAS)抑制人小细胞肺癌NCI-H446细胞系肺癌干细胞样细胞(LCSLCs)侵袭和转移作用。方法体外培养人小细胞肺癌NCI-H446细胞系细胞和LCSLCs。流式细胞术(FCM)检测CD133+表达。Trawell法检测LCSLCs侵袭和转移能力。Trawell法测定CAS对LCSLCs侵袭、转移能力的抑制作用。结果 LCSLCs呈现典型非黏附三维球状,并高表达CD133+。Trawell法结果显示LCSLCs具有很强的体外侵袭和转移能力,而CAS能显著抑制LCSLCs的体外侵袭和转移能力。结论 CAS能显著抑制LCSLCs的体外侵袭和转移能力。     

紫杉醇对人肝癌细胞SMMC-7721黏附及侵袭转移潜能的影响

目的探讨紫杉醇对人肝癌细胞株SMMC-7721的黏附及侵袭转移潜能的影响及机制。方法应用体外细胞．基质黏附实验检测不同浓度紫杉醇对SMMC-7721与Matrigel胶（人工基底膜胶）黏附性的影响：Transwell小室模型测定紫杉醇对细胞侵袭和迁移能力的影响;蛋白免疫印迹检测紫杉醇对细胞内黏着斑激酶（FAK）蛋白表达量的影响。结果不同浓度紫衫醇（10、20、40nmol／L）作用SMMC-7721细胞60min后,细胞与Matrigel胶黏附性下降;紫杉醇干预24h后,细胞的侵袭能力及迁移能力随紫杉醇浓度提高而明显减弱;细胞内FAK蛋白的表达量也随紫杉醇浓度增高而明显下降。结论紫杉醇能抑制肝癌细胞SMMC-7721的黏附及侵袭转移,其机制可能与其抑制肝癌细胞内FAK的表达相关。     

The selective 5-HT6 receptor antagonists SB-271046 and SB-399885 potentiate NCAM PSA immunolabeling of dentate granule cells, but not neurogenesis, in the hippocampal formation of mature Wistar rats

While there is now substantial evidence that 5-HT(6) antagonism leads to significantly improved cognitive ability, the mechanism(s) and/or pathway(s) involved are poorly understood. We have evaluated the consequence of chronic administration of the 5-HT(6) receptor antagonists SB-271046 and SB-399885 on neural cell adhesion molecule polysialylation state (NCAM PSA), a neuroplastic mechanism necessary for memory consolidation. Quantitative analysis of NCAM PSA immunopositive neurons in the dentate gyrus of drug-treated animals revealed a dose-dependent increase in polysialylated cell frequency following treatment with both SB-271046 and SB-399885. These effects could not be attributed to increased neurogenesis, as no difference in the rate of bromodeoxyuridine incorporation was apparent between the control and drug-treated groups. A substantial increase in the frequency of polysialylated cells in layer II of the entorhinal and perirhinal cortices was also observed, brain regions not previously associated with neurogenesis. Chronic treatment with SB-271046 or SB-399885 also significantly increased the activation of dentate polysialylation that is specific to learning. This effect does not occur with other cognition-enhancing drugs, such as tacrine, and this action potentially differentiates 5-HT(6) receptor antagonism as an unique neuroplastic mechanism for cognitive processes which may slow or reverse age/neurodegenerative related memory deficits.