STAT3基因沉默降低小鼠乳腺癌细胞的体外迁移能力

信号转导子与转录活化子3(STAT3)是一个具有信号转导和转录调控双重功能的转录因子,有文献报道STAT3在乳腺癌中的表达显著升高,并能促进乳腺癌的转移。为了深入探索STAT3在肿瘤发生发展中的作用和影响乳腺癌转移的分子机制,采用RNA干扰技术在小鼠乳腺癌细胞株4T1中沉默STAT3的表达。MTT实验结果显示STAT3沉默对4T1细胞的增殖能力没有影响;细胞迁移实验结果表明STAT3表达被沉默后4T1细胞的迁移能力明显被抑制;定量PCR结果显示,STAT3基因沉默后4T1细胞中VEGF和IL-6的mRNA水平下降,E-cadherin表达上升,mosin表达下降;信号通路检测显示STAT3基因表达沉默后MAPK的活化明显降低。研究表明STAT3在小鼠乳腺癌细胞的迁移过程中发挥重要作用,为以STAT3基因为靶向的治疗提供了一定的实验依据。     

N-乙酰氨基葡萄糖转移酶-V过表达对人肝癌细胞迁移及粘附分子表达的影响

为了探讨转染N-乙酰氨基葡萄糖转移酶Ⅴ(GnT-V)正义cDNA后对7721细胞迁移影响及其机制,我们研究了GnT-V/7721和pcDNA3/7721(对照组)两株细胞的迁移力及其与侵袭转移能力密切相关的细胞表面重要粘附分子整合蛋白和E-钙粘蛋白的表达情况.通过琼脂滴法检测两株细胞的迁移力;间接免疫荧光法测定细胞表面整合蛋白α5和β1的含量及用RT-PCR方法检测细胞整合蛋白α5和β1的mRNA水平;免疫细胞化学ABC法检测了细胞E-钙粘蛋白表达水平;Western杂交方法检测β-连环蛋白含量.结果发现,7721细胞经转染GnT-V cDNA后,迁移力明显增高;整合蛋白α5亚基的含量比对照组增加2.9倍;β1亚基未见明显变化.α5亚基mRNA水平为对照细胞的2.1倍,β1亚基的mRNA含量无明显改变.GnT-V/7721细胞E-钙粘蛋白及β-连环蛋白表达也有不同程度的升高.本文结果提示与N-糖链加工有关的GnT-V过表达,可促进7721细胞表面整合蛋白的表达以及E-钙粘蛋白β-连环蛋白的表达,从而增加肿瘤细胞的迁移能力.     

糖基因在髓性白血病耐药性中特征性改变

目的通过研究糖基因在髓性白血病中的差异表达,明确这些糖基因与白血病耐药的相关性,从而为预测和诊断髓性白血病耐药性,寻求逆转药物提供新策略和靶点。方法采用real-time PCR技术筛选髓性白血病细胞及其耐药细胞株中差异表达的糖基因,筛选出两组细胞差异表达3倍以上的糖基因,初步探索糖基因在髓性白血病耐药性中的特征性改变;采用流式细胞仪分析髓性白血病耐药细胞株与多种FITC标记植物凝集素的结合能力,表征比较细胞膜表面糖链的特征。结果 12个糖基因在NB4和NB4/ADR细胞株中表达具有显著的差异;高表达的糖基因与FITC标记植物凝集素的结合能力增强。结论髓性白血病细胞及其耐药细胞株中糖基因、细胞膜表面糖链特征均有显著差异,这些特征性改变与白血病多药耐药具有相关性。     

GPRC6A过表达前列腺癌细胞株构建及EMT相关研究

目的:构建GPRC6A基因过表达的前列腺癌LncapC4-2细胞株,检测细胞迁移和侵袭能力改变及EMT相关基因表达,为研究GPRC6A介导的EMT在前列腺癌发生发展过程中的作用奠定基础。方法:构建过表达GPRC6A的慢病毒载体pCDH-GPRC6A,人胚肾293T细胞包被病毒并感染Lncap C4-2细胞株,Puromycin筛选获得稳定过表达GPRC6A的细胞株。RT-PCR和Western blot验证细胞GPRC6A的过表达情况。划痕和transwell实验检测细胞迁移和侵袭能力,qPCR检测EMT相关基因表达。结果:成功构建GPRC6A表达的慢病毒载体,RT-PCR和Western blot检测LncapC4-2 GPRC6A过表达细胞株中GPRC6A的mRNA和蛋白质表达增高,与对照相比较,在过表达GPRC6A的LncapC4-2细胞中,细胞迁移和侵袭能力增强,并且EMT相关基因E-cadherin表达降低而SNAIL表达增高。结论:成功构建过表达的GPRC6A的前列腺癌细胞株,肿瘤细胞中的GPRC6A可能通过增强细胞迁移和侵袭能力并促进细胞EMT而参与前列腺癌发生发展过程。     

与骨髓基质细胞共培养前后的人急性髓系白血病细胞中N-糖链变化分析

骨髓基质细胞是骨髓微环境的重要组成部分,在促进造血干细胞生成血细胞的各个环节中起重要作用。作为细胞结构重要组成部分的糖链(如N-糖链),广泛参与了细胞之间的黏附、分化、增殖及信号转导等过程。为了探究骨髓微环境对血液细胞表面N-糖链变化的影响,该研究使用人骨髓来源的基质细胞HS27a与急性髓系白血病细胞KG1a共培养的体外模型,利用基质辅助激光解析电离飞行时间质谱(matrix-assistted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)技术从糖组学角度比较了与骨髓基质细胞共培养前后的KG1a细胞中N-糖链变化,并结合实时荧光定量PCR(Real-time PCR)和细胞凝集素染色对质谱结果加以验证。结果显示,与HS27a共培养后的KG1a中高甘露糖型Man10Glc NAc2Asn结构N-糖链相对强度降低,平分型、四天线复杂型和岩藻糖化的N-糖链相对强度增加,合成相应糖链的MGAT5[mannosyl(α-1,6)-glycoproteinβ-1,6-N-acetyl-glucosaminyltransferase]、MGAT3(mannosyl-glycoproteinβ-1,4-N-acetylglucosaminyltransferase)和FUT8(fucosyltransferase 8)基因水平表达上调。细胞染色结果证明,共培养后的KG1a对凝集素PHA-E+L(Phaseolus vulgaris Agglutinin)和LCA(Lens culinaris)的亲和作用增强。该研究从糖生物学角度揭示了骨髓来源的基质细胞对急性髓系白血病细胞在N-糖链变化上的影响。未来的研究重点将放在共培养前后急性髓系白血病细胞中显著差异糖链的生物学功能上,以了解N-糖链在骨髓微环境中所发挥的重要作用。     

整合素β1表达上调对肺癌细胞迁移和粘附能力的影响

目的:构建整合素β1的全基因表达载体,并探讨上调整合素β1蛋白表达对肺癌细胞生物学行为的影响.方法:根据GenBank数据库提供的整合素β1基因核苷酸序列,构建整合素β1的全基因表达载体,同时将空载体pCDNA3.1作为阴性对照.将载体转入感受态大肠杆菌,挑选阳性克隆,抽取重组栽体.2种重组栽体转染非小细胞肺癌细胞株PC-9细胞,用G418筛选后挑选单克隆并扩增获得稳定株.荧光显微镜、Real-time RT-PCR、Western blot检测整合素β1的基因及蛋白水平的表达情况.细胞划痕试验和粘附试验比较整合素β1对细胞迁移、粘附能力的影响.结果:G418筛选出稳定转染整合素β1全基因表达栽体和空载体的细胞,分别命名为PC-9/D6和PC-9/PCD.荧光显微镜见满视野带绿色荧光的细胞,PC-9/D6细胞整合素β1的mRNA、蛋白表达明显高于对照的PC-9/PCD细胞及母细胞PC-9.划痕试验和粘附试验表明整合素β1过表达的细胞株的迁移和粘附能力明显提高.结论:成功转染并筛选出整合素β1过表达细胞株,整合素β1过表达的细胞株的迁移和粘附能力明显升高.     

长链非编码RNA SPRY4-IT1对髓母细胞瘤增殖及侵袭转移的影响

目的：探讨长链非编码RNA SPRY4-IT1（LncRNA）对髓母细胞瘤增殖及侵袭转移的影响。方法：髓母细胞瘤Daoy细胞分为对照组和si-SPRY4-IT1组,分别利用脂质体Lipofectamine 2000将阴性对照荧光序列和SPRY4-IT1-siRNA转入细胞中,采用Real-time PCR检测转染后各组细胞中SPRY4-IT1的表达情况,CCK-8实验及平板克隆形成实验检测细胞增殖能力的变化,以细胞体外侵袭、迁移实验分别检测细胞侵袭、迁移能力的变化,Western blot检测SPRY4-IT1对基质金属蛋白酶（MMP）-2和MMP-9蛋白的影响。结果：si-SPRY4-IT1组SPRY4-IT1 mRNA表达水平、细胞体外增殖能力、细胞侵袭、迁移能力、细胞MMP-2蛋白表达均较对照组明显降低（P<0.05）,而MMP-9蛋白表达未见明显变化。结论：干扰长链非编码RNA SPRY4-IT1在髓母细胞瘤Daoy细胞中的表达能显著抑制细胞的增殖、侵袭和迁移能力。     

过表达外源FUT8增加乳腺癌细胞的迁移和侵袭能力

核心岩藻糖的修饰被认为是对糖蛋白进行转录后加工和功能调控的一种重要方式, 与肿瘤的发生发展密切相关, 但其在乳腺癌恶性转化过程中所发挥的生物学功能尚不明确. 本文构建了α1,6-岩藻糖基移转酶（FUT8）基因真核表达载体,将其转染人乳腺癌细胞Bcap-37, 实时PCR及Western印迹检测FUT8 mRNA和蛋白质表达, 通过细胞划痕实验和Transwell小室观察FUT8过表达对细胞体外迁移、侵袭能力的影响, 免疫共沉淀和Western印迹检测肿瘤细胞整合素、基质金属蛋白酶（matrix metalloproteinases, MMPs）家族相关蛋白质表达水平变化. 结果显示,外源FUT8基因在mRNA水平和蛋白质水平的表达均显著增加, FUT8过表达组细胞迁移和侵袭能力明显增强;上调FUT8基因表达可使Bcap-37细胞中核心岩藻糖基化的整合素-α3β1、MMP-2和MMP-9在蛋白质水平呈不同程度升高. 上述研究表明,FUT8可通过核心岩藻糖化修饰正性调控整合素-α3β1的功能, 诱导MMP-2、MMP-9的表达, 促进乳腺癌细胞的迁移和侵袭.     

硫化砷对人结肠癌细胞株HCT116生长及迁移能力的影响

目的:研究硫化砷对人结肠癌细胞株HCT116迁移能力的影响及其作用机制.方法:以HCT116细胞为研究对象,通过MTT法观察硫化砷对肿瘤细胞活性及增殖能力的影响,通过划痕实验观察硫化砷处理后细胞的迁移能力.Western B1ot、Real-time PCR检测硫化砷处理前后细胞内E-钙粘素、P53、Notch1蛋白的表达及相应mRNA水平的变化.结果:硫化砷对HCT116细胞有抑制增殖的作用,此作用随着剂量的增加而增强.选取无毒剂量的硫化砷(1 μM)处理HCT116细胞24h后,细胞的迁移能力降低了52.00％±7.55％.Western Blot及Real-time PCR结果显示,硫化砷处理后,HCT116细胞内的E-钙粘素蛋白水平及mRNA水平均明显升高,P53蛋白水平增高,但对Notch1蛋白及mRNA水平无明显影响.结论:一定剂量范围的硫化砷能抑制HCT116细胞增殖,并能降低其迁移能力.硫化砷降低HCT116细胞的迁移能力,其机制可能是通过上调P53蛋白的水平,从而增高E-钙粘素的表达实现的.     

核不均一核糖核蛋白R基因沉默抑制非小细胞肺癌细胞恶性转化

核不均一核糖核蛋白R(hnRNPR)是一种与mRNA生物学功能密切相关的RNA结合蛋白质,与多种肿瘤细胞的恶性转化相关。然而,在非小细胞肺癌(NSCLC)中的作用机制尚不清楚。本研究通过检索公共数据库发现,hnRNPR蛋白主要在肺癌细胞核中表达,hnRNPR mRNA在非小细胞肺癌组织中高表达,并且与肺腺癌患者的生存率呈负相关;hnRNPR的表达与非小细胞肺癌患者的性别、T分期显著相关(P<0.05)。构建hnRNPR基因沉默的非小细胞肺癌稳定细胞株,检测细胞功能变化,结果显示,hnRNPR基因沉默抑制了细胞增殖、迁移和侵袭能力以及上皮-间质转化(EMT),并将细胞周期阻滞在G₁期(P<0.01)。生物信息学分析显示,非小细胞肺癌中hnRNPR基因与9 310个基因的表达正相关(皮尔逊相关系数>0,P<0.05),与10 680个基因的表达负相关(皮尔逊相关系数<0,P<0.05)。综上所述,hnRNPR在非小细胞肺癌中高表达,可能作为剪接体的组分,通过调节相关基因的表达,促进了NSCLC细胞的恶性转化。     

Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis

Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo - the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, beta-1,6-GlcNAc-branching, alpha-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(beta-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis.     

Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.     

Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells

Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.     

Identification and phenotypic characterization of a subpopulation of T84 human colon cancer cells, after selection on activated endothelial cells

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of the cells in the host connective tissue. The differences in metastatic ability could be attributed to properties intrinsic of the various primary tumor types. Thus, the clonal selection of neoplastic cells during cancer progression results in cells better equipped for survival and formation of colonies in secondary sites. A cell line (T84SF) exhibiting an altered phenotypic appearance was selected from a colon cancer cell line (T84) by repetitive plating on TNFalpha-activated human endothelial cells and subsequent selection for adherent cells. Cell growth, motility, chemoinvasive abilities, tyrosine phosphorylation signaling, and the metastasis formation in nude mice of the two cell lines was compared. T84SF cells displayed in vitro an higher proliferation rate and a more invasive behavior compared to the parental cells while formed in vivo a greater number of metastatic colonies in nude mice. As concerns the signaling underlying the phenotypes of the selected cells, we examined the general tyrosine phosphorylation levels in both cell lines. Our results indicate that T84SF have an increased basal tyrosine phosphorylation of several proteins among which src kinase was identified. Treatment of cells with a specific inhibitor of src activity caused a greater in vitro inhibition of proliferation and invasive properties of T84 parental cells with respect to T84SF cells and diminished metastasis formation in vivo. Altogether, these data provide evidences that this new cell line may be valuable for identifying molecular mechanisms involved in the metastatic progression of colon cancer.     

Down-regulation of N-acetylglucosaminyltransferase V by tumorigenesis- or metastasis-suppressor gene and its relation to metastatic potential of human hepatocarcinoma cells

The effects of transfection of the metastasis suppressor gene nm23-H1 and cell-cycle related tumor-suppressor gene p16 on the activity of N-acetylglucosaminyltransferase V (GnT-V) and their relations to cancer metastatic potential were investigated. After transfection of nm23-H1 into 7721 human hepatocarcinoma cells and A549 human lung cancer cells, the activities of GnT-V were decreased by 28%-42% in the cells. In contrast, when p16 was transfected into these two cell lines, the decrease of GnT-V activity was only observed in A549 cells. This was probably to be due to the obvious expression of p16 gene in parental 7721 cells and the deletion of p16 in A549 cells. The decrease of GnT-V mRNA was only observed in nm23-H1-transfected cells, but not in p16-transfected A549 cells, suggesting that these two genes regulated GnT-V via different mechanisms. Horseradish peroxidase (HRP)-lectin staining showed that the 7721 cells transfected with nm23-H1 or the A549 cells transfected with p16 displayed a decreased intensity with HRP-leucoagglutinating phytohemagglutinin and increased intensity with HRP-concanavalin A, indicating the decline of beta1,6 N-acetylglucosamine branching structure on the asparagine-linked glycans of cell-surface and intracellular glycoproteins. The nm23-H1 transfected 7721 cells also displayed some changes in metastasis-related phenotypes, including the increase in cell adhesion to fibronectin (Fn), the decline in cell adhesion to laminin (Ln), and the decreased cell migration and invasion through matrigel. Transfection of antisense GnT-V cDNA into 7721 cells resulted in a decrease of GnT-V activity, an increase of cell adhesion to Fn or Ln, and a decrease in cell migration and invasion through matrigel. These phenotypes bore similarity to those of the 7721 cells transfected with nm23-H1. Our findings indicate that the down-regulation of GnT-V by nm23-H1 contributes to the alterations in metastasis-related phenotypes, and is an important molecular mechanism of metastasis suppression mediated by nm23-H1.     

Tissue transglutaminase expression is necessary for adhesion,metastatic potential and cancer stemness of renal cell carcinoma

Tissue transglutaminase (TG2) is the ubiquitously expressed member of transglutaminase family and shown to play a critical role in the development and progression of drug resistance malignancies. We have previously showed the association of TG2 upregulation with progression and metastasis of renal cell carcinoma (RCC) and low disease-free survival. In the present study we further investigate the role of TG2 in cell adhesion, migration and invasion of RCC by silencing TG2 expression in Caki-2 and A-498 primary site and Caki-1 and ACHN metastatic site RCC cell lines. Downregulation of TG2 expression led up to a 60% decrease in actin stress fiber formation and adhesion to β 1 integrin (ITGB1) substrates fibronectin, collagen type I and laminin in both primary and metastatic site RCC cell lines. In addition, treatment with siRNAs against TG2 impaired the migration capacity and cellular invasiveness of ITGB1 substrates in all 4 RCC cell lines. Lastly, the knockdown of TG2 in metastatic Caki-1 cells diminished the expression of CD44, CD73-and CD105 cancer stem cell-like markers. We conclude, for the first time, that TG2 expression is critical for cancer cell adhesion, migration, invasiveness and cancer cell-stemness during RCC progression and dissemination. Therefore, combined targeting of TG2 with drugs widely used in the treatment of RCC may be a promising therapeutic strategy for RCC.