关键词： cell proliferation   glioma   IQGAP1   metastasis   signaling pathway  

摘要： IQ motif containing GTPase activating protein 1 (IQGAP1) is a scaffold protein, which is aberrantly expressed in several tumor types and is closely associated with the development, metastasis and prognosis of cancer. Several studies have demonstrated that IQGAP1 has broad prospects in the basic and clinical research of tumors. The present study aimed to explore the effects of IQGAP1‑small interfering (si) NA on the proliferation and metastasis of U251 and U373 glioma cell lines, which markedly expressed IQGAP1. The human glioma cell lines (U251 and U373) were transfected with siRNA and transfection efficacy was confirmed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. Cell proliferation was detected using the Cell Counting kit‑8, and cell metastasis capabilities were detected using cell adhesion, migration and invasion assays. In addition, the expression levels of several tumor‑associated genes were determined by RT‑qPCR and western blotting. The results indicated that IQGAP1 was expressed at higher levels in glioma tissues compared with in normal brain tissues. IQGAP1‑siRNA significantly inhibited cell proliferation, and cell adhesion, migration and invasion. Furthermore, the expression levels of matrix metalloproteinase (MMP)2, Snail, MMP9, fibronectin 1 and Twist were suppressed, and E‑cadherin was upregulated in response to siRNA‑IQGAP1. The present study identified the function of IQGAP1 in glioma cell biology, and indicated that it may be considered a novel target for glioma treatment.

关键词： Akt PKB   insulin   insulin receptor   insulin receptor substrate 1 (IRS-1)   insulin resistance   mitogen-activated protein kinase (MAPK)   phosphatidylinositide 3-kinase (PI 3-kinase)   IQGAP1  

摘要： Insulin binds to the insulin receptor (IR) and induces tyrosine phosphorylation of the receptor and insulin receptor substrate-1 (IRS-1), leading to activation of the PKB/Akt and MAPK/ERK pathways. IQGAP1 is a scaffold protein that interacts with multiple binding partners and integrates diverse signaling cascades. Here we show that IQGAP1 associates with both IR and IRS-1 and influences insulin action. In vitro analysis with pure proteins revealed that the IQ region of IQGAP1 binds directly to the intracellular domain of IR. Similarly, the phosphotyrosine-binding domain of IRS-1 mediates a direct interaction with the C-terminal tail of IQGAP1. Consistent with these observations, both IR and IRS-1 co-immunoprecipitated with IQGAP1 from cells. Investigation of the functional effects of the interactions revealed that in the absence of IQGAP1, insulin-stimulated phosphorylation of Akt and ERK, as well as the association of phosphatidylinositol 3-kinase with IRS-1, were significantly decreased. Importantly, loss of IQGAP1 results in impaired insulin signaling and glucose homeostasis in vivo. Collectively, these data reveal that IQGAP1 is a scaffold for IR and IRS-1 and implicate IQGAP1 as a participant in insulin signaling.

关键词： 甲状腺癌   miRNA   靶基因   增殖  

摘要： 目的探讨miR-124对甲状腺癌细胞的作用机制。方法通过RT-PCR检测人甲状腺癌细胞K1、BCPAP、TPC-1和人甲状腺细胞Nthy-ori 3-1中miR-124的表达水平。MTT法检测甲状腺癌细胞K1转染miR-124 mimics、mimics control后的细胞存活率。根据靶基因预测软件The miRBase、Target Scan、Pic Tar预测miR-124的靶基因IQGAP1,构建wt-IQGAP1和mut-IQGAP1,采用双荧光素酶活性检测鉴定靶基因的正确性。Western印迹检测转染miR-124 mimics、mimics control后甲状腺癌细胞K1中IQGAP1的表达情况。甲状腺癌细胞K1中转染si IQGAP1和si IQGAP1 control,用MTT检测细胞增殖能力,Western印迹检测细胞中IQGAP1蛋白表达含量。结果甲状腺癌细胞K1、BCPAP、TPC-1中的miR-124的相对表达量与甲状腺细胞相比差异显著(P<0.05),三种甲状腺癌细胞中K1细胞的表达量最低。转染后12 h内,空白组、miR-124 mimics组和mimics control组的甲状腺癌K1细胞的存活数量差异不显著(P>0.05),从转染后12 h开始,miR-124 mimics组与对照组、mimics control组相比,甲状腺癌细胞生长抑制明显。预测miR-124的靶基因可能为IQGAP1,转染miR-124 mimics野生型IQGAP1中荧光素酶活性比突变型IQGAP1中荧光素酶活性显著降低,差异显著(P<0.05)。mimics control和wt-IQGAP1和mut-IQGAP1共转染组比较无明显差异(P>0.05)。转染miR-124 mimics组甲状腺癌K1细胞IQGAP1蛋白明显下降,miR-124负调控IQGAP1的表达。抑制甲状腺癌K1细胞中IQGAP1基因,细胞增殖能力减弱,细胞中IQGAP1蛋白表达减弱。结论 miR-124在甲状腺癌细胞中表达下调,miR-124可以抑制甲状腺癌细胞的增殖分化,其通过负调控靶基因IQGAP1抑制癌细胞增殖分化。

关键词： HECTD1   Cell movement   Cell spreading   Focal adhesions   IQGAP1   Ubiquitination  

摘要： Background: Cell migration including collective cell movement and individual cell migration are crucial factors in embryogenesis. During the spreading/migration of cells, several types of adhesive structures physically interacting with the extracellular matrix (ECM) or with another cell have been described and the formation and maturation of adhesion structures are coordinated, however the molecular pathways involved are still not fully understood. Results: We generated a mouse embryonic fibroblast line (MEF) from homozygous mutant (Hectd1(R/R), Hectd1(Gt(RRC200))) mouse of the E3 ubiquitin ligase for inhibin B receptor (Hectd1). Detailed examination of cell motion on MEF cells demonstrated that loss of Hectd1 resulted in accelerated cell spreading and migration but impaired directionality of migration. In Hectd1(R/R) cells paxillin and zyxin were largely mis-localized, whereas their expression levels were unchanged. In addition the formation of focal adhesions (FAs) was impaired and the focal complexes (FXs) were increased. We further identified HECTD1 as a key regulator of IQGAP1. IQGAP1 co-localized together with HECTD1 in the leading edge of cells. HECTD1 interacted with IQGAP1 and regulated its degradation through ubiquitination. Over-expression of IQGAP1 in control MEF phenocopied the spreading and migration defects of Hectd1(R/R) cells. In contrast, siRNA-mediated knockdown of IQGAP1 rescued the defects in cellular movement of Hectd1(R/R) cells. Conclusions: The E3 ligase activity of Hectd1 regulates the protein level of IQGAP1 through ubiquitination and therefore mediates the dynamics of FXs including the recruitment of paxillin and actinin. IQGAP1 is one of the effectors of HECTD1.

关键词： IQGAP1   glioblastoma multiforme (GBM)   scaffold protein   podosome/invadosome  

摘要： Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor. GBM is formed by a very heterogeneous astrocyte population, neurons, neovascularization and infiltrating myeloid cells (microglia and monocyte derived macrophages). The IQGAP1 scaffold protein interacts with components of the cytoskeleton, cell adhesion molecules, and several signaling molecules to regulate cell morphology and motility, cell cycle and other cellular functions. IQGAP1 overexpression and delocalization has been observed in several tumors, suggesting a role for this protein in cell proliferation, transformation and invasion. IQGAP1 has been identified as a marker of amplifying cancer cells in GBMs. To determine the involvement of IQGAP1 in the onco-biology of GBM, we performed immunohistochemical confocal microscopic analysis of the IQGAP1 protein in human GBM tissue samples using cell type-specific markers. IQGAP1 immunostaining and subcellular localization was heterogeneous;the protein was located in the plasma membrane and, at variable levels, in nucleus and/or cytosol. Moreover, IQGAP1 positive staining was found in podosome/invadopodia-like structures. IQGAP1(+) staining was observed in neurons (Map2(+) cells), in cancer stem cells (CSC;nestin(+)) and in several macrophages (CD31(+) or Iba1(+)). Our results indicate that the IQGAP1 protein is involved in normal cell physiology as well as oncologic processes.

关键词： IQGAP1   足细胞   细胞骨架重组   嘌呤霉素  

摘要： 目的:通过体外培养小鼠足细胞进行实验研究,采用分子生物学研究方法探讨IQGAP1对足细胞细胞骨架重组的调节作用。方法:嘌呤霉素(PAN)诱导的微小病变性肾病动物模型在临床表现和病理改变上与人类相比具有一定的相似性,而微小病变性肾病最具特征性的病理改变就是足突广泛融合,足细胞细胞骨架蛋白的重组在足突融合中起到关键作用。使用PAN50mg刺激小鼠足细胞后,运用实时定量real-timePCR和WesternBlot印迹法分别检测细胞内IQGAP1 mRNA和蛋白表达水平,激光共聚焦显微镜观察足细胞骨架F-actin的分布,F-actin肌动蛋白评分系统(CFS)对骨架重组进行定量分析,并进一步利用质粒转染技术和基因沉默技术分别观察上调和下调足细胞IQGAPI表达后对PAN诱导的足细胞骨架重组的影响。运用SPSS22.5统计软件处理和分析数据,计量资料采用均数土标准差(x±s)表示,参数资料采用单因素方差分析,LSD法进行组间比较,偏相关分析表示两变量之间的相关性,P<0.05表示差异有统计学意义。结果:重复三次试验,均顺利进行。(1)与对照组相比,PAN刺激组足细胞内IQGAP1 mRNA和蛋白表达明显下降(P<0.05),足细胞F-actin排列紊乱,CFS评分明显升高;(2)IQGAP1 siRNA进一步抑制IQGAP1表达后,PAN引起的上述足细胞F-actin重组更加明显(P<0.05);CFS评分更加升高(3)转染pCantag-myc-IQGAP1质粒促进IQGAP1表达后,PAN诱导的足细胞F-actin排列紊乱显著减轻,CFS评分明显下降(P<0.05)。结论:IQGAP1能抑制嘌呤霉素诱导的足细胞骨架重组,对维持足细胞正常骨架结构起着重要作用。

关键词： IQGAP1   thyroid cancer   proliferation   epithelial-mesenchymal transition   Wnt/beta-catenin  

摘要： Background: Thyroid cancer is the most common endocrine malignant disease with a high incidence rate. The expression of IQGAP1 is upregulated in various cancers, including thyroid cancer. However, the role and underlying mechanism of IQGAP1 in thyroid cancer are still not clear. Materials and methods: The expression of IQGAP1 in thyroid cancer tissues and cells was determined by reverse transcription polymerase chain reaction and Western blot analysis. Cells were transfected with different siRNAs using Lipofectamine 2000 or were treated with various concentrations of XAV939. The effects of IQGAP1 knockdown on proliferation and epithelial-mesenchymal transition (EMT) of thyroid cancer cells were determined by MTT assay and Western blot analysis. Animal experiments were performed to investigate the effects of IQGAP1 knockdown on the growth of tumors in vivo. Results: High IQGAP1 expression is found in thyroid cancer tissues and cells. Knockdown of IQGAP1 had inhibitory effects on cell proliferation and EMT, as well as on the Wnt/beta-catenin pathway. Additionally, inactivation of the Wnt/beta-catenin pathway by XAV939 or si-beta-catenin suppressed cell proliferation and EMT. Furthermore, suppression of the Wnt/beta-catenin pathway reversed the positive effects of pcDNA-IQGAP1 on cell proliferation and EMT in vitro. Moreover, downregulation of IQGAP1 suppressed tumor growth and EMT in SW579 tumor xenografts through the Wnt/a-catenin pathway in vivo. Conclusion: Our study demonstrated that knockdown of IQGAP1 inhibited cell proliferation and EMT through blocking the Wnt/a-catenin pathway in thyroid cancer.

关键词： 结直肠癌   miR-124   IQ结构域GTP酶激活蛋白1   细胞增殖   肿瘤转移  

摘要： 目的:结直肠癌是由正常细胞突变导致其增殖失控后肿瘤细胞扩散引起的疾病,其形成是一个复杂的多因素共同作用的结果,涉及多种基因、多种途径、多个步骤。据相关研究报道,结直肠癌的发病率及死亡率呈逐年升高趋势。microRNA是一类细胞内源性的非编码小RNA,在人体组织中广泛表达,参与对增殖、分化、死亡等基本细胞过程的调控。在肿瘤发展过程中,异常表达的miRNAs导致功能基因的表达失调,是引起肿瘤恶性生物学行为的重要原因。miR-124被发现在多种肿瘤中表达降低或缺失。体内外研究发现,恢复miR-124的表达能够抑制肿瘤细胞的生长、侵袭、EMT等恶性行为,提示其作为抑癌性miRNA的功能。因此,鉴定miR-124的靶基因是揭示以miR-124为中心的肿瘤调控网络的必要工作。IQGAP1(IQ Motif Containing GTPase Activating Protein 1)是 IQGAP 支架蛋白家族的一员,广泛表达于人体组织器官中。IQGAP1能与actin、E-cadherin等细胞骨架蛋白相互结合,在调节细胞粘附、细胞相互作用、细胞迁移等生物学行为中起重要作用。此外,IQGAP1还在MAPK、Wnt/β-catenin等信号途径的级联传导中起促进作用,影响细胞的增殖和分化。目前,在包括结直肠癌在内的多种肿瘤中均发现IQGAP1的异常高表达,且其表达水平与肿瘤的转移程度呈正相关,提示其在肿瘤进展中的重要作用。因此,我们拟探讨miR-124是否通过靶向调控IQGAP1而影响结直肠癌细胞的增殖和侵袭转移。方法:Real-time PCR检测结直肠癌组织中miR-124以及IQGAP1的表达水平,Western blot检测IQGAP1及其下游相关基因的蛋白表达水平,慢病毒感染方式建立稳定敲低IQGAP1的结直肠癌细胞株,CCK-8实验评价细胞的增殖能力,细胞克隆形成试验评价细胞生长能力,划痕实验评价细胞的迁移能力,Transwell侵袭实验评价细胞侵袭能力。生物信息学分析预测miR-124下游调控的靶基因,双荧光素酶报告试验进一步验证潜在靶向关系。结果:miR-124在结直肠癌中表达异常降低,而IQGAP1表达异常升高。恢复miR-124的表达能够显著降低ERK1/2的磷酸化水平并抑制β-catenin和IQGAP1的表达,体外功能试验证实恢复miR-124的表达均能够显著抑制结直肠癌细胞的增殖、克隆形成、侵袭和转移。双荧光素酶报告系统证实miR-124可以靶向结合IQGAP1。稳定敲低IQGAP1表达后证实敲低IQGAP1同样能够显著降低ERK1/2的磷酸化水平和抑制β-catenin的表达,体外功能试验证实敲低IQGAP1也能够显著抑制结直肠癌细胞的增殖、克隆形成、侵袭和转移。结论:在结直肠癌中miR-124通过影响MAPK以及Wnt/β-catenin信号的活性发挥抑癌作用;敲低IQGAP1同样能够通过影响MAPK以及Wnt/β-catenin信号的活性发挥抑癌作用;结合miR-124靶向结合IQGAP1的双荧光素酶报告结果,我们明确miR-124通过靶向调控IQGAP1抑制结直肠癌细胞的增殖和侵袭转移。

关键词： IQGAP1   食管癌   血管生成   机制   p-VEGFR2  

摘要： 目的:1.构建IQGAP1基因过表达和基因干扰的稳转细胞系及其对照细胞系。2.通过体内外实验分别检测IQGAP1基因对血管生成的影响。3.研究IQGAP1对血管生成影响的分子机制。方法:1.利用Lipofectamine 2000将绿色荧光蛋白(green fluorescent protein,GFP)标记的IQGAP1过表达质粒和它所对应的对照质粒分别转染到人食管癌细胞EC9706,用G418耐药单克隆细胞的筛选方法,等到稳定生长后,荧光显微镜下观察其细胞定位,并通过Western blot检测GFP-IQGAP1融合蛋白的表达,确定IQGAP1过表达的稳转细胞系是否构建成功。2.利用Lipofectamine 2000将IQGAP1干扰质粒及其对照质粒转染进人食管癌细胞KYSE150内,采用G418筛选耐药克隆,待其稳定生长后,荧光显微镜下观察KYSE150细胞GFP的表达,并通过Western blot检测对照组与干扰组IQGAP1蛋白的表达情况,确定IQGAP1干扰的稳转细胞系是否构建成功。3.分别通过体外HUVECs小管形成实验和体内的鸡胚尿囊膜实验来检测IQGAP1基因表达对肿瘤血管生成的影响。4.采用Western blot技术研究IQGAP1对食管癌细胞中VEGFA与其受体VEGFR2、p-VEGFR2血管生成相关因子表达的影响。5.通过Western blot技术研究IQGAP1对Akt和ERK活化的影响。6.在IQGAP1过表达组细胞中加入Akt和ERK抑制剂进行干预处理后,观察抑制剂对VEGFA和p-VEGFR2蛋白表达的影响及对血管生成的影响。结果:1.荧光显微镜下观察发现,在转染含GFP-IQGAP1重组质粒的EC9706细胞中观察到其为胞质定位;Western blot结果显示,在IQGAP1过表达细胞中有GFP-IQGAP1融合蛋白的表达,而在其对照组内则没有,说明IQGAP1过表达的稳转细胞系已构建成功。2.在荧光显微镜下可观察到,GFP在转染IQGAP1干扰质粒及对照质粒的KYSE150细胞中为全细胞定位;对支架蛋白IQGAP1在干扰组细胞内的表达量与其在对照组细胞内的表达量通过Western blot进行比较,发现其表达量显著降低,有统计学意义(P<0.0001)。3.体外HUVECs小管形成的实验结果显示,与对照组相比较,IQGAP1过表达组小管生成数更多,而且所生成小管的管腔结构更加完整,两者差异有统计学意义(P<0.0001),表明IQGAP1过表达在体外可以促进血管的生成;体内鸡胚尿囊膜的实验结果也显示,IQGAP1过表达组生成的血管数量较对照组多,并且所生成血管的形态舒展,颜色较为鲜红,经统计学分析,差异有统计学意义(P<0.001),说明IQGAP1过表达可以促进体内血管的生成;体内外实验结果一致,表明IQGAP1过表达后会促进肿瘤血管的生成。***1干扰组采用体外HUVECs小管生成实验,结果显示,与对照组相比较,发现IQGAP1基因干扰后小管生成数较少,生成小管的管腔结构也不完整,差异有统计学意义(P<0.0001),表明IQGAP1干扰后在体外可以抑制血管的生成;体内鸡胚尿囊膜实验结果显示,IQGAP1基因干扰后所生成的血管数量较对照组少,生成的血管色泽灰暗并且主血管较少,经数据统计得,两者之间的差异有统计学意义(P<0.001);上述体内外实验结果一致,表明干扰IQGAP1基因表达后能够明显抑制肿瘤的血管生成。5.在IQGAP1过表达的细胞中,经Western blot检测发现,VEGFA与p-VEGFR2蛋白表达量与其在对照细胞内的表达量相比都升高,差异有统计学意义(P<0.0001),而VEGFR2蛋白的表达量则在IQGAP1过表达组与其对照组细胞中无明显差异,表明IQGAP1可能通过VEGFA与VEGFR2相互作用后促进VEGFR2发生磷酸化,进而促进血管的生成。6.在IQGAP1干扰组细胞中,经Western blot检测发现,VEGFA与p-VEGFR2蛋白的表达量与其在对照细胞内相比,表达量都降低,差异有统计学方面的意义(P<0.0001),而VEGFR2的表达量则在IQGAP1干扰组与其对照组中无明显差异,表明IQGAP1可能通过阻断了VEGFA与其受体VEGFR2的相互作用进而抑制血管生成的过程。7.经Western blot检测发现,IQGAP1过表达组细胞中p-Akt、p-ERK蛋白的表达量与其在对照组的结果比较,发现两者的表达量都升高,差异有统计学的意义(P<0.0001);而IQGAP1干扰组细胞p-Akt、p-ERK的表达量均低于其在对照组内的表达量,差异有统计学意义(P<0.0001);说明IQGAP1对肿瘤细胞血管生成的发生机制的调