关键词： MIB1   ST7   IQGAP1   胰腺癌  

摘要： 目的:胰腺癌是一种起病隐匿、发展迅速的消化道恶性肿瘤。目前,除根治性切除外,胰腺癌尚无有效的治疗方法。胰腺癌的遗传异质性与其高度恶性的生物学行为密切相关。MIB1(E3 ubiquitin ligase mind bomb 1)是一种E3泛素连接酶,参与各种细胞生物活动过程。我们前期试验证实MIB1在胰腺癌中表现为高表达,但MIB1在胰腺癌进展中的作用仍不清楚。本研究旨在探讨MIB1调控胰腺癌发生发展中的作用,并阐明其作用机制。方法:(1)使用TCGA数据库评估胰腺癌组织和胰腺正常组织中MIB1的m RNA水平。(2)用组织芯片和IHC检测胰腺癌标本中MIB1的蛋白水平。(3)收集我院胰腺癌组织标本并进行Western blot分析胰腺癌组织中MIB1蛋白水平。(4)在Bx PC-3和SW1990细胞中用两个不同的sh RNA沉默MIB1的表达。并通过MTS实验和克隆形成实验以及裸鼠成瘤实验探究敲低MIB1基因后对胰腺癌细胞的增殖和侵袭的影响。(5)通过过表达MIB1探究其对Bx PC-3和SW1990胰腺癌细胞增殖和侵袭的影响。(6)采用免疫共沉淀(co-IP)技术和蛋白质谱分析技术寻找并验证与MIB1相互作用的蛋白ST7。(7)运用邻近连接试验(Proximity Ligation Assay,PLA)以证实MIB1和ST7在Bx PC-3细胞中的相互作用。(8)利用si RNA/sh RNA沉默或质粒过表达MIB1及ST7后,用Western Blot、RT-PCR实验分别检测MIB1和ST7的蛋白和转录水平的变化。(9)用蛋白泛素化免疫共沉淀技术验证MIB1能否直接参与ST7的泛素化,并检测调控后的ST7蛋白半衰期变化。(10)构建MIB1的RING结构突变体,验证RING结构的E3连接酶活性。(11)运用胰腺癌组织芯片明确MIB1及ST7的相关关系。(12)沉默或不沉默以及过表达ST7探究其对胰腺癌细胞的增殖和侵袭能力的影响。(13)单独或联合沉默MIB1和ST7后,在体内和体外实验探究其对胰腺癌细胞的增殖作用影响。(14)用沉默或不沉默ST7的Bx PC-3进行RNA-seq分析,对差异表达的基因进行KEGG通路富集分析,寻找显著调控的通路及基因。(15)单独或联合沉默IQGAP1和ST7后,利用Western Blot、RT-PCR实验检测两者蛋白及m RAN水平的变化;利用MTS实验探究其对胰腺癌细胞的生长增值能力。结果:(1)TCGA数据库评估胰腺癌组织和胰腺正常组织中MIB1的m RNA水平,发现MIB1在16%的胰腺癌中表达上调。(2)用组织芯片(n=25例正常胰腺组织,n=35例胰腺导管腺癌)和IHC检测胰腺癌标本中MIB1的蛋白水平,发现胰腺癌组织中MIB1蛋白水平明显高于正常胰腺组织(P<0.001)。(3)对我院标本的Western blot分析证实,胰腺癌组织(n=12)的MIB1蛋白水平高于癌旁组织(n=12)。(4)体内体外实验证实沉默MIB1后抑制胰腺癌细胞(PANC-1和Bx PC-3)的增殖和侵袭。(5)体内体外实验证实过表达MIB1基因后促进了胰腺癌细胞(PANC-1和Bx PC-3)的增殖和侵袭。(6)蛋白质谱分析寻找到感兴趣的互作蛋白ST7,并进一步用co-IP证实MIB1与ST7的相互作用。(7)PLA证实了MIB1和ST7在Bx PC-3细胞中的直接相互作用。(8)发现MIB1基因敲除增加了胰腺癌细胞Bx PC-3、SW1990和ASPC-1细胞中ST7的蛋白水平,但不增加ST7的m RNA水平。相反,MIB1的过度表达降低了胰腺癌细胞中ST7蛋白的水平,且ST7的m RNA水平保持不变。(9)运用蛋白半衰期实验检测发现,MIB1的抑制延长了Bx PC-3细胞中ST7蛋白的半衰期。(10)MIB1-ΔRING突变体的表达不改变Bx PC-3细胞中ST7的泛素化水平,但过表达野生型MIB1则增加了ST7的泛素化水平。(11)评估发现胰腺癌组织芯片(n=35个样本)中ST7和MIB1的蛋白水平呈负相关(r=-0.3535,P=0.0372)。(12)MTS和克隆形成实验表明,两种不同的sh RNA沉默ST7,显著促进了Bx PC-3和SW1990细胞的生长及体外侵袭能力。(13)ST7沉默促进肿瘤生长,MIB1沉默抑制Bx PC-3细胞的增殖。ST7和MIB1的联合沉默在体外和体内都减弱了MIB1下调的抗增殖作用。(14)RNAseq分析及对差异表达基因的KEGG途径富集分析表明,ST7基因敲除上调了许多涉及肌动蛋白细胞骨架途径的基因。其中,ST7的沉默显著上调了IQGAP1的表达。(15)Western blotting和q RT-PCR分析证实ST7基因敲除增加了IQGAP1

关键词： IQGAP1   MMP-2   侵袭   NF-κB  

摘要： 目的:1.探究IQGAP1(IQ motif containing GTPase activating protein 1)对食管鳞癌细胞迁移和侵袭的影响。2.探究IQGAP1对MMP-2基因表达和活性的影响。3.探究IQGAP1影响食管鳞癌细胞迁移和侵袭以及MMP-2基因表达和活性的分子机制。方法:1.将GFP-IQGAP1质粒和对照组空载质粒转染至EC9706细胞中,将IQGAP1短发卡RNA质粒和阴性对照组转染至KYSE150细胞中,通过Western blot实验验证IQGAP1高表达细胞系和IQGAP1基因敲降细胞系的建立是否成功。2.采用MTT实验和平板克隆实验检测IQGAP1高表达或基因敲降对食管鳞癌细胞增殖能力的影响;通过划痕愈合实验、Transwell实验检测IQGAP1高表达或基因敲降对食管鳞癌细胞迁移和侵袭能力的影响。3.通过Western blot实验和Real-Time PCR实验检测IQGAP1高表达或基因敲降细胞系中MMP-2蛋白及m RNA的表达水平;采用明胶酶谱实验检测IQGAP1高表达或基因敲降细胞系中MMP-2的活性。4.通过Western blot实验、细胞免疫荧光实验检测IQGAP1对NF-κB信号通路的影响以及PDTC对NF-κB信号通路的影响;采用NF-κB抑制剂PDTC作用于IQGAP1高表达细胞系,通过划痕愈合实验、Transwell实验检测PDTC对食管鳞癌细胞迁移、侵袭能力的影响;通过Western blot实验、Real-Time PCR实验和明胶酶谱实验检测PDTC对食管鳞癌细胞中MMP-2表达及活性的影响。结果:*** blot实验结果显示IQGAP1高表达细胞系能表达GFP-IQGAP1融合蛋白,而对照组细胞无。IQGAP1基因敲降细胞系的IQGAP1表达水平明显低于对照组细胞。***实验和平板克隆实验结果显示IQGAP1高表达细胞系的增殖能力明显高于对照组细胞(P<0.05)。IQGAP1基因敲降细胞系的增殖能力明显低于对照组细胞P<0.05)。划痕愈合实验、Transwell实验结果显示,与对照组细胞相比,IQGAP1高表达组细胞具有更强的迁移和侵袭能力(P<0.05),而IQGAP1基因敲降组细胞迁移和侵袭能力减弱(P<0.05)。*** blot实验、Real-Time PCR实验及明胶酶谱实验结果显示与对照组细胞相比,IQGAP1高表达细胞系具有更高的MMP-2表达水平和功能活性(P<0.05)。而IQGAP1低表达细胞系的MMP-2表达水平和功能活性降低(P<0.05)。*** blot实验结果显示,IQGAP1高表达细胞系中NF-κB(p65)表达增高(P<0.05)。IQGAP1基因敲降细胞系中NF-κB(p65)表达降低(P<0.05)。免疫荧光显示IQGAP1高表达细胞系中,NF-κB(p65)核定位水平增多,IQGAP1基因敲降细胞系中NF-κB(p65)核定位水平降低。Western blot实验、划痕愈合实验、Transwell实验结果显示,PDTC处理IQGAP1高表达细胞后,NF-κB(p65)表达水平降低,细胞的侵袭、迁移能力受到抑制。免疫荧光显示,PDTC处理IQGAP1高表达细胞后,NF-κB(p65)核定位水平降低。Western blot实验、Real-Time PCR实验和明胶酶谱实验结果显示,PDTC处理IQGAP1高表达细胞后,MMP-2表达水平和功能活性受到抑制。结论:***1促进食管鳞癌细胞增殖、迁移和侵袭。***1促进食管鳞癌细胞MMP-2的表达及功能活性,提示IQGAP1可能通过调控MMP-2来影响肿瘤细胞的迁移和侵袭。***1通过NF-κB信号通路来调控食管鳞癌细胞的迁移、侵袭能力,以及MMP-2的表达和活性。

关键词： 动脉粥样硬化   炎症   IQGAP1   miR-124  

摘要： 动脉粥样硬化(atherosclerosis,AS)的主要病理基础是缺血性心脑血管病,如:冠心病、脑梗死、外周血管病等,较为常见,严重危害人们的身体健康。迄今为止,动脉粥样硬化的发病机制尚不明确。所以,有关动脉粥样硬化的病理机制及冠心病、心绞痛等心肌缺血性疾病的潜在治疗靶点成为了大家关注的重点之一。人们普遍认为,动脉粥样硬化的发展经历了脂质斑块、纤维斑块和动脉粥样斑块的形成,其继发于斑块出血、斑块破裂、血栓形成、钙化等变化。动脉粥样硬化是血管壁对外部损伤的非正常反应,并具有慢性炎性疾病的特征。目前,动脉粥样硬化的致病因素繁多,涉及到多个致病机制,炎症反应有可能是动脉粥样硬化各发病机制的枢纽。IQ结构域GTP酶激活蛋白1(IQ motif containing GTPase activating proteins1,IQGAP1)是一种支架蛋白,在多种疾病中,包括动脉粥样硬化,均显著高表达。另有研究表明miR-124(micro RNA 124)在动脉粥样硬化病人中呈现低表达。而生物信息学则提示IQGAP1是miR-124的一个潜在靶点。所以本课题以动脉粥样硬化的血管内皮细胞及其分泌的炎症因子为重点,深入研究动脉粥样硬化发生发展的分子机制。目的:研究IQGAP1和miR-124在动脉粥样硬化中对血管内皮细胞活化的调控作用。研究方法:1.通过Target Scan Human数据库检索micro RNA 124的调控靶标;然后通过双荧光素酶报告实验(Dual Luciferase reporter assay,DR)验证miR-124能否直接与其靶标结合;2.体外培养人冠状动脉内皮细胞(Human Coronary Artery Endothelial Cells,HCAEC),使用氧化低密度脂蛋白(Oxidized Low Density Lipoprotein,ox-LDL)刺激正常的HCAEC 24h,建立ox-LDL诱导的内皮细胞损伤模型,分为对照组和模型组,在显微镜下观察细胞形态学的变化,确定造模成功;3.体外培养人冠状动脉内皮细胞,通过脂质体转染miR-124 mimics和inhibitor,继续培养细胞,构建miR-124过表达与沉默模型。提取内皮细胞的总RNA和总蛋白,使用荧光定量PCR(Quantitative Real-time PCR,q RT-PCR)和蛋白质免疫印迹法(Western blot),分别检测下游的IQGAP1以及炎症相关蛋白TNF-α,IL-6和IL-1β的表达水平;转染p EGFP-C2-IQGAP1和p EGFP-C2-NC,构建IQGAP1过表达和沉默模型,并检测炎症相关蛋白。4.流式细胞术(Flow Cytometry,FCM)检测miR-124过表达组和沉默组内皮细胞的凋亡情况及细胞周期进程;5.体外培养人冠状动脉内皮细胞,通过脂质体转染IQGAP1 mimics和inhibitor,继续培养细胞,构建IQGAP1沉默与过表达模型;6.流式细胞仪检测IQGAP1过表达组和沉默组内皮细胞的凋亡情况及细胞周期进程,并进行Western blot检测下游炎症因子TNF-α,IL-6和IL-1β的蛋白表达变化。结果:1.通过Target Scan Human数据库检索到miR-124能够作用于IQGAP1 m RNA的3'-UTR的非编码区,且荧光素酶报告结果证实IQGAP1是miR-124的直接靶标;2.相比于对照组,模型组内皮细胞在显微镜下明显活性降低;通过Western blot检测,结果表明模型组的IQGAP1表达明显升高(P<0.01),具有统计学意义;3.与miR-124沉默组相比,过表达组的IQGAP1 m RNA水平明显低于沉默组(P<0.05),有统计学意义。TNF-α,IL-6和IL-1β的表达水平均明显升高,两组间均存在显著差异(P<0.01);流式细胞仪结果表明过表达组显著促进HCAEC的凋亡,阻滞了细胞周期;4.与IQGAP1沉默组相比,过表达组的下游炎症因子的表达都显著升高,(P<0.01),有统计学意义;流式细胞仪结果表明沉默组显著促进HCAEC的凋亡,阻滞了细胞周期。结论:***1能够促进人冠状动脉内皮细胞中炎症因子的分泌,促进细胞周期进程,减少凋亡;***-124能够抑制人冠状动脉内皮细胞中炎症因子的分泌,抑制细胞周期进行,促进凋亡;***-124通过靶向抑制IQGAP1从而抑制冠状动脉内皮细胞活化,起到了抗炎的作用。

关键词： receptor tyrosine kinase   phosphorylation   scaffold protein   protein phosphorylation   mass spectrometry (MS)   phosphotyrosine signaling   IQGAP1   receptor tyrosine kinase   phosphotyrosine   IQGAP2  

摘要： IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

关键词： Age-related macular degeneration   choroidal neovascularization   IQGAP1   Vascular endothelial growth factor   Vascular endothelial growth factor receptor 2   Rac1  

摘要： Loss of visual acuity in neovascular age-related macular degeneration (nAMD) occurs when factors activate choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelium into the sensory retina and develop into choroidal neovascularization (CNV). Active Rac1 (Rac1GTP) is required for CEC migration and is induced by different AMD-related stresses, including vascular endothelial growth factor (VEGF). Besides its role in pathologic events, Rac1 also plays a role in physiologic functions. Therefore, we were interested in a method to inhibit pathologic activation of Rac1. We addressed the hypothesis that IQGAP1, a scaffold protein with a Rac1 binding domain, regulates pathologic Rac1GTP in CEC migration and CNV. Compared to littermateIqgap1(+/+),Iqgap1(-/-)mice had reduced volumes of laser-induced CNV and decreased Rac1GTP and phosphorylated VEGFR2 (p-VEGFR2) within lectin-stained CNV. Knockdown of IQGAP1 in CECs significantly reduced VEGF-induced Rac1GTP, mediated through p-VEGFR2, which was necessary for CEC migration. Moreover, sustained activation of Rac1GTP induced by VEGF was eliminated when CECs were transfected with an IQGAP1 construct that is unable to bind Rac1. IQGAP1-mediated Src activation was involved in initiating Rac1 activation, CEC migration, and tube formation. Our findings indicate that CEC IQGAP1 interacts with VEGFR2 to mediate Src activation and subsequent Rac1 activation and CEC migration. In addition, IQGAP1 binding to Rac1GTP results in sustained activation of Rac1, leading to CEC migration toward VEGF. Our study supports a role of IQGAP1 and the interaction between IQGAP1 and Rac1GTP to restore CECs quiescence and, therefore, prevent vision-threatening CNV in nAMD.

关键词： Transgelin   IQGAP1   17-AAG   Scaffold proteins   Heat shock proteins  

摘要： Several studies have been conducted on the transgelin (TAGLN) protein and its critical role in cancer biology. However, the regulation of this protein and the way in which this regulation is correlated with the functions of IQ motif-containing GTPase-activating protein 1 (IQGAP1) in MDA-MB231 cells, remain unclear. We generated stable TAGLN-knockdown and TAGLN-overexpressing cells. These cells, along with their control counterparts, were cultured in the presence or absence of 17-AAG. The different cell groups were then subjected to functional assays to assess proliferation, chemotaxis, and invasion. TAGLN regulation was found to affect the efficacy of 17-AAG. The ability of TAGLN to influence the levels of IQGAP1 and its binding partners altered the critical functions of breast cancer cells. Therefore, the altered functionality of MDA-MB-231 cells, as a consequence of TAGLN regulation, is correlated with IQGAP1 signaling.

关键词： Cell signalling   Metastasis  

摘要： Background Hepatitis B virus (HBV) has a crucial role in the progression of hepatocellular carcinoma (HCC). Tumour cells must develop anoikis resistance in order to survive before metastasis. This study aimed to investigate the mechanism of IQGAP1 in HBV-mediated anoikis evasion and metastasis in HCC cells. Methods IQGAP1 expression was detected by immunohistochemistry, real-time PCR and immunoblot analysis. Lentiviral-mediated stable upregulation or knockdown of IGAQP1, immunoprecipitation, etc. were used in function and mechanism study. Results IQGAP1 was markedly upregulated in HBV-positive compared with HBV-negative HCC cells and tissues. IQGAP1 was positively correlated to poor prognosis of HBV-associated HCC patients. IQGAP1 overexpression significantly enhanced the anchorage-independent growth and metastasis, whereas IQGAP1-deficient HCC cells are more sensitive to anoikis. Mechanistically, we found that HBV-induced ROS enhanced the association of IQGAP1 and Rac1 that activated Rac1, leading to phosphorylation of Src/FAK pathway. Antioxidants efficiently inhibited IQGAP1-mediated anoikis resistance and metastasis. Conclusions Our study indicated an important mechanism by which upregulated IQGAP1 by HBV promoted anoikis resistance, migration and invasion of HCC cells through Rac1-dependent ROS accumulation and activation of Src/FAK signalling, suggesting IQGAP1 as a prognostic indicator and a novel therapeutic target in HCC patients with HBV infection.

关键词： IQGAP1   胰腺祖细胞   RNA干扰   细胞增殖  

摘要： 为探讨沉默IQ结构域三磷酸鸟苷合酶-激活蛋白1(IQ motif containing GTPase-activating protein 1,IQGAP1)基因的表达对胰腺祖细胞增殖的影响,本研究将靶向IQGAP1基因的小干扰RNA(siRNA)转染胰腺祖细胞,qPCR、Western blotting验证IQGAP1基因表达的下调,通过细胞计数试剂盒8(cell counting kit-8,CCK8)法检测IQGAP1基因沉默对胰腺祖细胞增殖的影响,流式细胞术检测细胞周期进程,qPCR、Western blotting检测细胞周期蛋白D2(cyclin D2,CCND2)的表达水平。结果显示,干扰片段有效沉默IQGAP1基因的表达,IQGAP1蛋白表达水平降低了(71.30±0.08)%;与对照组相比,沉默IQGAP1基因的表达使胰腺祖细胞增殖能力降低了(45.37±0.02)%,细胞周期阻滞在G0/G1期,CCND2基因表达水平明显降低。表明沉默IQGAP1基因可能通过抑制CCND2基因表达,抑制胰腺祖细胞的增殖。

关键词： IQGAP1   Senescence   Intercellular communication   Cellular bridges (CBs)   Tunneling nanotubes (TNTs)   Vascular smooth muscle cells (VSMCs)  

摘要： Cell senescence - an irreversible proliferation arrest - is one of the possible cellular responses to stress. There is a vast variety of stimuli, extrinsic and intrinsic, known to induce senescence, and several molecular pathways involved in the process;yet much still remains to be explained. Senescent cells can communicate with neighboring cells through secreted factors such as cytokines and chemokines. Several years ago it was shown that cells can also communicate in a more direct manner by an exchange of proteins via cellular bridges (CBs). Recent studies show that in senescent cells the intensity of such transfer increases. The research also revealed that Cdc42 and actin polymerization are indispensable for this process to occur. Here, we evaluate the hypothesis that, apart from actin and Cdc42, also IQGAP1 could be involved in direct intercellular communication. Our results showed that direct transfer occurred preferentially between senescent cells and that IQGAP1 was not essential for this process. Interestingly, cells harboring mutated IQGAP1 had altered morphology and were characterized by decreased proliferation, increased time of division and appearance of some senescence markers (increased activity of senescence-associated beta-galactosidase and induction of senescence-associated secretory phenotype). Our findings suggest that IQGAP1 dysfunction can induce senescence.

关键词： colorectal cancer   ERK1   2   IQGAP1   metastasis   PLS1  

摘要： Tumor metastasis is the dominant cause of death in colorectal cancer (CRC) patients, and it often involves dysregulation of various cytoskeletal proteins. Plastin 1 (PLS1) is an actin-bundling protein that has been implicated in the structure of intestinal epithelial microvilli;however, its role in CRC metastasis has not yet been determined. In this study, we demonstrated that PLS1 is highly expressed in 33.3% (45/135) of CRC patients and is correlated with lymph node metastasis and poor survival. In in vitro and in vivo experiments, PLS1 induced the migration and invasion of CRC cells and the metastases to the liver and lung in mice. Moreover, the expressions of key factors for CRC metastases, matrix metalloproteinase (MMP) 9 and 2, were enhanced by PLS1, which was dependent on phosphorylating ERK1/2 activated by IQGAP1/Rac1 signaling. The connection between these signals and PLS1 was further confirmed in CRC tissues of patients and the metastatic nodules from a mouse model. These findings suggest that PLS1 promotes CRC metastasis through the IQGAP1/Rac1/ERK pathway. Targeting PLS1 may provide a potential approach to inhibit the metastasis of CRC cells.