关键词： 肺炎衣原体   基因芯片   血管平滑肌细胞   细胞粘附   细胞迁移   IQGAP1shRNA  

摘要： 目的： 利用肺炎衣原体(Chlamdia pneumoniae, ***)体外感染大鼠血管平滑肌细胞(Vascular smooth muscle cell, VSMC)模型,分析***感染对VSMC(?)细胞迁移相关基因表达的影响：观察***感染对VSMC粘附和迁移能力的影响;探讨IQGAP1在***感染诱导VSMC粘附和迁移中的作用。 方法： ***增殖培养后体外感染大鼠VSMC;2.应用基因芯片技术分析***感染大鼠VSMC12h后,VSMC内细胞迁移相关基因表达的改变;3.运用MTT去观察***感染对VSMC粘附能力的影响;4.应用Transwell实验观察***感染对VSMC迁移能力的影响;5.利用RT-PCR技术检测***感染的VSMC内IQGAP1mRNA的表达水平;6.采用Western blot技术检测***感染的VSMC为IQGAP1蛋白的表达水平;7.应用RNA干扰技术使IQGAP1表达水平下调,RT-PCR技术检测IQGAP1mRNA的表达,以确定干扰效果;8.运用MTT法观察RNA干扰下调IQGAP1基因后对***感染诱导的VSMC粘附的影响;9.应用Transwell实验观察RNA干扰沉默IQGAP1表达后对***感染诱导的VSMC迁移的影响。 结果： ***感染组与正常对照组均应用Affymetrix Rat Genome2302.0Array,通过与大鼠31000个探针进行杂交,分析了31042个转录体和变异体,其中包括28000个己知基因。芯片杂交结果通过计算机扫描、定量和分析后,共发现差异表达基因35个,其中上调基因12个,下调基因23个;与细胞迁移密切相关的TLR2基因上调、TGF-B3和AKAP12基因下调。基因本体论分析结果显示,35个差异显著的基因中,30个基因可以在生物学过程、细胞组成和分子功能中找到注释,4个基因未搜索到其功能分类;代谢通路分析结果显示,7个基因参与细胞周期相关的信号通路、补体激活的经典途径相关信号通路、氧化应激相关信号通路和前列腺素合成调节相关信号通路;***感染VSMC16h后,细胞粘附于Matrigel的能力明显增强。MTT比色结果显示,***感染组的吸光度(Absorbance, A)值明显高于正常对照组(P<0.001);Cpn感染组的细胞粘附率为162.342%±4.691%(P<0.001);***感染VSMC19h后,***感染组的细胞迁移数目明显多于正常对照组(P<0.01);细胞迁移指数为152.653%±7.493%(P<0.01);***感染VSMC2h后,IQGAP1(?)带亮度与正常对照组差别不大;随着感染时间延长,IQGAP1条带亮度逐渐增强;***感染12h时IQGAP1条带亮度最强;之后,IQGAP1条带亮度又逐渐减弱,直至感染48h时,条带亮度与正常对照组基本相同;5. Western blot结果显示,***感染VSMC后,随着感染时间延长,IQGAP1的表达水平也整体表现出先逐渐升高而后又逐渐降低的趋势;***感染VSMC16h时IQGAP1的表达水平最高;随后,IQGAP1的表达水平又逐渐降低;6.重组质粒转染VSMC后24h,荧光显微镜下可观察到pGPH1/GFP/Neo-IQGAP1Scrambled shRNA转染组(阴性对照组)和pGPH1/GFP/Neo-IQGAP1shRNAs转染组细胞均发出清晰的绿色荧光,而无质粒转染对照组和正常对照组细胞中则未见有绿色荧光;转染48h后,各组细胞中的绿色荧光较24h时增强;RT-PCR结果显示,无质粒转染对照组和阴性对照组细胞内IQGAP1mRNA表达水平与正常对照组无显著差异;而IQGAP1mRNA在pGPH1/GFP/Neo-IQGAP1shRNA转染组中的表达水平与正常对照组相比明显降低： 7.经MTT比色后发现,pGPH1/GFP/Neo-IQGAP1shRNAs转染组的A值明显低于对照组(P<0.001),细胞粘附率为68.543%±1.197%(P<0.001);阴性对照组细胞粘附于Matrigel的能力与正常对照组相比无明显变化(P>0.05);***感染转染pGPH1/GFP/Neo-IQGAP1shRNAs的VSMC后,该组的A值明显低于***感染组(P<0.001),其细胞粘附率为104.991%±1.225%(P<0.001);8.重组质粒pGPHl/GFP/Neo-IQGAP1shRNAs转染VSMC后,细胞迁移数目明显低于正常对照组(P<0.01),细胞迁移指数为69.927%±4.838%(P<0.01);阴性对照组细胞的迁移数目与正常对照组相比无明显差异(P>0.05);***

摘要： Purpose: This study was designed to explore the role of IQGAP1 in the invasiveness of thyroid cancer and its potential as a novel prognostic marker and therapeutic target in this cancer. Experimental Design: We examined IQGAP1 copy gain and its relationship with clinicopathologic outcomes of thyroid cancer and investigated its role in cell invasion and molecules involved in the process. Results: We found IQGAP1 copy number (CN) gain >= 3 in 1 of 30 (3%), 24 of 74 (32%), 44 of 107 (41%), 8 of 16 (50%), and 27 of 41 (66%) of benign thyroid tumor, follicular variant papillary thyroid cancer (FVPTC), follicular thyroid cancer (FTC), tall cell papillary thyroid cancer (PTC), and anaplastic thyroid cancer, respectively, in the increasing order of invasiveness of these tumors. A similar tumor distribution trend of CN >= 4 was also seen. IQGAP1 copy gain was positively correlated with IQGAP1 protein expression. It was significantly associated with extrathyroidal and vascular invasion of FVPTC and FTC and, remarkably, a 50%-60% rate of multifocality and recurrence of BRAF mutation-positive PTC (P = 0.01 and 0.02, respectively). The siRNA knockdown of IQGAP1 dramatically inhibited thyroid cancer cell invasion and colony formation. Coimmunoprecipitation assay showed direct interaction of IQGAP1 with E-cadherin, a known invasion-suppressing molecule, which was upregulated when IQGAP1 was knocked down. This provided a mechanism for the invasive role of IQGAP1 in thyroid cancer. In contrast, IQGAP3 lacked all these functions. Conclusions: IQGAP1, through genetic copy gain, plays an important role in the invasiveness of thyroid cancer and may represent a novel prognostic marker and therapeutic target for this cancer. Clin Cancer Res;16(24);6009-18. (C) 2010 AACR.

关键词： Growth factors   Arteriosclerosis   Signal transduction   Vascular disease   Smooth muscle  

关键词： apoptosis   ECyd   focused proteomics   IQGAP1   RNase L  

摘要： Mammalian intracellular ribonuclease L ( RNase L) is a latent endoribonuclease that functions against viral infections as an apoptosis-inducing protein, and its activity requires intracellular 5'-end- triphosphorylated-2',5' oligoadenylates (2-5A) as an activator. Previously, we showed that RNase L can be activated in human cancer cell line HT1080 by an RNA polymerase I inhibitor, 1-(3-C-ethynyl-beta-d-ribo-pentofuranosyl) cytosine (3'-ethynylcytidine;ECyd). In ECyd-treated cells, knockdown of the RNase L resulted in a marked decrease in c-jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting apoptosis. We investigate RNase L binding partners by focused proteomic approach using immunoprecipitation with anti-RNase L IgG and mass spectrometry. We found that the IQ motif-containing Ras GTPase-activating-like protein 1 (IQGAP1) can associate with RNase L, and that phosphorylation occurs on the IQGAP1. ECyd-induced JNK phosphorylation and apoptosis were inhibited when IQGAP1 was knocked down with a small interfering RNA. These results raise the interesting possibility that the RNase L-IQGAP1 association may regulate JNK phosphorylation in RNase L-madiated apoptosis. It is likely IQGAP1 works as a regulator in apoptosis.

关键词： Normal Liver   SNU387 Cell   Hepatic Adenoma   Liver Cancer Cell Line   Calponin Homology Domain  

摘要： Background: IQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC. Methods: IQGAP1 and IQGAP2 protein expression was assessed by Western blotting and immunohistochemistry. IQGAP mRNA was measured by quantitative RT-PCR. The methylation status of the Iqgap2 promoter was determined by pyrosequencing of bisulfite-treated genomic DNA. Results: IQGAP1 and IQGAP2 expression was reciprocally altered in 6/6 liver cancer cell lines. Similarly, immunohistochemical staining of 82 HCC samples showed that IQGAP2 protein expression was reduced in 64/82 (78.0%), while IQGAP1 was present in 69/82 (84.1%). No IQGAP1 staining was detected in 23/28 (82.1%) normal livers, 4/4 (100.0%) hepatic adenomas and 23/23 (100.0%) cirrhosis cases, while IQGAP2 was increased in 22/28 (78.6%), 4/4 (100.0%) and 23/23 (100.0%), respectively. Although the Iqgap2 promoter was not hypermethylated in HCC at any of the 25 CpG sites studied (N = 17), IQGAP2 mRNA levels were significantly lower in HCC specimens (N = 23) than normal livers (N = 6). Conclusions: We conclude that increased IQGAP1 and/or decreased IQGAP2 contribute to the pathogenesis of human HCC. Furthermore, downregulation of IQGAP2 in HCC occurs independently of hypermethylation of the Iqgap2 promoter. Immunostaining of IQGAP1 and IQGAP2 may aid in the diagnosis of HCC, and their pharmacologic modulation may represent a novel therapeutic strategy for the treatment of liver cancer.

关键词： Macrophages   Ischemia   Vasculogenesis   Inflammation   Angiogenesis   Blood flow   Necrosis   Muscle regeneration  

摘要： Background: Neovascularization is an important repair mechanism in response to ischemic injury and is dependent on inflammation, angiogenesis and reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is a key regulator for actin cytoskeleton and motility. We previously demonstrated that IQGAP1 mediates vascular endothelial growth factor (VEGF)-induced ROS production and migration of cultured endothelial cells (ECs);however, its role in post-ischemic neovascularization is unknown. Methodology/Principal Findings: Ischemia was induced by left femoral artery ligation, which resulted in increased IQGAP1 expression in Mac3(+) macrophages and CD31(+) capillary-like ECs in ischemic legs. Mice lacking IQGAP1 exhibited a significant reduction in the post-ischemic neovascularization as evaluated by laser Doppler blood flow, capillary density and a-actin positive arterioles. Furthermore, IQGAP1(-/-) mice showed a decrease in macrophage infiltration and ROS production in ischemic muscles, leading to impaired muscle regeneration and increased necrosis and fibrosis. The numbers of bone marrow (BM)-derived cells in the peripheral blood were not affected in these knockout mice. BM transplantation revealed that IQGAP1 expressed in both BM-derived cells and tissue resident cells, such as ECs, is required for post-ischemic neovascularization. Moreover, thioglycollate-induced peritoneal macrophage recruitment and ROS production were inhibited in IQGAP1(-/-) mice. In vitro, IQGAP1(-/-) BM-derived macrophages showed inhibition of migration and adhesion capacity, which may explain the defective macrophage recruitment into the ischemic tissue in IQGAP1(-/-) mice. Conclusions/Significance: IQGAP1 plays a key role in post-ischemic neovascularization by regulating, not only, ECs-mediated angiogenesis but also macrophage infiltration as well as ROS production. Thus, IQGAP1 is a potential therapeutic target for inflammation-and angiogenesis-dependent ischemic cardiovascular d