关键词： 胰腺肿瘤   IQ结构域GTP酶激活蛋白1   癌性锚蛋白重复序列   预后   病理特征  

摘要： 目的:探讨胰腺癌组织中IQ结构域GTP酶激活蛋白1(IQGAP1)、癌性锚蛋白重复序列(Gankyrin)的表达及临床意义。方法:回顾性分析2011年1月—2014年1月武威市人民医院行胰腺癌根治术治疗的95例胰腺癌患者的病历及随访资料,免疫组织化学染色法检测正常胰腺组织、胰腺癌组织及癌旁组织中IQGAP1和Gankyrin的表达,分析IQGAP1和Gankyrin表达与胰腺癌临床病理参数的关系,采用Kaplan-Meier生存曲线分析不同IQGAP1和Gankyrin表达患者的总生存率的差异,Cox比例风险回归模型分析患者预后的影响因素。结果:与癌旁组织和正常胰腺组织相比,胰腺癌组织中IQGAP1和Gankyrin表达均上调(P<0.05)。IQGAP1和Gankyrin表达均与胰腺癌TNM分期、分化程度和糖类抗原199(CA199)表达相关(P<0.05)。IQGAP1阳性、Gankyrin阳性患者的5年生存率均明显低于阴性患者(P<0.05);Cox比例风险回归分析结果显示,TNM分期、IQGAP1表达、Gankyrin表达和CA199表达均是胰腺癌患者预后的独立危险因素(HR=1.250、13.316、3.542、4.089,P<0.05)。结论:胰腺癌组织中IQGAP1和Gankyrin表达上调,IQGAP1和Gankyrin表达与胰腺癌患者生存以及预后相关,可能作为胰腺癌患者的预后预测因子,辅助胰腺癌的诊断和临床治疗。

关键词： circ‐   IQGAP1   miR‐   671‐   5p   Osteoarthritis   TCF4  

摘要： Objective To explore the function of circular RNA IQ motif-containing GTPase-activating protein 1 (circ-IQGAP1) in interleukin (IL)-1 beta-induced osteoarthritis (OA) model and to explore whether circ-IQGAP1 can modulate microRNA-671-5p (miR-671-5p) and transcription factor 4 (TCF4) to regulate chondrocyte apoptosis, inflammatory injury, and extracellular matrix degradation. Methods The cartilage tissues were collected from 32 OA patients or normal subjects. Human chondrocyte CHON-001 cells were challenged via different doses of IL-1 beta for 24 hours. CHON-001 cells were transfected with circ-IQGAP1 overexpression vector, TCF4 overexpression vector, small interfering RNA (siRNA) for circ-IQGAP1, miR-671-5p mimic, miR-671-5p inhibitor or corresponding negative controls. Circ-IQGAP1, miR-671-5p and TCF4 abundances in cartilage tissues or CHON-001 cells were examined via quantitative reverse transcription polymerase chain reaction (qRT-PCR) or western blot. Cell viability was investigated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). Cell apoptosis was measured by flow cytometry. The inflammatory injury was analyzed by the secretion levels of inflammatory cytokines (IL-6, IL-8 and tumor necrosis factor-alpha [TNF-alpha]) by enzyme-linked immunosorbent assay (ELISA). The extracellular matrix degradation was evaluated by expression of aggrecan and matrix metalloproteinase 13 (MMP13) via western blot. The target relationship of miR-671-5p and circ-IQGAP1 or TCF4 was analyzed via dual-luciferase reporter and RNA immunoprecipitation (RIP) analyses. Results Circ-IQGAP1 abundance was enhanced in the cartilage tissues from OA patients compared with normal subjects (n = 32), and its expression was increased in CHON-001 cells after treatment of IL-1 beta in a dose-dependent pattern. MiR-671-5p expression was decreased in the cartilage tissues from OA patients (n = 32) and IL-1 beta-challenged CHON-001 cells. MiR-671-5p expression was negatively associ

关键词： IQ结构域GTP酶活化蛋白1   糖尿病肾病   足细胞凋亡   免疫组织化学法  

摘要： 目的探讨IQ结构域GTP酶活化蛋白1(IQGAP1)在2型糖尿病肾病患者足细胞凋亡中的作用及机制探究。方法采用免疫组织化学法完成两组肾脏组织中IQGAP1、p-ERK1/2、ERK1/2表达水平;采用SPSSPearson相关性分析软件分析IQGAP1与p-ERK1/2、ERK1/2表达及分布关系。结果实验组糖尿病肾病组织中IQGAP1阳性率低于对照组(P<0.05);实验组糖尿病肾病组织中p-ERK1/2、ERK1/2阳性率均高于对照组(P<0.05);免疫组化结果表明:实验组糖尿病肾病组织中足细胞裂隙变窄,足突缩小及伴有节段性融合,可见足细胞凋亡小体形成,IQGAP1分布在肾小球足细胞、系膜细胞、血管内皮细胞中;对照组肾脏组织中未见肾脏病变改变,IQGAP1表达水平较高;SPSSPearson相关性分析结果表明:实验组肾脏组织中IQGAP1与p-ERK1/2、ERK1/2蛋白阳性率呈正相关性(P<0.05)。结论IQGAP1在2型糖尿病肾病患者中呈低表达,且表达水平与p-ERK1/2、ERK1/2存在相关性,可能由于p-ERK1/2、ERK1/2能引起足细胞凋亡,从而引起大量蛋白尿,导致糖尿病肾病的发生。

关键词： AMD1   FTO   hepatocellular carcinoma   IQGAP1   N6‐   methyladenosine   polyamination   stemness  

摘要： Background S-adenosylmethionine decarboxylase proenzyme (AMD1) is a key enzyme involved in the synthesis of spermine (SPM) and spermidine (SPD), which are associated with multifarious cellular processes. It is also found to be an oncogene in multiple cancers and a potential target for tumor therapy. Nevertheless, the role AMD1 plays in hepatocellular carcinoma (HCC) is still unknown. Methods HCC samples were applied to detect AMD1 expression and evaluate its associations with clinicopathological features and prognosis. Subcutaneous and orthotopic tumor mouse models were constructed to analyze the proliferation and metastasis of HCC cells after AMD1 knockdown or overexpression. Drug sensitive and tumor sphere assay were performed to investigate the effect of AMD1 on HCC cells stemness. Real-time quantitative PCR (qRT-PCR), western blot, immunohistochemical (IHC) and m6A-RNA immunoprecipitation (Me-RIP) sequencing/qPCR were applied to explore the potential mechanisms of AMD1 in HCC. Furthermore, immunofluorescence, co-IP (Co-IP) assays, and mass spectrometric (MS) analyses were performed to verify the proteins interacting with AMD1. Results AMD1 was enriched in human HCC tissues and suggested a poor prognosis. High AMD1 level could promote SRY-box transcription factor 2 (SOX2), Kruppel like factor 4 (KLF4), and NANOG expression of HCC cells through obesity-associated protein (FTO)-mediated mRNA demethylation. Mechanistically, high AMD1 expression increased the levels of SPD in HCC cells, which could modify the scaffold protein, Ras GTPase-activating-like protein 1 (IQGAP1) and enhance the interaction between IQGAP1 and FTO. This interaction could enhance the phosphorylation and decrease the ubiquitination of FTO. Conclusions AMD1 could stabilize the interaction of IQGAP1 with FTO, which then promotes FTO expression and increases HCC stemness. AMD1 shows prospects as a prognostic predictor and a therapeutic target for HCC.

关键词： IQGAP1   GTPase   Cancer metastasis   Cytoskeleton  

摘要： The metastatic spread of tumor cells to distant anatomical locations is a critical cause for disease progression and leads to more than 90 % of cancer-related deaths. IQ motif-containing GTPase-activating protein 1 (IQGAP1), a prominent regulator in the cancer metastasis process, is a scaffold protein that interacts with components of the cytoskeleton. As a critical node within the small GTPase network, IQGAP1 acts as a binding partner of several small GTPases, which in turn function as molecular switches to control most cellular processes, including cell migration and invasion. Given the significant interaction between IQGAP1 and small GTPases in cancer metastasis, we briefly elucidate the role of IQGAP1 in regulating cancer metastasis and the varied interactions existing between IQGAP1 and small GTPases. In addition, the potential regulators for IQGAP1 activity and its interaction with small GTPases are also incorporated in this review. Overall, we comprehensively summarize the role of IQGAP1 in cancer tumorigenicity and metastasis, which may be a potential anti-tumor target to restrain cancer progression.

关键词： Bacteria   Cellular microbiology   Cytoskeleton   Pathogens  

摘要： Campylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1. The pathogen Campylobacter jejuni invades intestinal cells after secreting protein effectors into the host cell cytosol via the flagellum. Here, Negretti et al. show that one of these effectors, CiaD, binds to host protein IQGAP1, thus leading to unconstrained activity of small GTPase Rac1, which modulates actin reorganization and bacterial internalization.

关键词： cell adhesion   cell extensions   collagen   phagocytosis   small GTPases  

摘要： Many adult connective tissues undergo continuous remodeling to maintain matrix homeostasis. Physiological remodeling involves the degradation of collagen fibers by the intracellular cathepsin-dependent phagocytic pathway. We considered that a multidomain, small GTPase activating protein, IQGAP1, which is involved in the generation of cell extensions, is required for collagen phagocytosis, possibly arising from its interactions with cdc42 and the actin-binding protein Flightless I (FliI). We examined the role of IQGAP1 in collagen phagocytosis by human gingival fibroblasts (HGFs) and by IQGAP1+/+ and IQGAP1-/- mouse embryonic fibroblasts. IQGAP1 was strongly expressed by HGFs, localized to vinculin-stained cell adhesions and sites where cell extensions are initiated, and colocalized with FliI. Immunoprecipitation showed that IQGAP1 associated with FliI. HGFs showed 10-fold increases of collagen binding, 6-fold higher internalization, and 3-fold higher beta 1 integrin activation between 30 and 180 min after incubation with collagen. Compared with IQGAP1+/+ fibroblasts, deletion of IQGAP1 reduced collagen binding (1.4-fold), collagen internalization (3-fold), beta 1 integrin activation (2-fold), and collagen degradation (1.8-fold). We conclude that IQGAP1 affects collagen remodeling through its regulation of phagocytic degradation pathways, which may involve the interaction of IQGAP1 with FliI.

关键词： AMP-activated kinase (AMPK)   scaffold protein   signaling   protein–protein interaction   metformin   metabolic regulation   homeostasis   calcium   calmodulin (CaM)   IQGAP1  

摘要： AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the a1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.

关键词： 分枝杆菌   PDIM   IQGAP1   VEGF  

摘要： 研究背景:众所周知,结核病是一种危害严重的传染性,引起结核病的是结核分枝杆菌,是一种细胞内的病原菌。结核分枝杆菌既不产生内毒素,也不分泌外毒素和侵袭性酶,它的致病性主要与细菌细胞壁上高含量的脂质成分有关。目的:探讨骨架蛋白IQGAP1(IQ motif-containing GTPase-activating protein 1)在分枝杆菌细胞壁脂质PDIM介导感染和肉芽肿形成中的作用,揭示IQGAP1调控p38MAPK和VEGF表达对分枝杆菌感染与致病的机制;并进一步研究骨架蛋白IQGAP1的小分子抑制剂对分枝杆菌PDIM介导致病的影响,为寻找和鉴定新的结核病的治疗靶点提供实验依据。方法:研究工作主要通过结合体外Raw264.7巨噬细胞感染实验和小鼠体内感染实验完成。1.揭示IQGAP1对分枝杆菌感染早期炎症因子表达的影响及p38MAPK信号在该过程中的作用。(1)Raw264.7巨噬细胞培养传代后,随机分为3组:无菌PBS处理作为对照(Mock)、野生型海分枝杆菌感染组(wide-type,WT)、海分枝杆菌细胞壁毒力成分PDIM缺失突变株(ΔPDIM)感染组。western blot检测感染后0.5 h、1 h、2 h、4 h时IQGAP1的蛋白表达。(2)感染4 h时,q RT-PCR检测促炎因子(TNF-α、IL-6)和抑炎因子(TGF-β、IL-10)的表达;western blot检测IQGAP1、TNF-α、TGF-β及p38MAPK和NF-κBp65的磷酸化水平。(3)设计合成针对IQGAP1的si RNA序列和过表达质粒(GV219＿Iqgap1)并分别转染Raw264.7巨噬细胞,再分别感染WT或?PDIM菌株,q RT-PCR检测上述促炎或抑炎因子的表达;western blot检测IQGAP1、TNF-α、TGF-β及p38MAPK和NF-κBp65的磷酸化水平。Confocal检测IQGAP1的si RNA干扰或过表达对细菌感染过程NF-κBp65入核的影响。(4)Raw264.7细胞分别感染WT和?PDIM菌株,q RT-PCR检测p38MAPK磷酸化级联反应的关键激酶如MKK3和MKK6及失活激酶的磷酸化酶如MKP1和MKP5的表达;Raw264.7细胞分别转染IQGAP1的si RNA和过表达质粒后再检测上述分子的表达;在上述两步实验结果基础上,western blot检测WT和?PDIM菌株感染后MKK3磷酸化水平及过表达或抑制IQGAP1后MKK3磷酸化水平的变化。(5)ΔPDIM菌感染Raw264.7细胞,再用p38MAPK抑制剂SB203580处理细胞,western blot检测不同时间点NF-κBp65磷酸化表达;用ΔPDIM菌和Gossypetin(MKK3/MKK6的特异抑制剂)共孵育Raw264.7细胞,western blot检测MKK3磷酸化水平。(6)构建海分枝杆菌的小鼠尾静脉感染模型,然后第7天时小鼠腹腔注射骨架蛋白抑制剂——Nocodazole(每2天一次),连续注射七次后处死小鼠,比较小鼠尾巴大体病变,收集小鼠尾组织研磨涂板计算菌载量,HE染色观察比较组织病理学变化;同时western blot检测IQGAP1等蛋白的表达变化。2.探讨分枝杆菌感染过程中诱导IQGAP1对VEGF表达的意义与机制。(1)建立上述相同的Raw264.7感染模型,感染4 h时,western blot和ELISA分别检测细胞及上清液中VEGF的蛋白表达。(2)将IQGAP1的si RNA序列和GV219＿Iqgap1质粒分别转染Raw264.7巨噬细胞,再分别感染WT或?PDIM菌株,western blot检测VEGF的表达。(3)用VEGF-receptor的特异性抑制剂阿西替尼(Axitinib)处理Raw264.7细胞,然后western blot检测VEGF及IQGAP1的表达。(4)收取小鼠尾静脉感染模型中的尾组织,western blot检测VEGF的表达;免疫组化染色观察IQGAP1和VEGF的表达和分布。(5)结核分枝杆菌感染的小鼠、兔子和猴子的肺组织及临床结核病人样本中IQGAP1和VEGF的表达与分布。结果:***1通过调控p38MAPK信号通路影响分枝杆菌感染早期炎症因子的表达。(1)Raw264.7巨噬细胞感染模型上,WT菌株感染组IQGAP1蛋白水平明显上调(**P<0.01),而在ΔPDIM组表达微弱。(2)q RT-PCR检测发现WT菌株感染组中抑炎因子(TGF-β、IL-10)的表达显著高于ΔPDIM组(**P<0.01),而促炎因子(TNF-α、IL-6)显著低于ΔPDIM组(**P<0.01)。Western blot检测发现ΔPDIM组中

关键词： ASH2L   IQGAP1   基因沉默   幽门螺杆菌   胃癌   生物学效应   转录组测序  

摘要： 目的本实验室前期的实验研究表明在幽门螺杆菌(Helicobacter pylori,Hp)感染的条件下,AGS细胞中ASH2L表达水平增高,且ASH2L和IQGAP1存在互作关系。本研究拟通过沉默ASH2L和IQGAP1基因以探索其对人胃癌AGS细胞的增殖、迁移、侵袭以及凋亡等生物学效应的影响并分析二者在Hp感染相关疾病中的作用机制。方法1.沉默ASH2L和IQGAP1基因对胃腺癌AGS细胞生物学效应的影响:用si RNA技术沉默胃腺癌AGS细胞中ASH2L基因、IQGAP1基因的表达,利用实时荧光定量PCR和Western blot检测沉默效果;用cck-8检测胃癌细胞的增殖情况;划痕愈合实验和transwell实验检测胃癌细胞迁移和侵袭能力的变化;TUNEL实验和流式细胞术检测胃癌细胞凋亡的情况。2.转录组测序(RNA-sequencing,RNA-seq)技术研究沉默ASH2L和IQGAP1基因与Hp感染在胃癌发生发展中的作用和机制:分别下调胃腺癌细胞AGS和正常胃粘膜上皮细胞GES-1中的ASH2L基因、IQGAP1基因的表达后,加入Hp感染因素处理,分别制备Hp感染和Hp未感染的AGS和GES-1细胞的三个生物学重复RNA样品,利用RNA-seq技术对转录组进行高通量测序,对测序数据进行生物信息学分析,将差异基因进行相应的富集分析(GO、KEGG),再应用在线软件进行蛋白质的互作(Protein-Protein Interaction,PPI)网络分析,以探讨ASH2L与IQGAP1对细胞生物学效应影响的机制,分析Hp感染诱导与胃癌发生的可能机制以及ASH2L基因和IQGAP1基因在Hp感染激活的NF-κB信号通路中的作用和机制。结果1.沉默ASH2L和IQGAP1基因对AGS细胞生物学效应的影响结果(1)与对照组比较,ASH2L沉默组的ASH2L基因以及IQGAP1沉默组IQGAP1基因m RNA水平以及蛋白表达明显下调(P值均小于0.05)。(2)在分别沉默ASH2L和IQGAP1基因后,与对照组比较,AGS细胞增殖能力减弱、迁移能力减弱、侵袭能力减弱、凋亡率升高(P值均小于0.05)。2.转录组测序数据分析结果(1)ASH2L和IQGAP1基因在各组细胞中的沉默效率均高于75%,送检的30个RNA样本均符合测序标准。所有组别均富集到相关差异基因,差异基因的表达差异倍数|log2Fold Change|>1。(2)沉默ASH2L基因和沉默IQGAP1基因对胃腺癌AGS细胞生物学效应作用机制的分析:分析在AGS细胞中沉默ASH2L基因和沉默IQGAP1基因的转录组测序数据,利用韦恩分析,发现沉默两个基因后共同影响到的差异表达基因有829个,其中共同上调差异基因有160个,共同下调656个。进一步利用GO富集分析发现,共同作用于上皮细胞迁移的调节相关基因有ANXA3、HBEGF、ITGA3、ETS1、SCARB1、VEGFA、KLF4、ADGRA2、BCAS3、TDGF1、PTPRM、ACVRL1、ANGPT4、HRG;共同作用于内质网应激下的内源性凋亡信号通路的相关基因有CHAC1、CASP4、ATF4、XBP1、ERN1、TRIB3、PMAIP1。KEGG富集结果显示,沉默两个基因后引起的差异表达基因共同参与的生物学作用相关的信号通路有:m TOR signaling pathway、Apoptosis、Calcium signaling pathway、MAPK signaling pathway、TGF-beta signaling pathway、VEGF signaling pathway。(3)Hp感染对正常胃粘膜上皮细胞GES-1癌变作用机制的分析:分析Hp感染和Hp未感染GES-1细胞的差异表达基因库,同时分析AGS细胞与GES-1细胞中的差异表达基因库,进一步利用韦恩分析,发现两个差异基因库中共同的差异表达基因有1059个,其中共同上调差异基因有160个,共同下调656个。KEGG富集到与癌症发生相关的共同信号通路包括Pathways in cancer、Erb B signaling pathway、p53 signaling pathway、TGF-beta signaling pathway、MAPK signaling pathway、Wnt signaling pathway。(4)ASH2L基因和IQGAP1基因在Hp感染激活的NF-κB信号通路中的作用和机制的分析:在AGS细胞中沉默AHS2L基因,发现ASH2L与ALPK1基因的表达均显著下调,而在加入Hp感染后,ASH2L与ALPK1基因的表达下调不再显著;在AGS细胞中沉默IQGAP1基因,IQGAP1与ALPK1基因的表达均显