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

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.