关键词： 甲状腺乳头状癌   环状RNA   翻译   IQGAP1   上皮间质转化  

摘要： 目的:甲状腺癌是发病率最高的内分泌系统肿瘤,近来发病率明显上升,约95%以上的甲状腺癌病理类型为高分化的分化型甲状腺癌(Differentiated thyroid carcinoma,DTC),乳头状癌(Papillary thyroid carcinoma,PTC)为DTC中最常见。绝大多数PTC患者经过早期发现及规范手术,术后进行TSH抑制治疗,并视病情辅予放射性碘等治疗预后通常较好,但仍有部分患者出现肿瘤早期进展或者术后早期出现复发,甚至出现远处转移,降低无病生存率。环状RNA(circ RNA)是一种呈环形结构的RNA特殊剪切形式,近年来随着转录组测序以及翻译组高通量测序的普及,已有研究报道circ RNA并不完全是非编码RNA,circ RNA可能含有开放阅读框(Open reading frame,ORF),通过内部核糖体进入位点(Internal ribosome entry site,IRES)结合核糖体,从而翻译多肽,并由多肽影响细胞的增殖、迁移、侵袭及上皮间质转化等生物学行为。Circ PTPRM源于PTPRM(Protein Tyrosine Phosphatase Receptor Type M,PTPR-μ),由其第9-14外显子环形剪切而成。有研究证实circ PTPRM可影响肝癌细胞的增殖、迁移、侵袭。通过软件预测及评估,circ PTPRM存在内部核糖体进入位点及开放阅读框,可编码187个氨基酸的多肽circ PTPRM-187aa。本文旨在研究circ PTPRM-187aa在PTC中的作用,寻找circ PTPRM-187aa与下游分子之间的关联,并试图分析下游信号通路变化,明确该多肽对PTC生物学行为产生影响的作用机制。方法:首先选取临床组织样品,利用转录组测序数据分析circ RNA在PTC组织及配对癌旁组织中的表达情况,随后利用翻译组测序数据从中筛选出存在高翻译潜能的circ RNA。利用跨环状接点的特性设计PCR引物及RNase R酶评估筛选到的circ PTPRM环形结构及耐稳定性。利用q RT-PCR实验分析中国医科大学附属第一医院甲状腺外科临床获得的100对PTC组织和配对癌旁组织,观察circ PTPRM在组织中的表达情况并分析与临床病理资料的关系。q RT-PCR及荧光原位杂交观察circ PTPRM在Nthy-ori3-1细胞系和四种PTC细胞系的表达情况及结构定位。利用si RNA技术下调circ PTPRM的表达,q RT-PCR实验确认干扰效率后通过CCK-8和平板克隆实验检测PTC细胞增殖能力的变化,利用划痕实验及Transwell实验检测下调circ PTPRM对PTC细胞迁移、侵袭能力的影响,最后采用Western blot实验检测下调circ PTPRM对PTC细胞EMT相关蛋白的影响。q RT-PCR实验检测各外源性过表达circ PTPRM构型的过表达效率,Western blot实验检测各组过表达构型翻译多肽的能力。随后CCK-8和平板克隆实验检测各外源性过表达circ PTPRM构型对PTC细胞增殖能力的影响,利用划痕实验及Transwell实验检测各组PTC细胞的迁移、侵袭能力的影响,Western blot实验检测PTC细胞EMT相关蛋白的变化。使用凝胶电泳及串联质谱检测circ PTPRM翻译多肽的特异性序列;并使用免疫荧光实验检测FLAG标签融合蛋白的细胞定位。免疫沉淀及质谱实验检测与circ PTPRM-187aa发生作用的蛋白,并用Western blot实验进一步确认,并对比临床病理资料。Western blot实验检测CHX处理及过表达circ PTPRM对IQGAP1的表达量变化;转染HA标记的泛素并行免疫沉淀及Western blot实验检测IQGAP1泛素化修饰的变化。GSEA分析IQGAP1及circ PTPRM源基因PTPRM可能调控的信号通路,并分析IQGAP1与相应信号通路中关键蛋白的表达关系。Western blot实验检测单纯下调IQGAP1及共转染上调circ PTPRM对RAC1、CDC42、以及EMT相关蛋白表达的影响。结果:转录组测序中发现720个circ RNA在PTC组织与癌旁组织中呈现差异表达,其中上调301个,下调419个。通过对circ RNA的ORF、IRES结构进行预测,翻译组接头读取数、转录组差异4项数据综合评估,得出7个具有高翻译潜能的circ RNA,其中circ PTPRM差异最显著。核酸凝胶电泳证实circ PTPRM不存在于基因组DNA中,而是RNA环形剪切的产物;q RT-PCR实验证实circ PTPRM相比m RNA更耐受线性RNA消化酶的消化及温度、时间的自然降解。组织q

关键词： PTPRQ   IQGAP1   结直肠癌   去磷酸化   增殖   迁移  

摘要： 目的:初步探究PTPRQ促进结直肠癌细胞增殖和迁移的分子机制,为结直肠癌的诊断和防治提供靶点和治疗策略。方法:采用免疫组织化学分析PTPRQ在结肠腺癌中的表达,分析其表达与临床病理特点和预后的关系。采用免疫沉淀和质谱,分析与PTPRQ相互作用的蛋白。免疫共沉淀验证PTPRQ和IQGAP1蛋白间的相互作用,免疫组织化学分析两者在结肠腺癌中表达的相关性。采用si RNA干扰IQGAP1的表达,CCK8和划痕实验观察IQGAP1沉默是否会逆转PTPRQ的效应。采用通用型抗酪氨酸磷酸化抗体检测PTPRQ对IQGAP1酪氨酸磷酸化水平的影响。结果:共收集结肠腺癌组织样本94例和癌旁组织样本86例。与癌旁组织相比,PTPRQ在结肠腺癌组织中的表达显著增高（P＜0.0001）。PTPRQ的表达与年龄、性别、结直肠癌的TNM分期等无关,没有统计学差异。与PTPRQ低表达者相比较,PTPRQ高表达的患者预后更差（P=0.039）。免疫共沉淀和质谱结果显示,PTPRQ与IQGAP1存在蛋白间相互作用。免疫组织化学结果显示,PTPRQ和IQGAP1在结肠腺癌组织中的表达呈正相关（R=0.6114,P＜0.001）。与对照si RNA相比,沉默IQGAP1的表达后,显著下调了PTPRQ过表达细胞的增殖速度和迁移能力,进一步的磷酸化检测发现,与对照组相比,PTPRQ过表达的HCT116细胞中,IQGAP1的酪氨酸磷酸化水平显著下降。结论:PTPRQ在结肠腺癌中高表达,且与预后不良相关;PTPRQ通过与IQGAP1相互作用促进结直肠癌细胞增殖和迁移;PTPRQ导致IQGAP1酪氨酸去磷酸化。

摘要： 2071

关键词： IQGAP1   MIB1   pancreatic cancer   ST7  

摘要： Despite recent progress in cancer treatment, the prognosis of patients with pancreatic cancer still remains poor. Pancreatic tumors are reported to display high molecular heterogeneity. Elucidating the molecular mechanisms underlying pancreatic cancer progression is essential for improving patient treatment and survival. The overexpression of E3 ubiquitin ligase mind bomb 1 (MIB1) was previously described in pancreatic cancer cells, where it enhanced tumor cell proliferation. However, the role of MIB1 in pancreatic cancer progression remains elusive. In the present study, we confirmed that MIB1 expression is elevated in pancreatic cancer tissues and that high levels of MIB associate with unfavorable prognosis. Overexpression of MIB1 enhanced proliferation and invasion of pancreatic cancer cells both in vitro and in vivo. We further investigated the molecular mechanisms downstream of MIB1 and observed for the first time that MIB1 targets suppressor of tumorigenicity 7 protein (ST7), previously described as suppressor of tumorigenicity, for proteasomal degradation. Furthermore, we found that ST7 suppressed tumor growth by downregulating IQ motif containing GTPase activating protein 1 (IQGAP1) in pancreatic tumor cells. Thus, these data show that MIB1 promotes pancreatic cancer progression by inducing ST7 degradation followed by downregulation of IQGAP1 in pancreatic cancer cells. In conclusion, our research shows that the MIB1/ST7/IQGAP1 axis is essential for pancreatic cancer progression, and MIB1 inhibition may serve as a novel therapeutic strategy in patients with pancreatic cancer.

关键词： DP1   GPCR   IQGAP1   Interactome   Signaling   Trafficking  

摘要： Background: Mechanisms governing localization, trafficking and signaling of G protein-coupled receptors (GPCRs) are critical in cell function. Protein-protein interactions are determinant in these processes. However, there are very little interacting proteins known to date for the DP1 receptor for prostaglandin D-2. Methods: We performed LC-MS/MS analyses of the DP1 receptor interactome in HEK293 cells. To functionally validate our LC-MS/MS data, we studied the implications of the interaction with the IQGAP1 scaffold protein in the trafficking and signaling of DP1. Results: In addition to expected interacting proteins such as heterotrimeric G protein subunits, we identified proteins involved in signaling, trafficking, and folding localized in various cell compartments. Endogenous DP1-IQGAP1 co-immunoprecipitation was observed in colon cancer HT-29 cells. The interaction was augmented by DP1 agonist activation in HEK293 cells and GST-pulldown assays showed that IQGAP1 binds to intracellular loops 2 and 3 of DP1. Co-localization of the two proteins was observed by confocal microscopy at the cell periphery and in intracellular vesicles in the basal state. PGD2 treatment resulted in the redistribution of the DP1-IQGAP1 co-localization in the perinuclear vicinity. DP1 receptor internalization was promoted by overexpression of IQGAP1, while it was diminished by IQGAP1 knockdown with DsiRNAs. DP1-mediated ERK1/2 activation was augmented and sustained overtime by overexpression of IQGAP1 when compared to DP1 expressed alone. IQGAP1 knockdown decreased ERK1/2 activation by DP1 stimulation. Interestingly, ERK1/2 signaling by DP1 was increased when IQGAP2 was silenced, while it was impaired by IQGAP3 knockdown. Conclusions: Our findings define the putative DP1 interactome, a patho-physiologically important receptor, and validated the interaction with IQGAP1 in DP1 function. Our data also reveal that IQGAP proteins may differentially regulate GPCR signaling. General significanc

摘要： In response to oncogenic signals, Alternative Splicing (AS) regulators such as SR and hnRNP proteins show altered expression levels, subnuclear distribution and/or post-translational modification status, but the link between signals and these changes remains unknown. Here, we report that a cytosolic scaffold protein, IQGAP1, performs this task in response to heat-induced signals. We show that in gastric cancer cells, a nuclear pool of IQGAP1 acts as a tethering module for a group of spliceosome components, including hnRNPM, a splicing factor critical for the response of the spliceosome to heat-shock. IQGAP1 controls hnRNPM's sumoylation, subnuclear localisation and the relevant response of the AS machinery to heat-induced stress. Genome-wide analyses reveal that IQGAP1 and hnRNPM co-regulate the AS of a cell cycle-related RNA regulon in gastric cancer cells, thus favouring the accelerated proliferation phenotype of gastric cancer cells. Overall, we reveal a missing link between stress signals and AS regulation.

关键词： 甲状腺癌   miR-506-3p   IQ结构域GTP酶激活蛋白(IQGAP)1   细胞增殖   迁移   侵袭  

摘要： 目的探讨miR-506-3p靶向IQ结构域GTP酶激活蛋白(IQGAP)1调控甲状腺癌细胞增殖、迁移和侵袭的分子机制。方法采用qRT-PCR和Western印迹检测甲状腺癌细胞SW579、FTC-133、KAT-18和正常甲状腺上皮细胞TEC中miR-506-3p、IQGAP1 mRNA和IQGAP1蛋白的表达;建立miR-506-3p过表达或IQGAP1抑制表达的SW579细胞株。采用噻唑蓝(MTT)法检测细胞增殖活力,Transwell法检测细胞迁移和侵袭能力,Western印迹检测细胞周期蛋白(Cyclin)D1、P21、P27、基质金属蛋白酶(MMP)-2、MMP-9和MMP-14蛋白表达水平。采用双荧光素酶报告基因实验验证miR-506-3p对IQGAP1的靶向作用。结果与正常甲状腺上皮细胞TEC相比,甲状腺癌细胞SW579、FTC-133和KAT-18中miR-506-3p表达显著降低,而IQGAP1表达显著升高。miR-506-3p过表达或IQGAP1抑制表达均可抑制SW579细胞的增殖、迁移和侵袭。IQGAP1是miR-506-3p的靶基因,miR-506-3p可负向调控IQGAP1表达。IQGAP1过表达可显著逆转miR-506-3p对SW579细胞的增殖、迁移和侵袭的抑制作用。结论miR-506-3p可通过下调IQGAP1表达抑制甲状腺癌细胞的增殖、迁移和侵袭。

关键词： PUMA蛋白   IQGAP1蛋白   胰腺癌   临床病理特征  

摘要： 目的探讨PUMA、IQGAP1蛋白在胰腺癌中的表达及与临床病理、预后生存的相关性。方法选取2016年10月至2018年10月本院收治的胰腺癌患者胰腺癌组织标本、远离胰腺肿瘤3 cm的癌旁组织标本各74份,比较两组织中PUMA、IQGAP1蛋白的表达情况,并探讨其与病理特征的关系。随访2年,了解74例患者生存率,采用ROC曲线分析不同PUMA、IQGAP1表达的胰腺癌患者预后2年生存情况,采用Logistic回归模型分析影响胰腺癌患者2年预后的独立危险因素。结果胰腺癌组织中的PUMA阳性率显著低于癌旁组织,IQGAPl阳性率显著高于癌旁组织,差异均有统计学意义(P<0.05)。TNM分期为Ⅲ~Ⅵ期、有淋巴结转移、分化程度为中-低分化的胰腺癌患者PUMA阴性率、IQGAPl阳性率更高(P<0.05)。74例胰腺癌患者2年生存率为16.22%。PUMA阴性表达、IQGAPl阳性表达的胰腺癌患者死亡率高于PUMA阳性表达、IQGAPl阴性表达的患者,差异有统计学意义(P<0.05)。生存分析显示PUMA(表达阳性)、IQGAPl(表达阴性)者生存期较长,Keplan-meier生存曲线明显不同(P<0.05)。TNM(Ⅲ~Ⅵ期)、淋巴结转移(有)、分化程度(中-低分化)、PUMA(表达阴性)、IQGAPl(表达阳性)为影响胰腺癌患者预后生存的独立危险因素(P<0.05)。结论 PUMA阴性、IQGAPl阳性为影响胰腺癌患者预后生存的危险因素,临床可通过加强监测二者表达情况,以评估胰腺癌患者病情进展情况及预后。

关键词： IQGAP1   Microvascular endothelium   Cytoskeleton   Acute lung injury   Rap1   ICAM-1  

摘要： Pulmonary microvascular barrier dysfunction is a hallmark feature of acute lung injury (ALI). IQGAP1 is a ubiquitously expressed scaffolding protein known to regulate cancer metastasis, angiogenesis, and barrier stability. However, the function of IQGAP1 in lipopolysaccharide (LPS)-induced microvascular endothelial hyperpermeability remains poorly understood. In the present study, we demonstrated that IQGAP1 was markedly upregulated in LPS-induced ALI models and rat pulmonary microvascular endothelial cells (RPMVECs). Lentivirus-mediated knockdown of IQGAP1 significantly attenuated the formation of actin stress fibers, phosphorylation of myosin light chain (MLC), and disruption of VE-cadherin, thereby protecting the RPMVECs barrier failure from LPS damage. In addition, IQGAP1 depletion reduced the reactive oxygen species (ROS)-mediated increase in intracellular adhesion molecule-1 (ICAM-1) in RPMVECs stimulated with LPS. Mechanistically, we found that the upregulation of IQGAP1 affected the activity of Rap1 and the downstream phosphorylation of Src. In conclusion, these findings reveal an essential mechanism by which increased IQGAP1 in LPS-treated RPMVECs promotes barrier dysfunction and ICAM-1 upregulation, at least in part by regulating Rap1/Src signalling, indicating that IQGAP1 may be a potential therapeutic target to prevent endothelial hyperpermeability and inflammation in ALI.