关键词： IQGAP1   氧化应激   mtDNA   焦亡   动脉粥样硬化  

摘要： 研究背景 动脉粥样硬化（Atheroscleros,AS）是一种慢性炎症性疾病,简单来说一般包括三个病理过程—脂质沉积、炎症浸润、泡沫细胞形成最终导致血管内径变窄,是世界上最常见的引起死亡的原因之一。AS的形成与内皮细胞,单核细胞,巨噬细胞以及平滑肌细胞的功能有着密切关系,在形成早期,血液和动脉内膜之间内皮单层发生改变,这是AS的起始部位;且内皮细胞易受高脂血症、氧化应激、趋化因子、DNA异位等损伤模式分子的影响引起炎症反应,这进一步导致内环境的紊乱促进了内皮细胞功能活化。然而,目前药物治疗AS仍然具有一定的局限性。越来越多的研究表明抑制内皮细胞焦亡可能成为治疗AS的一种新思路,但其具体发生机制仍不清楚。焦亡是一种不同于细胞凋亡和坏死的程序性细胞死亡过程,与炎症小体的形成密切相关并伴随着促炎因子的大量释放。IQGAP蛋白家族存在于许多生物体中,已在人类中鉴定出三种IQGAP1、IQGAP2和IQGAP3,其中IQGAP1在组织中广泛表达,是最具特征的IQGAP蛋白质,在真核生物中具有进化保守性,有助于形成调节细胞骨架动力学、细胞内信号传导和细胞间相互作用的复合物;同时,在调节细胞粘附、信号传导和迁移中起重要作用。随着研究的逐渐深入,日益增多的证据表明IQGAP1作用于心肌细胞影响心脏重塑,心室壁变薄和收缩力下降;同时对血管生成过程中内皮细胞的增殖和迁移有着重要作用,这与线粒体活性氧（mt ROS）的产生有关。重要的是,随着mt ROS在细胞内累积可能会导致线粒体损伤进而发生Caspase-1-NLRP3经典途径焦亡。新的发现表明线粒体损伤导致线粒体DNA（mtDNA）泄露到细胞质,激活细胞质中DNA感受器cGAS-STING,并触发分子链反应以诱导I型干扰素（IFN-I）,启动和维持炎症反应。在心血管领域中发现mtDNA激活的cGAS-STING信号在心肌细胞焦亡中同样被广泛关注,但在AS中未有确切报道。我们的探索性研究IQGAP1是否通过调节线粒体功能导致mtDNA泄露激活cGAS-STING信号通路诱导内皮细胞焦亡以及特异性小分子抑制剂RU.521和C-176的治疗作用,以期对AS的发生机制和治疗提供了一个新的视野。 研究目的 1.研究动脉粥样硬化模型中IQGAP1,cGAS,STING对内皮细胞焦亡的影响。 2.阐明IQGAP1对动脉粥样硬化进程中活性氧ROS影响及其部分调控机制。 3.检测线粒体损伤后细胞质中ds DNA泄露并进行定量和组织定位分析。 4.明确IQGAP1在线粒体损伤过程中调节mtDNA释放激活DNA感受器cGAS-STING信号通路。 5.小分子抑制剂RU.521和C-176通过特异性阻断cGAS和STING信号通路,从而抑制IQGAP1促进的动脉粥样硬化,探索可能存在的的药物靶点。 研究方法 1.购买40只ApoE-/-C57BL/J和40只Ldlr-/-C57BL/J,两种小鼠各随机分为四组正常饮食组（Cont）,高脂饮食组（HFD）,高脂饮食+AAV-Sh RNA-IQGAP1（AAV-Si-IQGAP1）组,高脂饮食+AAV-Sh RNA-NC（AAV-NC）组。在适应一周后注射腺病毒及其对照,两种小鼠分别使用相对应的高脂饲料连续喂养12周,每间隔两周记录小鼠体重。使用HE,油红切片,油红大体染色对主动脉组织进行病理检测并检测血清生化指标LDL,TC,TG,HDL。使用IHC、IF和Westernblot观察焦亡相关分子NLRP3,GSDMD-N,Caspase-1和cGAS,STING信号通路蛋白表达,qRT-PCR观察炎症因子IL-1β,IL-18和DNA感受器cGAS,STING以及抗氧化应激GPX-1,SOD-1 m RNA的表达情况。 2.在小鼠组织中通过线粒体追踪标记Mito-Tracker与ds DNA抗体进行双重定位染色,观察mtDNA的释放情况的定性分析。同时,对小鼠主动脉组织进行利用mtDNA提取试剂盒进行q-PCR定量分析。利用TUNEL染色观察主动脉组织内皮细胞焦亡情况,通过透射电镜观察线粒体损伤。 3.在体外通过棕榈酸（PA,0.3mmol/L）刺激人脐静脉内皮细胞（HUVECs）模拟内皮细胞损伤模型。通过Mito SOX染色,DCFH-DA流式细胞术检测PA诱导HUVECs细胞中活性氧（ROS）的产生。通过Western blot观察PA诱导的HUVECs中焦亡相关蛋白NLRP3,GSDMD-N,Caspase-1,IL-1β,IL-18的表达情况,扫描电镜观察焦亡细胞膜“打孔”现象。 4.在体外分别构建IQGAP1的过表达质粒和阴性对照（pc DNA3.1-3x Flag-IQGAP1,pc DNA3.1-3x Flag-NC）,小干扰RN

关键词： Hepatocellular carcinoma   PD-L1   M2 macrophages   CD8(+)T cells   Immune escape   MISP   IQGAP1   STAT3  

摘要： Background: Hepatocellular carcinoma (HCC) is a prevailing cancer affecting human health. M2 macrophages are essential in mediating immune responses in tumors. This study investigated the action of M2 macrophages in immune escape of HCC. Methods: Mitotic spindle positioning (MISP), IQ motif containing GTPase activating protein 1 (IQGAP1) and programmed cell death-1 (PD-L1) levels in primary HCC/tumor-adjacent tissues were determined by Western blot, followed by correlation analysis. M2 macrophage and CD3(+)CD8(+)T cell percentages were estimated by flow cytometry. Hep3B and HepG2 cells were treated with M2 macrophage conditioned medium (M2-CM) and M2 macrophage-derived extracellular vesicles (M2-EVs) and/or co-cultured with CD8(+)T cells, followed by assessment of cell viability and apoptosis. TNF- alpha and INF-gamma levels were measured by ELISA. MISP and IQGAP1 over-expression plasmids were transfected into HCC cells to explore their role in immune escape. The interactions among MISP, IQGAP1, STAT3, and PD-L1 were analyzed by co-immunoprecipitation. The mechanism of M2-EVs in HCC immune escape was verified in nude mice. Results: MISP/IQGAP1/PD-L1 were upregulated in HCC tissues. MISP negatively-correlated with IQGAP1/PD-L1 and IQGAP1 positively-correlated with PD-L1. M2 macrophages were reduced but CD8(+)T cells were increased in HCC tissues with high MISP expression. M2-CM or M2-EVs inhibited the killing ability of CD8(+)T cells, increased HCC cell viability, impeded HCC cell apoptosis, induced CD8(+)T cell apoptosis, downregulated TNF- alpha and INF-gamma, and upregulated PD-L1. M2-EVs facilitated HCC cell immune escape by potentiating IQGAP1 nuclear trans-location and activating STAT3 phosphorylation through MISP downregulation. In vivo experiments further verified the action of M2-EVs through MISP. Conclusion: M2-EVs promote HCC cell immune escape by upregulating PD-L1 through the MISP/IQGAP1/STAT3 axis.

关键词： Atherosclerosis   IQGAP1   Oxidative stress   MtDNA sensors   Pyroptosis  

摘要： Atherosclerosis (AS) is the most common cardiovascular disease and has limited therapeutic options. IQ motifcontaining GTPase-activating protein 1 (IQGAP1) is an important scaffolding protein regulating mitochondrial function influencing endothelial cell activity. Evidence suggests that mitochondrial damage can lead to leakage of mtDNA into the cytoplasm to activate the DNA sensor cGAS-STING to mediate pyroptosis. However, whether IQGAP1 induces NLRP3-mediated endothelial cell pyroptosis by regulating mitochondrial function and activating the DNA sensor cGAS-STING, and its underlying mechanisms remain unclear. In vivo, ApoE-/- C57BL/J and Ldlr-/- C57BL/J mice were pre-injected with adeno-associated virus (AAV) by the tail vein to specifically silence IQGAP1 expression and were fed a high-fat diet (HFD) for 12 weeks. IQGAP1 knockdown reduced mtDNA release and decreased the expression of DNA receptors and pyroptosis-related molecules as determined by immunohistochemistry and immunofluorescence. In vitro, palmitic acid (0.3 mmol/L) was incubated with human umbilical vein endothelial cells (HUVECs) for 24 h. Overexpression of IQGAP1 in HUVECs, flow cytometry, and mitochondrial superoxide staining revealed increased levels of ROS. Moreover, the mitochondrial tracker with dsDNA co-localization showed the release of mtDNA into the cytoplasm increased, which activated the DNA receptor cGAS-STING. Protein blotting and TUNEL staining revealed that IQGAP1 promoted NLRP3mediated pyroptosis. Furthermore, cGAS or STING small-molecule inhibitors RU.521 or C-176 reverse IQGAP1-promoted HUVECs from undergoing NLRP3-mediated pyroptosis. These results suggest that IQGAP1 promotes oxidative stress and mtDNA release, activates the DNA sensor cGAS-STING, and leads to NLRP3mediated pyroptosis. The present study provides new insights into the mechanisms underlying AS and identifies new pharmacological targets for treatment.

关键词： Colorectal cancer   Vemurafenib resistance   Metastasis   LY2835219   ARF1-IQGAP1 interaction  

摘要： Introduction: Acquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation. Objectives: We identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study. Methods: DIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQdomain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of *** biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays. Results: We found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer Conclusion: This study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that

关键词： annular pancreas   cell migration   IQGAP1   malformation   NRCAM  

摘要： Background: Annular pancreas (AP) is a congenital defect of unknown cause in which the pancreas encircles the duodenum. Theories include abnormal migration and rotation of the ventral bud, persistence of ectopic pancreatic tissue, and inappropriate fusion of the ventral and dorsal buds before rotation. The few reported familial cases suggest a genetic ***: We conducted exome sequencing in 115 affected infants from the California birth defects ***: Seven cases had a single heterozygous missense variant in IQGAP1, five of them with CADD scores >20;seven other infants had a single heterozygous missense variant in NRCAM, five of them with CADD scores >20. We also looked at genes previously associated with AP and found two rare heterozygous missense variants, one each in PDX1 and ***: IQGAP1 and NRCAM are crucial in cell polarization and migration. Mutations result in decreased motility which could possibly cause the ventral bud to not migrate normally. To our knowledge, this is the first study reporting a possible association for IQGAP1 and NRCAM with AP. Our findings of rare genetic variants involved in cell migration in 15% of our population raise the possibility that AP may be related to abnormal cell migration.

关键词： ZC3H13   IQGAP1   m6A modification   Papillary thyroid   carcinoma  

摘要： Purpose: The purpose of this study was to clarify the effect of ZC3H13 on the growth of papillary thyroid carcinoma (PTC). Methods: Firstly, we used qRT-PCR and Western blot to compare the difference in the expression of ZC3H13 between normal thyroid epithelial cells and PTC cell lines. Then, ZC3H13 overexpression/knockout thyroid cancer cells were constructed by lentivirus transfection, and the effects of overexpression of ZC3H13 on the proliferation, migration and invasion of PTC cells were detected by CCK8 and transwell experiments. Lastly, MeRIP-qPCR, RIP and o Actinomycin D were used to verify that ZC3H13 regulated the expression of downstream target gene IQGAP1 through m6A modification. Results: ZC3H13 expression was decreased in PTC cell lines BCPAP, KTC-1, k1, HTH83, and TPC1. Proliferation, invasion, and migration of PTC cells were inhibited by overexpressed ZC3H13 but increased by knockdown of ZC3H13. IQGAP1 expression was suppressed by ZC3H13 overexpression but enhanced by ZC3H13 knockdown. In ZC3H13-overexpressed PTC cells, the m6A level of IQGAP1 mRNA was increased, and the IQGAP1 mRNA expression was decreased with the increasing time of Actinomycin D treatment. YTHDF2 enriched more IQGAP1 mRNA than IgG and knockdown of YTHDF2 reversed the effect of ZC3H13 overexpression on IQGAP1 mRNA stability. The xenograft tumor experiment in nude mice confirmed that the overexpression of ZC3H13 inhibited tumor growth, while overexpression of IQGAP1 could reverse the inhibitory effect of ZC3H13 overexpression on tumor growth. Conclusion: ZC3H13 mediates IQGAP1 mRNA degradation by promoting m6A modification of IQGAP1 mRNA, this provides a prospective therapeutic target for PTC. (c) 2023, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://***/licenses/bync-nd/4.0/).

关键词： Cell migration   Focal Adhesion   IQGAP1   Hax1   Microtubules  

摘要： Cell migration is a highly orchestrated process requiring the coordination between the cytoskeleton, cell membrane and extracellular matrix adhesions. Our previous study demonstrated that Hax1 interacts with EB2, a microtubule end-binding protein, and this interaction regulate cell migration in keratinocytes. However, little is known about the underlying regulatory mechanism. Here, we show that Hax1 links dynamic focal adhesions to regulate cell migration via interacting with IQGAP1, a multidomain scaffolding protein, which was identified by affinity purification coupled with LC-MS/MS. Biochemical characterizations revealed that C-terminal region of Hax1 and RGCT domain of IQGAP1 are the most critical binding determinants for its interaction. IQGAP1/Hax1 interaction is essential for cell migration in MCF7 cells. Knockdown of HAX1 not only stabilizes focal adhesions, but also impairs the accumulation of IQGAP in focal adhesions. Further study indicates that this interaction is critical for maintaining efficient focal adhesion turnover. Perturbation of the IQGAP1/Hax1 interaction in vivo using a membrane-permeable TAT-RGCT peptide results in impaired focal adhesion turnover, thus leading to inhibition of directional cell migration. Together, our findings unravel a novel interaction between IQGAP1 and Hax1, suggesting that IQGAP1 association with Hax1 plays a significant role in focal adhesion turnover and directional cell migration.

关键词： Extracellular vesicles   CRISPR/Cas9   liver cancer   sorafenib resistance   cancer stem cells  

摘要： BackgroundSorafenib resistance poses therapeutic challenges in HCC treatment, in which cancer stem cells (CSCs) plays a crucial role. CRISPR/Cas9 can be utilized as a potential technique to overcome the drug resistance. However, a safe, efficient and target specific delivery of this platform remains challenging. Extracellular vesicles (EVs), the active components of cell to cell communication, hold promising benefits as delivery *** Herein we report the normal epithelial cell -derived EVs engineered with HN3(HLC9-EVs) show competing tumor targeting ability. Anchoring HN3 to the membrane of the EVs through LAMP2, drastically increased the specific homing of HLC9-EVs to GPC3(+)Huh-7 cancer cells rather than co-cultured GPC3(-)LO2 cells. Combination therapy of HCC with sorafenib and HLC9-EVs containing sgIF to silence IQGAP1 (protein responsible for reactivation of Akt/PI3K signaling in sorafenib resistance) and FOXM1 (self-renewal transcription factor in CSCs attributed to sorafenib resistance), exhibited effective synergistic anti-cancer effect both in vitro and in vivo. Our results also showed that disruption of IQGAP1/FOXM1 resulted in the reduction of CD133(+) population that contribute to the stemness of liver cancer *** By reversing sorafenib resistance using combination therapeutic approach with engineered EVs encapsulated CRISPR/Cas9 and sorafenib, our study foreshadows a path for a better, accurate, reliable and successful anti-cancer therapy in the future.