关键词： 皮肤黑色素瘤   成纤维细胞   基质细胞   活化转录因子3   白介素6  

摘要： 背景与目的皮肤黑色素瘤是起于皮肤表皮基底层的黑色素细胞的皮肤恶性肿瘤。黑色素瘤极易发生转移,是皮肤三大恶性肿瘤中最致命的一种。临床上对于黑色素瘤,尤其是发生转移的皮肤黑色素瘤病例的治疗一直是一个很棘手的问题。黑色素瘤细胞通常是由被激活的细胞外信号调节激酶(ERK)途径进行调节,该信号由BRAF激活突变所诱导。BRAF是RAF家族的一种蛋白激酶,其功能位于RAS下游,通过磷酸化MEK激活ERK来帮助刺激细胞生长和存活。临床上用于治疗皮肤恶性黑色素瘤的BRAF抑制剂可短暂有效,但是大多数患者会产生耐药性。肿瘤微环境(tumor microenvironment,TME)指的是肿瘤在发生发展过程中所处的局部环境,构成这一局部环境的细胞成分复杂,包括但不限于免疫细胞、血细胞、内皮细胞、脂肪细胞和细胞基质。肿瘤基质是肿瘤微环境的重要组成部分,主要由成纤维细胞和细胞外基质构成。在过去的几十年里,越来越多的研究成果证实TME在决定疾病进展和治疗结果方面的作用变得越来越明显。肿瘤细胞与微环境之间的相互作用机制非常复杂,但可分为两大类:即接触式和非接触式途径。其中接触式途径涉及细胞-细胞和细胞外基质粘附分子的接触依赖性机制。而在非接触式途径中,诸多可溶性分子,如生长因子,趋化因子和细胞因子,以及可溶性的亚细胞器(包括微泡和外泌体)等在整个过程中起到至关重要的作用,最终,这些相互作用通过分泌和旁分泌机制,完成与恶性肿瘤细胞的相互作用。肿瘤微环境肿瘤基质细胞中最主要的细胞成分为成纤维细胞。最近,越来越多的相关研究成果显示,肿瘤基质组织与肿瘤抗药性的产生有很大关系,提示癌症治疗应该包括靶向基质细胞来抑制肿瘤的策略。ATF3(activating transcription factor 3,ATF3)为 ATF/CREB 家族重要成员之一。作为重要的转录因子,ATF3在不同组织中表现的功能可能存在差异。ATF3基因在肿瘤发展中可能会表现出促癌基因和抑癌基因正反两方面作用。本课题组前期研究结果显示在人表皮角质形成细胞中,上调ATF3表达水平会抑制p53基因依赖的细胞衰老,从而促进皮肤鳞状细胞癌的发生发展。人真皮成纤维细胞中亦有ATF3基因表达,但对其功能的研究甚为少见,目前为止只有一篇报道指出低表达ATF3的成纤维细胞可转化为癌症相关成纤维细胞表形,并最终促进了皮肤鳞状细胞癌的发展。但是人真皮成纤维细胞ATF3与皮肤黑色素瘤之间的关系尚未见报道。为填补这一空白,本研究首先拟通过对皮肤黑色素瘤临床病例标本进行检测,明确ATF3在临床黑色素瘤基质细胞中表达情况。随后利用ATF3过表达和敲除等手段,借助共培养体系和条件培养基等方法,通过体外体内实验检测原代人真皮成纤维细胞对皮肤黑色素瘤细胞生长与迁移等生物学行为的影响,并针对一系列指标进行检测以明确机制。最后本研究将尝试验证药物诱导ATF3增高表达的人真皮成纤维细胞对人皮肤黑色素瘤细胞的影响,以期将来为皮肤黑色素瘤的治疗提供新的思路和方案。方法1.对皮肤恶性黑色素瘤临床病例标本ATF3表达水平的检测首先收集病理诊断为皮肤黑色素瘤的临床病例肿瘤石蜡标本,利用免疫组织化学染色技术对石蜡切片进行染色,后利用RNAscope原位杂交染色技术,对免疫组织化学染色结果进行验证。随后利用免疫荧光双重染色技术,检测人正常真皮成纤维细胞、人皮肤良性黑色素痣以及人皮肤黑色素瘤肿瘤基质组织中成纤维细胞ATF3表达水平,并对结果进行统计学分析。2.人真皮成纤维细胞ATF3对恶性黑色素瘤细胞生长与迁移影响体外及体内实验检测首先分离原代人真皮成纤维细胞并培养。利用已有针对人ATF3基因过表达和CRISPR/Cas9基因敲除的慢病毒和逆病毒来制备相应的病毒,并用病毒感染人真皮成纤维细胞已达到ATF3过表达及敲除。利用qRT-PCR以及Western Blot等技术对ATF3表达效果进行验证。利用共培养体系,以及收集ATF3不同表达水平的成纤维细胞条件培养基来培养黑色素瘤细胞,然后利用CCK-8、细胞克隆形成实验、transwell以及细胞划痕实验等方法,检测ATF3过表达或敲除的人真皮成纤维细胞对皮肤黑色素瘤生长与迁移能力的影响。采用8周nude/nude裸鼠,随机分为二组,每组三只,将ATF3过表达的人真皮成纤维细胞与皮肤黑色素瘤细胞混合,利用裸鼠皮下成瘤实验检测其对黑色素瘤细胞体内成瘤能力的影响。3.人真皮成纤维细胞ATF3对恶性黑色素瘤细胞生长与迁移影响分子机制的检测利用qRT-PCR技术,对过表达或敲除ATF3基因的人真皮成纤维细胞相应炎性因子等基因变化进行检测,筛选备选下游基因。利用ELISA技术,对条件培养基中相应炎性因子分泌蛋白水平变化进行检测,以验证qRT-PCR结果。利用候选基因相应的重组蛋白或者抑制剂,结合ATF3基因

关键词： ZnO NPs   ATF3   凋亡   DNA损伤   p53  

摘要： 纳米氧化锌（Zn O nanoparticles,Zn O NPs）是一类新型高性能的多功能无机材料,被广泛应用于日用品、电子设备、橡胶、食品药品等领域,其对环境和职业暴露人群的潜在风险逐渐引起人们广泛关注。呼吸道是Zn O NPs主要暴露途径之一,Zn O NPs可引起呼吸道上皮细胞炎症、凋亡、DNA损伤等不良结局。Zn O NPs对细胞的毒效应主要取决于Zn O NPs的暴露程度（外因）和细胞的反应（内因）,然而目前对Zn O NPs发挥毒效应过程中的关键响应基因及其功能的认识尚不全面。我们前期研究发现Zn O NPs显著诱导ATF3表达,本学位论文对ATF3蛋白在Zn O NPs致人支气管上皮细胞毒效应中的作用进行深入研究。本课题所采用的Zn O NPs平均粒径为34.0 nm,呈六方纤锌矿晶体结构,具有较好分散性。首先利用CCK-8检测Zn O NPs处理条件下人支气管上皮（HBE）细胞活力变化。结果显示,5μg/m L浓度Zn O NPs显著降低HBE细胞活力,随着浓度的增加,细胞活力进一步下降。进一步利用荧光探针检测HBE细胞在Zn O NPs处理下细胞内总活性氧（ROS）生成情况。结果显示,5μg/m L浓度Zn O NPs作用下HBE细胞内ROS含量显著升高。利用γH2AX染色观察HBE细胞在Zn O NPs处理下DNA损伤情况。结果显示,5μg/m L浓度Zn O NPs引起HBE细胞内DNA双链断裂。以上结果说明,Zn O NPs可引起HBE细胞的细胞毒性效应以及遗传毒性效应。实时荧光定量PCR以及蛋白质免疫印迹结果显示,Zn O NPs处理后细胞内ATF3表达显著上调。活性氧清除剂（NAC）预处理后,Zn O NPs引起ATF3升高的水平得到回复,说明ATF3受Zn O NPs诱导的氧化应激调控。Nrf2信号通路是Zn O NPs引起氧化应激反应的关键信号通路,于是我们探索Nrf2对ATF3表达的调控。抑制HBE细胞内源Nrf2表达后,ATF3 m RNA水平以及蛋白质水平均显著降低;荧光素酶报告基因实验结果显示,Nrf2调控ATF3启动子的转录活性。进一步,我们研究了ATF3在Zn O NPs毒效应中的作用。我们发现,敲低ATF3后,Zn O NPs刺激引起的γH2AX形成显著增加,而细胞活力显著升高、细胞凋亡受到抑制。以上结果提示ATF3抑制Zn O NPs引起的DNA损伤,并促进Zn O NPs引起的细胞凋亡。p53是调节DNA损伤修复的关键蛋白,免疫共沉淀（Co-IP）结果证实ATF3与p53存在相互作用。并且ATF3可抑制Zn O NPs引起的p53蛋白的降解,这说明ATF3通过稳定p53促进DNA损伤修复。综上,本论文发现:1)Zn O NPs通过Nrf2信号通路促进HBE细胞中ATF3表达;2)HBE细胞中敲低ATF3导致Zn O NPs引起的DNA损伤显著增加;3)HBE细胞中敲低ATF3抑制Zn O NPs引起的细胞凋亡。本论文证明ATF3是支气管上皮细胞响应Zn O NPs暴露的关键基因,可抑制Zn O NPs诱导的DNA损伤,这将有助于阐明Zn O NPs发挥毒效应的分子机制,并可能为Zn O NPs造成的健康风险提供防护依据和手段。

关键词： Alcoholic liver disease   RNA-seq   Liver   NMN   ATF3   ERK1/2   Sirtuin   HIGH-RESOLUTION METABOLOMICS   P38 MAPK   ETHANOL   ATF3   ACTIVATION   STRESS   BIOSYNTHESIS   METABOLISM   KINASE   MITOCHONDRIA  

摘要： Background: Chronic alcohol consumption is a significant cause of liver disease worldwide. Several biochemical mechanisms have been linked to the initiation and progression of alcoholic liver disease (ALD) such as oxidative stress, inflammation, and metabolic dysregulation, including the disruption of NAD+/NADH. Indeed, an ethanol-mediated reduction in hepatic NAD(+) levels is thought to be one factor underlying ethanol-induced steatosis, oxidative stress, steatohepatitis, insulin resistance, and inhibition of gluconeogenesis. Therefore, we applied a NAD(+) boosting supplement to investigate alterations in the pathogenesis of early-stage ALD. Methods: To examine the impact of NAD(+) therapy on the early stages of ALD, we utilized nicotinamide mononucleotide (NMN) at 500 mg/kg intraperitoneal injection every other day, for the duration of a Lieber-DeCarli 6-week chronic ethanol model in mice. Numerous strategies were employed to characterize the effect of NMN therapy, including the integration of RNA-seq, immunoblotting, and metabolomics analysis. Results: Our findings reveal that NMN therapy increased hepatic NAD(+) levels, prevented an ethanol-induced increase in plasma ALT and AST, and changed the expression of 25% of the genes that were modulated by ethanol metabolism. These genes were associated with a number of pathways including the MAPK pathway. Interestingly, our analysis revealed that NMN treatment normalized Erk1/2 signaling and prevented an induction of Atf3 overexpression. Conclusions: These findings reveal previously unreported mechanisms by which NMN supplementation alters hepatic gene expression and protein pathways to impact ethanol hepatotoxicity in an early-stage murine model of ALD. Overall, our data suggest further research is needed to fully characterize treatment paradigms and biochemical implications of NAD(+)-based interventions.

关键词： 慢性阻塞性肺疾病   ATF3   ATF4   γ-GCS表达  

摘要： 目的:探究慢性阻塞性肺疾病(COPD)患者肺组织中活化转录因子3(ATF3)与活化转录因子4(ATF4)对γ-谷氨酰半胱氨酸合酶(γ-GCS)表达的影响,为预防及诊断COPD提供临床资料。方法:63例COPD确诊患者(COPD组)及同期在体检中心进行健康体检的正常居民(正常组)63例,比较2组患者治疗前后ATF3mRNA、ATF4mRNA、γ-GCSm RNA表达含量、ATF3、ATF4、γ-GCS蛋白表达含量及COPD组患者ATF3、ATF4、γ-GCS信使RNA和蛋白及心肺功能指标的相关性。结果:COPD组治疗前ATF3mRNA、ATF4mRNA、γ-GCSmRNA、ATF3蛋白、ATF4蛋白、γ-GCS蛋白与正常组比较,差异有统计学意义(P<0.05),COPD组治疗后ATF4 mRNAγ-GCS蛋白与正常组比较差异有统计学意义(P<0.05);同时COPD组患者ATF3、ATF4、γ-GCS信使RNA和蛋白与心肺功能相关指标相关性明显,差异有统计学意义(P<0.05)。结论:COPD患者肺组织中ATF3与ATF4可影响γ-GCS的表达,并且可直接反应患者的心肺功能,临床上可以通过对患者肺组织中ATF3与ATF4对γ-GCS表达的影响,及时的预防及诊断COPD。

关键词： Mitochondrial oxidative stress   Activating transcription factor 3   Inflammation   Brain microvascular endothelial cells   Lipolysis   Triglyceride-rich lipoproteins   RNA-Seq   TUMOR-NECROSIS-FACTOR   FATTY-ACID OXIDATION   ACTIVATING TRANSCRIPTION   LIPOLYSIS PRODUCTS   ENDOTHELIAL-CELLS   GENE-EXPRESSION   FACTOR-3 ATF3   MESSENGER-RNA   UP-REGULATION   INDUCTION  

摘要： Elevation of blood triglycerides, primarily triglyceride-rich lipoproteins (TGRL), is an independent risk factor for cardiovascular disease and vascular dementia (VaD). Accumulating evidence indicates that both atherosclerosis and VaD are linked to vascular inflammation. However, the role of TGRL in vascular inflammation, which increases risk for VaD, remains largely unknown and its underlying mechanisms are still unclear. We strived to determine the effects of postprandial TGRL exposure on brain microvascular endothelial cells, the potential risk factor of vascular inflammation, resulting in VaD. We showed in Aung et al., J Lipid Res., 2016 that postprandial TGRL lipolysis products (TL) activate mitochondrial reactive oxygen species (ROS) and increase the expression of the stress-responsive protein, activating transcription factor 3 (ATF3), which injures human brain microvascular endothelial cells (HBMECs) in vitro. In this study, we deployed high-throughput sequencing (HTS)-based RNA sequencing methods and mito stress and glycolytic rate assays with an Agilent Seahorse XF analyzer and profiled the differential expression of transcripts, constructed signaling pathways, and measured mitochondrial respiration, ATP production, proton leak, and glycolysis of HBMECs treated with TL. Conclusions: TL potentiate ROS by mitochondria which activate mitochondrial oxidative stress, decrease ATP production, increase mitochondrial proton leak and glycolysis rate, and mitochondria DNA damage. Additionally, CPT1A1 siRNA knockdown suppresses oxidative stress and prevents mitochondrial dysfunction and vascular inflammation in TL treated HBMECs. TL activates ATF3-MAPKinase, TNF, and NRF2 signaling pathways. Furthermore, the NRF2 signaling pathway which is upstream of the ATF3-MAPKinase signaling pathway, is also regulated by the mitochondrial oxidative stress. We are the first to report differential inflammatory characteristics of transcript variants 4 (ATF3-T4) and 5 (ATF3-T5) of th

关键词： AK4   Bladder cancer   Proliferation   Invasion   Therapeutic target   ADAPTIVE-RESPONSE   LUNG-CANCER   POLYMORPHISMS   GENES   ATF3   EXPRESSION   MORTALITY   MARKER   RISK  

摘要： Bladder cancer is the second most common urological cancer worldwide with low early diagnosis and high mortality. Since the time of diagnosis directly affects survival rate, early detection and precise biomarkers of bladder cancer are very important. Adenylate kinase 4 (AK4) is a key enzyme involved in cellular metabolism and multiple cancer development; however, the potential role of AK4 in bladder tumorigenesis is still unclear. Immunohistochemistry assay was conducted to evaluate the expression level of AK4 in 107 human bladder cancer tissues. Overall survival and recurrence-free survival were used to assess the prognosis of patients. Colony formation and MTT assays [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] were performed to measure the proliferation capacity of tumor cells. Cell scratch assays and transwell assays were performed to measure the invasion capacity of tumor cells. The expression level of involved genes was measured by reverse transcription-polymerase chain reaction and western blot assays. The animal model was used to examine the effects of indicated protein on tumorigenesis and invasion in vivo. Herein, our study demonstrated that increased AK4 expression in patients with bladder cancer was associated with a poor prognosis. We further found that inhibition of AK4 in bladder cancer cell line T24 and 5637 can obviously inhibit the proliferation of cancer cells. Transwell assay results showed that down-regulated AK4 was related to the decreased metastasis of T24 and 5637 cells. In addition, AK4-shRNA transfected obviously inhibited tumor growth and metastasis in mice compared with the scramble group. Taken together, the results provide strong evidence of the involvement of AK4 in the progression of bladder cancer and suggest that it could have high potential as a therapeutic target of disease.

关键词： ACTIVATING TRANSCRIPTION FACTOR-3   DIET-INDUCED THERMOGENESIS   BINDING PROTEIN CHREBP   ADIPOSE-TISSUE   GENE-EXPRESSION   ABDOMINAL OBESITY   GLUCOSE   FAT   ENERGY   CELL  

摘要： Billions of people have obesity-related metabolic syndromes such as diabetes and hyperlipidemia. Promoting the browning of white adipose tissue has been suggested as a potential strategy, but a drug still needs to be identified. Here, genetic deletion of activating transcription factor 3 (ATF3(-/-)) in mice under a high-fat diet (HFD) resulted in obesity and insulin resistance, which was abrogated by virus-mediated ATF3 restoration. ST32da, a synthetic ATF3 inducer isolated from Salvia miltiorrhiza, promoted ATF3 expression to downregulate adipokine genes and induce adipocyte browning by suppressing the carbohydrate-responsive element-binding protein-stearoyl-CoA desaturase-1 axis. Furthermore, ST32da increased white adipose tissue browning and reduced lipogenesis in HFD-induced obese mice. The anti-obesity efficacy of oral ST32da administration was similar to that of the clinical drug orlistat. Our study identified the ATF3 inducer ST32da as a promising therapeutic drug for treating diet-induced obesity and related metabolic disorders.

关键词： flavivirus   infection   inflammation   innate immunity   macrophage   cholesterol 25-hydroxylase   PATTERN-RECOGNITION RECEPTORS   CHOLESTEROL 25-HYDROXYLASE   25-HYDROXYCHOLESTEROL   INNATE   APOPTOSIS   ACTIVATION   TRANSCRIPTION   MECHANISMS   SREBPS   ATF3  

摘要： Zika virus (ZIKV)(3) is an enveloped, single-stranded, positive-sense RNA virus of the Flaviviridae family that has emerged as a public health threat because of its global transmission and link to microcephaly. Currently there is no vaccine for this virus. Conversion of cholesterol to 25-hydroxycholesterol by cholesterol 25-hydroxylase (CH25H) has been shown to have broad antiviral properties. However, the molecular basis of induction of CH25H in humans is not known. Elucidation of signaling and transcriptional events for induction of CH25H expression is critical for designing therapeutic antiviral agents. In this study, we show that CH25H is induced by ZIKV infection or Toll-like receptor stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines, including IL-1 beta, tumor necrosis factor alpha, and IL-6, and this induction depends on the STAT1 transcription factor. Additionally, we observed that cAMP-dependent transcription factor (ATF3) weakly binds to the CH25H promoter, suggesting cooperation with STAT1. However, ZIKV-induced CH25H was independent of type I interferon. These findings provide important information for understanding how the Zika virus induces innate inflammatory responses and promotes the expression of anti-viral CH25H protein.

关键词： Forensic pathology   Skin contusion   Vital reaction   ATF3   BTG2   MOLECULAR PATHOLOGY   WOUND AGE   EXPRESSION   INFLAMMATION   TRYPTASE   SYSTEM   CD15  

摘要： The detection of vitality of wounds, especially when the wounds are inflicted very close to the time of death, is one of the most challenging issues in forensic pathology. This study investigated expression levels of ATF3 and BTG2 in mouse and human skin wounds. Protein levels examined by western blot showed that there was no significant change in ATF3 and BTG2 between wounded and intact skins. However, mRNA levels demonstrated higher expression of ATF3 and BTG2 in ante-mortem contused mouse skins, compared with the intact and postmortem contused skins. Increased ATF3 and BTG2 in the level of mRNA could also be detected until 96 h and 48 h after death, respectively. Human wounded skin samples from forensic autopsy cases were also examined. Increased ATF3 mRNA levels were detected until 48 h after autopsy in 5 of 6 cases. However, no differences were observed between wounded and intact skins for BTG2. These findings suggest that the detection of mRNA levels of ATF3, but not BTG2, can be considered as a potential marker for vital reaction of skin contusion. Postmortem human samples should be used in order to validate the availability of markers screened by animal experiment. (C) 2019 Elsevier B.V. All rights reserved.