关键词： Silica   Silicosis   Inflammation   ATF3   RNA-Seq  

摘要： Background: Millions of workers are exposed to dust containing silica. Chronic and over-exposure to silica will lead to silicosis, which is an irreversible and sometimes fatal lung disease. The disordered physiological processes of silicosis consist of accumulation of silica particles in the alveoli of the lung. Then, the ingestion of the silica particles by macrophages was followed by an inflammatory response. Up till now, the chest radiographs remain the key tool in diagnosing and assessing the extent of silicosis. However, concerns exist regarding the sensitivity and specificity of the technique. Therefore, there is still a need to develop a biomarker for silicosis for early detection of silicosis. Method: In this study, RNA-Seq was applied to detect the gene expression changes when silica was exposed to macrophages at different time intervals. RNA-Seq provides a broader dynamic range, increased specificity and sensitivity, and easier detection of rare and low-abundance transcripts. Bioinformatics tools such as the Database for Annotation, Visualization and Integrated Discovery (DAVID) and Gene Functional Classification Tool and Search Tool for the Retrieval of Interacting Genes (STRING) were applied for data analysis. Quantitative PCR was used to validate the results. Results: Our results showed that regulation of transcription factors was the dominant activated pathway in early exposure of silica to macrophages, followed by inflammatory responses which were the main mechanisms in silicosis. One of the findings was the upregulation of activating transcription factor 3 (ATF3) during silica exposure. When ATF3 expression was inhibited by siRNA, the production of cytokines IL-1 beta, IL-6 and TNF was further increased. Conclusion: This indicated that ATF3 may be a potential early diagnostic biomarker for silicosis and ATF3 acts as a repressor in inflammatory responses induced by silica.

摘要： Mice lacking genes involving in the DNA-damage response (DDR) are often tumor prone owing to genome instability caused by oncogenic challenges. Previous studies demonstrate that activating transcription factor 3 (ATF3), a common stress sensor, can activate the tumor suppressor p53 and regulate expression of p53 target genes upon DNA damage. However, whether ATF3 contributes to the maintenance of genome stability and tumor suppression remains unknown. Here we report that Atf3-deficient (Atf3(-/-)) mice developed spontaneous tumors, and died significantly earlier than wild-type (Atf3(+/+)) mice. Consistent with these results, Atf3(-/-) mouse embryonic fibroblasts (MEFs) had more aberrant chromosomes and micronuclei, and were genetically unstable. Whereas we demonstrated that ATF3 activated p53 and promoted its pro-apoptotic activity in mouse thymi and small intestines, the chromosomal instability caused by Atf3 deficiency was largely dependent on the regulation of p53 by ATF3. Interestingly, loss of Atf3 also promoted spontaneous tumorigenesis in Trp53(+/-) mice, but did not affect tumor formation in Trp53(-/-) mice. Our results thus provide the first genetic evidence linking ATF3 to the suppression of the early development of cancer, and underscore the importance of ATF3 in the maintenance of genome integrity.

关键词： 结肠癌   ATF3基因   Wnt/β-catenin信号通路   上皮-间质转化  

摘要： 目的激活转录因子3(activating transcription factor 3,ATF3)是转录因子ATF/CREB(cyclic AMP-responsive element binding protein)家族中的一员,在正常细胞中表达低,但是通过信号刺激可以诱导其短时间快速表达发挥作用。本研究旨在探讨ATF3在结肠癌中的表达及临床病理特征相关性,揭示ATF3过表达对结肠癌HCT116细胞Wnt/β-catenin信号通路和EMT的影响。方法利用免疫组化法分析ATF3在结肠癌组织中的表达及其临床病理特征相关性。分别向HCT116细胞转染空载和ATF3过表达质粒:采用细胞增殖检测试剂盒8(cell counting kit 8,CCK8)法检测ATF3对细胞增殖的影响;利用划痕实验观察ATF3对细胞迁移的影响;采用蛋白质印迹法检测ATF3过表达对Wnt/β-catenin信号通路和EMT过程中蛋白的影响。结果 ATF3在正常结肠黏膜上皮中高表达,在癌组织及转移癌中低表达,χ~2=27.2,P<0.001。细胞实验显示,转染前两组细胞增殖差异无统计学意义,t=0.60,P=0.566;与对照组相比,观察组48h的吸光度值降低,t=4.21,P=0.003。转染质粒48h观察组划痕愈合速度变慢;转染48h观察组ATF3蛋白表达量明显多于对照组;观察组β-catenin、c-myc、Cyclin D1、TCF7L2和N-cadherin蛋白表达减少,GSK-3β和E-cadherin增多。结论 ATF3过表达可以抑制结肠癌细胞的增殖和迁移;ATF3过表达可以抑制Wnt/β-catenin信号通路和上皮-间质转化,抑制结肠癌的发生及转移;ATF3可能在结肠癌的发生发展起着重要的作用。

关键词： Podocyte   Apoptosis   Injury   Activating transcription factor 3   Nuclear factor of activated T cell  

摘要： Podocyte injury and loss contribute to proteinuria, glomerulosclerosis, and eventually kidney failure. Activating transcription factor 3 (ATF3) is a stress inducible transcription factor that is transiently expressed following stimulation. However, we show for the first time an induction of ATF3 in podocytes from patients with chronic kidney disease, including minimal change disease, focal segmental glomerulosclerosis, and diabetic nephropathy. The role of ATF3 induction in podocytes under chronic conditions is currently unknown. Compared with the control (C57 or BKS), ATF3 expression was elevated in animal model of proteinuria (LPS-treated C57 mice) and the model of diabetic nephropathy (db/db mice). Similarly, ATF3 was increased in high glucose (HG)-treated, lipopolysaccharide (LPS)-treated, or Ionomycin-treated podocytes in vitro. Overexpression of ATF3 increased podocyte apoptosis and decreased expression of podocin, the cell marker of podocyte;in contrast, ATF3-small interfering RNA knockdown reduced podocyte apoptosis and increased podocin expression. The translocation of ATF3 to the nucleus was increased upon stimulation. ATF3 directly modulates the regulation of NFATc1 gene promoter activity and alters the expression of Wnt6 and Fzd9, direct target genes of NFATc1 signaling. The ATF3 binding site of NFATc1 gene promoter is located at the region 671-775 base pairs upstream of the transcription start site. These results indicate a novel inducible axis of ATF3-NFAT in podocyte injury and loss.

关键词： 肿瘤   转移   ATF3   VEGF  

摘要： 化疗作为肿瘤的重要治疗手段,通过直接杀伤肿瘤细胞可达到抗肿瘤的目的.但化疗在抗肿瘤同时,也有促转移的作用.本文主要从化疗促肿瘤转移相关基因、通路等方面对化疗促肿瘤转移机制进行总结.

关键词： 猪血凝性脑脊髓炎病毒   ATF3   细胞凋亡   细胞周期  

摘要： 猪血凝性脑脊髓炎(Porcine hemagglutinating encephalomyelitis,PHE)是仔猪的一种急性、高度接触性传染病,其病原是猪血凝性脑脊髓炎病毒(Porcine hemagglutinating encephalomyelitis virus,PHEV),主要感染1～3周龄的哺乳仔猪,主要以呕吐衰竭和明显的神经症状为特征。转录激活因子3(activating transcription factor 3,ATF3)是转录因子ATF/CREB家族成员之一,ATF3是一个适应反应基因,在静息细胞中低表达,在受到刺激时表达量会迅速升高。前期mRNA芯片分析结果表明,小鼠大脑及小鼠神经胶质瘤细胞(N2a)感染PHEV后,ATF3均呈现高表达。但ATF3高表达的机制及其对神经细胞的影响尚不清楚。本实验首先对N2a细胞感染PHEV后不同时间段的ATF3表达水平进行了检测,同时对不同滴度PHEV感染N2a细胞后的ATF3表达水平进行了检测,以分析病毒载量与ATF3表达水平之间的关系;其次,合成ATF3siRNA,构建ATF3过表达载体,分别转染N2a细胞,建立稳定表达的细胞系,然后接种PHEV,分析PHEV的mRNA的表达水平,并检测细胞周期和细胞凋亡,以探究ATF3是否影响病毒的复制,探究在PHEV感染神经细胞过程中ATF3对宿主细胞发挥的作用。本试验在探究PHEV感染神经细胞的机制中选择N2a细胞作为模型,根据试验内容和试验目的将N2a细胞接种于六孔板进行分组培养,将PHEV吸附N2a细胞1 h后,通过RT-PCR和Western Blot方法,于感染后2 h、12 h、24 h,检测神经细胞内ATF3表达水平的变化。N2a细胞转染siRNA和表达载体后接种PHEV,设置5组:对照组、ATF3干扰接毒组、阴性对照接毒组、ATF3过表达接毒组、空载接毒组,每组3个重复,利用RT-PCR技术检测PHEV mRNA表达情况,探究ATF3是否影响病毒的复制;通过Annexin V-FITC/PI双染法检测12 h细胞凋亡,利用PI单染法检测12 h细胞周期,以测定ATF3对PHEV感染的神经细胞的凋亡和细胞周期的影响。结果表明,ATF3在正常N2a细胞中表达水平较低,随着感染时间的增加,PHEV mRNA表达量和ATF3 mRNA表达量均出现先升高后降低的趋势,二者之间呈正相关;随着感染时间的增加ATF3的蛋白表达量持续升高;随着病毒滴度的降低,ATF3表达量也随之下降转染了ATF3siRNA后,ATF3表达显著受到抑制;转染了ATF3过表达载体后,ATF3表达显著升高。利用RT-PCR技术检测PHEV mRNA表达水平,利用流式细胞术检测细胞凋亡和细胞周期,结果发现,ATF3干扰接毒组细胞与阴性对照接毒组相比,PHEV mRNA表达量无明显差异;早期凋亡升高;G1期阻滞加重。ATF3过表达接毒组细胞与空载接毒组细胞相比,早期凋亡率下降;S期阻滞减缓。综上所述,ATF3在PHEV感染N2a细胞的过程中与病毒的复制呈正相关,PHEV会影响ATF3的表达,但ATF3并不会影响PHEV的复制,ATF3在PHEV感染N2a细胞的过程中会起到抗凋亡和促进细胞周期的作用。

关键词： 活化转录因子(ATF)   Toll样受体(TLR)   哺乳动物雷帕霉素靶蛋白(mTOR)   缺氧诱导因子(HIF)   缺血再灌注损伤(IRI)   巨噬细胞  

摘要： 目的:探究髓系细胞ATF3调节小鼠肝脏缺血再灌注损伤后免疫活化的机制。方法:使用缺血再灌注损伤模型分析ATF3基因敲除鼠的肝脏损伤及巨噬细胞浸润情况,检测指标包括血清ALT,肝脏切片HE、Ly6G、CD11b和TUNEL染色等;实时定量PCR技术被用来检测肝脏及细胞中细胞因子产生情况,指标有TNF-a,IL-1?,IL-6和TGF-?;采用Weatern Blot技术检测组织或细胞中m TOR,HIF-1α,p70S6K,TLR4,PHD1等蛋白表达情况。采用m TOR靶向抑制实验验证其免疫调节功能;采用p70S6K si RNA转染试验验证其在m TOR信号通路中的作用;采用HIF-1a si RNA抑制实验验证其在ATF3介导的炎性免疫调节中的功能。结果:ATF3敲除后相比较于对照组明显加重了缺血再灌注诱导的肝损伤。ATF3敲除后促进了巨噬细胞/中性粒细胞的侵润和肝细胞的凋亡。ATF3敲除促进了m TOR,p70S6K,TLR4和HIF-1a蛋白的表达,但是降低了PHD1的表达。m TOR靶向抑制实验、p70S6K si RNA抑制实验和HIF-1a si RNA抑制试验结果都显示明显逆转了ATF3敲除诱导发生的肝损伤,炎性细胞因子TNF-a,IL-1?,IL-6表达降低,同时TGF-?表达升高。肝细胞的凋亡情况以及肝功能情况也明显得到改善。结论:在小鼠肝脏缺血再灌注损伤中,髓系细胞中ATF3基因通过下调m TOR/p70S6K/HIF-1α信号通路的活性参与损伤后的炎性免疫调控,并发挥对肝脏的保护作用。

关键词： DON   细胞周期   ATF3   EGR1   组蛋白乙酰化  

摘要： 脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON),又称呕吐毒素(vomitoxin),属于单端孢霉烯族B类化合物,主要由禾谷镰刀菌等真菌产生,污染小麦、豆粕和玉米等谷类作物及其制品,对误食的人和动物产生广泛的毒性效应。DON的毒性作用主要表现为抑制DNA和蛋白质的合成、核糖体应激反应、炎症反应、抑制细胞增殖和产生细胞周期阻滞等。已有研究显示DON诱导人脐静脉内皮细胞、人结直肠癌细胞和人肠上皮细胞产生G2/M期周期阻滞,但其分子机制仍不清楚。实验室前期转录组学数据提示DON可促进细胞周期调控相关的两个重要转录因子EGR1和ATF3上调表达,本研究围绕EGR1与ATF3在DON诱导细胞G2/M期阻滞中的作用机制展开研究,主要取得以下研究结果:1、DON诱导HepG2细胞产生G2/M期阻滞分别用2μg/mL和4μg/mL DON处理HepG2细胞6 h、12 h、24 h和48 h,并通过流式细胞仪检测DON处理前后细胞周期的变化,结果显示DON作用下G2/M期的细胞比例显著增加,且4μg/mL DON比2μg/mL DON作用更强烈,但DON处理不同时间的阻滞效果差异不明显。2、EGR1在DON诱导细胞G2/M期阻滞中发挥关键作用qRT-PCR和Western blot实验结果显示DON显著上调EGR1和细胞周期抑制蛋白p21的表达。EGR1敲降后DON对p21的诱导作用和细胞G2/M期阻滞作用均受到抑制,说明EGR1是DON诱导细胞G2/M期阻滞的关键因子。3、ATF3?Zip2a/2b在DON促进EGR1上调表达中发挥重要作用ATF3?Zip2a/2b是ATF3缺失bZIP区域2a/2b段的蛋白变体,2μg/mL DON分别处理HepG2细胞6 h、12 h、24 h和48 h后,ATF3?Zip2a/2b上调表达。进一步研究显示,过表达ATF3全长蛋白抑制DON对EGR1的诱导作用,过表达ATF3?Zip2a/2b变体增强DON对EGR1的诱导作用,表明ATF3?Zip2a/2b在DON诱导EGR1表达过程中发挥重要作用。4、DON通过H3K27ac和H3K9ac促进ATF3?Zip2a/2b的表达2μg/mL DON处理下组蛋白乙酰化酶P300和PCAF强烈上调。染色质免疫共沉淀结果显示,DON处理下P300对应的H3K27ac和PCAF对应的H3K9ac在ATF3启动子区富集程度增强。在siPCAF/siGCN5或siCBP/siP300的HepG2细胞中,DON不能诱导ATF3?Zip2a/2b上调表达,说明DON通过增强ATF3启动子区H3K27ac和H3K9ac修饰程度促进ATF3?Zip2a/2b上调表达。本课题明确了EGR1和ATF3在DON诱导HepG2细胞G2/M期阻滞中的重要作用,阐明了DON通过增强ATF3启动子区组蛋白H3K27ac和H3K9ac的修饰程度来诱导ATF3?Zip2a/2b表达,进而促进EGR1上调表达,导致HepG2细胞G2/M期阻滞的分子机制。研究结果对明晰DON的分子毒理具有重要理论意义。

关键词： 激活转录因子3   结肠癌   Wnt/β-catenin信号通路   上皮-间质转化  

摘要： 背景结直肠癌(CRC)是全世界常见的消化道恶性肿瘤之一。近年来,在我国结肠癌的发病率和死亡率有明显的升高趋势,严重威胁着人们的健康。激活转录因子3(ATF3)是转录因子家族中的一员,在正常细胞中表达低,但是通过信号刺激可以诱导其在短时间内快速表达并发挥作用,也被称为是适应性反应基因。有研究表明,ATF3与多种肿瘤的发生发展有关,但ATF3对结肠癌细胞增殖和迁移及分子机制尚不完全清楚。目的探讨ATF3与结肠癌增殖和转移的关系,揭示过表达和干扰ATF3表达水平对结肠癌细胞系中Wnt/β-catenin信号通路和EMT的影响。方法1.免疫组织化学方法检测包含49例人结肠癌病理组织芯片(TMA),分析ATF3蛋白在结肠癌组织中的表达及其临床病理特征相关性。*** Blot检测ATF3在结肠癌细胞系中的基础表达水平,选择低表达水平的细胞转染质粒进行过表达实验,在高表达的细胞中进行干扰其表达实验。***-PCR方法检测ATF3在结肠癌细胞系中的mRNA表达水平。***-8法检测ATF3对结肠癌细胞增殖的影响。5.细胞划痕实验和Transwell观察ATF3对结肠癌细胞迁移的影响。*** blot检测ATF3蛋白表达,分析其对结肠癌细胞增殖和迁移的分子机制。结果***3在结肠癌组织及癌旁淋巴结中高表达率分别为42.9%、10.0%,明显低于正常结肠组织中ATF3高表达率61.2%,P<0.01。***3在HCT116细胞和HCT8细胞中表达量分别是0.41±0.05、0.53±0.10,而在SW480和RKO细胞中的表达量分别是1.25±0.12、1.69±0.10,显示ATF3在结肠癌细胞系中表达有差异,F=116.64,P<0.01。3.在细胞中转染ATF3过表达质粒,HCT116细胞和HCT8细胞48 h实验组和对照组的吸光度值分别为(1.47±0.10、1.77±0.12)、(0.43±0.09、0.64±0.07);在细胞中转染ATF3的siRNA,RKO细胞和SW480细胞48h测实验组和对照组的吸光度值分别为(1.52±0.21、0.96±0.14)、(0.61±0.10、0.40±0.07);以上实验组结果与其相应对照组比较,都有明显差异,P<0.05。4.在细胞中转染ATF3过表达质粒,HCT116细胞和HCT8细胞48 h计数实验组和对照组的穿膜细胞数分别为:(35.0±15.2、79.3±15.9)、(33.5±13.1、66.6±8.8),两两比较,P<0.01;在细胞中转染ATF3的siRNA,在RKO细胞和SW480细胞48h计数实验组和对照组的穿膜细胞数分别为:(114.8±10.2、79.6±9.4)、(35.7±7.2、15.1±7.4),两两比较,P<0.05。***/β-catenin信号通路中,HCT116细胞和HCT8细胞中转染ATF3过表达质粒,β-catenin、GSK-3β、TCF7L2蛋白表达量;RKO细胞和SW480细胞中转染ATF3的siRNA,ATF3、β-catenin、GSK-3β、TCF7L2蛋白表达量,以上实验组结果与各自对照组相比,都有明显差异,P<0.05或P<0.01;上皮-间质转化(EMT)相关蛋白实验中,HCT116细胞和HCT8细胞转染ATF3过表达质粒,E-cadherin、N-cadherin蛋白表达量;SW480细胞中转染ATF3的siRNA,E-cadherin、N-cadherin蛋白表达量,以上实验组结果与各自对照组相比,都有明显差异,P<0.05。结论ATF3可以抑制结肠癌细胞的增殖和迁移,可能是通过调控Wnt/β-catenin信号通路和EMT,抑制了结肠癌的发生发展。

关键词： transcription   DNA repair   ubiquitination   proteasome   transcription arrest   TCR   ATF3   CSA   CSB   MDM2  

摘要： Cockayne syndrome (CS) is caused by mutations in CSA and CSB. The CSA and CSB proteins have been linked to both promoting transcription-coupled repair and restoring transcription following DNA damage. We show that UV stress arrests transcription of approximately 70% of genes in CSA- or CSB-deficient cells due to the constitutive presence of ATF3 at CRE/ATF sites. We found that CSB, CSA/DDB1/CUL4A, and MDM2 were essential for ATF3 ubiquitination and degradation by the proteasome. ATF3 removal was concomitant with the recruitment of RNA polymerase II and the restart of transcription. Preventing ATF3 ubiquitination by mutating target lysines prevented recovery of transcription and increased cell death following UV treatment. Our data suggest that the coordinate action of CSA and CSB, as part of the ubiquitin/pro-teasome machinery, regulates the recruitment timing of DNA-binding factors and provide explanations about the mechanism of transcription arrest following genotoxic stress.