关键词： ATF3   舌鳞状细胞癌   IFI6   IFI27   CRISPR/Cas9  

摘要： 背景与目的鳞状细胞癌(Squamous cell carcinoma,SCC)是头颈部恶性肿瘤的主要组织学类型,占这些部位恶性肿瘤的90%-95%,头颈部鳞状细胞癌(Head and neck squamous cell carcinoma,HNSCC)是全球排名第六的常见恶性肿瘤,其中口腔鳞状细胞癌(Oral squamous cell carcinoma,OSCC)占 HNSCC的25%,且舌鳞状细胞癌(Tongue squamous cell carcinoma,TSCC)所占比例较高。TSCC是一种具有高度侵袭性的恶性肿瘤,容易发生局部复发和远处转移。此外,TSCC在发生发展过程中常无特异性症状从而给早期诊断制造了巨大困难,最终导致预后不良。即使采用放化疗及手术等联合治疗,其5年的生存率仍不理想。恶性肿瘤的发生发展是一个多因素的过程,其中环境因素、遗传易感性、不良生活方式(如酒精和烟草)和病毒感染(人类乳头瘤病毒)在内的诸多因素与TSCC的发生有关,但TSCC发生发展的详细分子机制尚不清楚。因此,现迫切需要进一步探索发现TSCC发生发展的相关分子机制,为临床早期诊断及治疗TSCC提供新的策略。转录激活因子3(Activating transcription factor 3,ATF3)为ATF/CREB(Activating transcription factor/cyclic amp response element-binding)家族成员之一,是调节细胞应激反应的关键转录因子,在不同的组织中表达水平和功能不同。近年来ATF3在肿瘤细胞中的作用得到了广泛关注,在不同的肿瘤类型和细胞环境中,ATF3既可作为癌基因又可作为抑癌基因并发挥相应功能。最近的研究发现ATF3是miR-488和miR-431的下游靶点,在TSCC的进展中发挥重要作用,但ATF3在TSCC细胞中的下游效应迄今尚未得到深入研究。本研究旨在探索ATF3在TSCC肿瘤细胞中的功能及其下游信号通路,为开发治疗TSCC等肿瘤的新疗法提供新的思路。材料与方法***3在TSCC组织及细胞中的表达本课题首先采用H&E染色和免疫组织化学技术(Immunohistochemistry,IHC)检测了不同分化程度的TSCC组织以及正常舌组织中ATF3的表达。然后,通过细胞免疫荧光技术对TSCC细胞系CAL27和SCC-9以及正常口腔角质形成细胞(Human oral keratinocytes,HOK)进行ATF3染色分析,进一步明确ATF3在不同细胞系中的表达情况。最后,利用癌症基因组图谱(The cancer genome atlas,TCGA)数据库集中分析了HNSCC和TSCC中ATF3基因表达水平。***3对TSCC细胞增殖和迁移的影响为了研究ATF3对TSCC细胞功能的具体影响,本研究采用RNA指导的Cas9核酸酶基因编辑技术(Clustered regularly interspaced short palindromic repeats,CRISPR/Cas9)敲除TSCC细胞系CAL27和SCC-25中的ATF3基因,并通过慢病毒感染过表达TSCC细胞系SCC-9和SCC-4中的ATF3基因。利用CCK-8技术检测ATF3基因敲除或过表达对TSCC细胞系增殖能力的影响;通过Transwell实验和细胞划痕实验检测敲除或过表达ATF3基因对TSCC细胞系迁移能力的影响。此外,通过Western blot技术检测了AKT和ERK信号通路在ATF3基因敲除或过表达时的激活情况。***3调控TSCC细胞增殖和迁移的分子机制为了探索ATF3基因调控TSCC细胞增殖和迁移的分子机制,本课题利用RNA-seq分析了敲除ATF3基因的CAL27细胞的mRNA基因表达谱。通过GO分析和基因测序获得敲除ATF3基因的CAL27细胞中相关信号通路和差异性表达的基因。通过qRT-PCR和ChIP分析确定在TSCC细胞中ATF3下游的两个靶点干扰素诱导蛋白6(Interferon-induced protein6,IFI6)和干扰素诱导蛋白27(Interferon-induced protein27,IFI27)。并通过IHC和TCGA数据库进一步确认IFI6和IFI27在TSCC组织中高表达。为了分析ATF3是否通过IFI6或IFI27调控TSCC细胞的增殖与迁移,本课题利用基因编辑技术敲低或过表达TSCC细胞中IFI6和IFI27基因,通过CCK-8、Transwell和细胞划痕等实验检测敲低或过表达IFI6和IFI27基因对TSCC细胞(己敲除或过表达ATF3基因)的增殖和迁移等功能的影响,并通过Western blot技术检测了 AKT和ERK信号通路

关键词： 高钾离子   铁死亡   ATF3   内质网应激  

摘要： 研究目的:本研究主要探究肿瘤高钾微环境下对肿瘤细胞铁死亡的影响,阐明可能的分子机制,为肿瘤微环境治疗提供潜在的靶点。研究方法:首先配置高渗高钾培养基,在DMEM培养基中加入不同浓度的氯化钾;同时又配置相同浓度的等渗培养基以排除单纯加氯化钾引起渗透压改变的影响。不同浓度的高钾培养基联合铁死亡诱导剂(Erastin;RSL3)为实验组,常规钾离子浓度的培养基联合铁死亡诱导剂为对照组;CCK-8法、细胞克隆及Edu增殖实验法检测高浓度钾对肿瘤细胞铁死亡的影响;RNA测序技术筛选高钾导致铁死亡抵抗的潜在通路;全波长酶标仪检测高钾及GSK2606414处理后的细胞内GSH、MDA相对含量;流式细胞仪检测肿瘤细胞内ROS及钙离子相对水平;WB分析ATF3、PERK、IRE1及ATF6α的蛋白表达;构建ATF3敲除质粒检测ATF3对铁死亡的影响;将高钾预处理一周的细胞植入小鼠皮下,体内验证高钾对铁死亡的影响;免疫组化验证高浓度钾对ATF3及铁死亡指标COX2表达的影响。结果:1、CCK-8结果显示高钾可以显著挽救Erastin引起的铁死亡,当钾离子浓度达35mM时效果最甚(P值<0.001);但不能挽救RSL3引起的铁死亡。2、细胞克隆及Edu增殖实验结果显示,Erastin诱导后,高钾处理组细胞依旧有增殖能力。3、RNA测序结果揭示,Erastin处理后细胞内质网相关基因显著上调,而高钾联合Erastin处理组内质网并没有显著上调,提示高钾可能通过内质网影响铁死亡。4、使用不同内质网抑制剂并联合Erastin处理,发现抑制内质网PERK相关通路可显著挽救Erastin引起的铁死亡(P值<0.001)。5、通过测序结果分析高钾可能通过内质网相关蛋白ATF3来影响铁死亡。Western blot结果显示,使用Erastin后,细胞内ATF3水平显著升高,联合高浓度钾后则显著下调。6、敲除ATF3后显著提高细胞对铁死亡的耐性(P值<0.001),过表达ATF3则促进肿瘤细胞铁死亡(P值<0.001)。7、体内结果显示,高钾预处理细胞可显著挽救PE引起的铁死亡(P值<0.001);同时组化结果显示高钾预处理组能显著降低PE引起COX2及ATF3的表达。研究结论:高浓度钾离子通过抑制ATF3表达抑制肿瘤细胞铁死亡

关键词： 镧   学习记忆   内质网应激   铁死亡   激活转录因子3  

摘要： 目的:稀土元素（Rare earth elements,REEs）是元素周期表中的第IIIB族中17种的金属元素,原子序数分别为21、39和57～71。研究表明REEs通过食物链进入体内,对机体神经系统产生损害,稀土地区儿童的智商水平和记忆力明显低于正常儿童。镧（Lanthanum,La）在自然界中相对活泼,常被用来研究REEs的神经毒作用机制。铁死亡（Ferroptosis）是铁依赖的调节性细胞死亡,由脂质过氧化驱动,是细胞内代谢紊乱和氧化还原状态失衡的结果。激活转录因子3（Activating transcription factor 3,ATF3）是由应激因素诱导的转录因子,其表达可被细胞应激迅速诱导,进而调控下游基因表达转录。还原型烟酰胺腺嘌呤二核苷酸磷酸（Nicotinamide adenine dinucleotide phosphate,NADPH）通常参与机体内多种代谢反应,作为供氢体参与氧化还原反应。NADPH氧化酶4（NADPH oxidase 4,NOX4）促进细胞生成ROS。下调胱氨酸/谷氨酸的反向转运体（Cystine/glutamate antiporter system,System Xc）表达水平,导致半胱氨酸和谷胱甘肽（Glutathione,GSH）耗竭。发生内质网应激时,ATF3被诱导表达上调。本研究探讨La是否通过影响内质网应激水平,促进ATF3表达上调,进而上调NOX4,促进细胞生成ROS,同时下调System Xc,致半胱氨酸和GSH耗竭,造成脂质过氧化增强,从而促进铁死亡,发挥神经毒性作用。本研究将为La所致神经毒性损害以及其他神经系统疾病的预防与治疗提供新的研究资料。研究方法:采用实验动物中心购买的实验用清洁级健康成年Wistar大鼠,雌鼠和雄鼠分别为16只,体重为245±10 g,雌雄1∶1的比例合笼。雌鼠完全随机分组,包括对照组、低、中和高剂量组,分组后进行染毒,从雌鼠孕期第0天开始经饮水染毒,对照组大鼠饮蒸馏水,低剂量、中剂量和高剂量的LaCl组孕鼠分别饮用含0.125%、0.25%和0.5%LaCl的蒸馏水溶液,至仔鼠离乳后终止染毒。Morris水迷宫试验测试仔鼠学习记忆能力;电镜观察仔鼠海马组织神经元的超微结构改变;普鲁士蓝染色观察仔鼠海马铁蛋白情况;免疫荧光法观察仔鼠海马ATF3蛋白表达情况;试剂盒检测仔鼠海马中ROS、MDA、GSH/GSSG和Fe含量;Western blot检测仔鼠海马中内质网应激相关蛋白（PERK、GRP78、ATF4）、ATF3、NOX4、SLC7A11、GPX4和线粒体电子传递链复合物蛋白（NDUFB8、SDHB、UQCRC2和ATP5A）表达情况。取新生24 h内Wistar大鼠,体外培养原代神经元,待成熟后,分别给予含0、0.025、0.05和0.1m M LaCl的培养基,染毒时间24 h,随即检测相关指标。采用神经元特异性烯醇化酶（Neuron-specific enolase,NSE）对神经元进行鉴定;试剂盒法检测原代神经元MDA和Fe水平;Western blot法检测原代神经元中PERK、GRP78、ATF4、ATF3、NOX4、SLC7A11、GPX4、NDUFB8、SDHB、UQCRC2和ATP5A蛋白表达情况。结果:***水迷宫的定位航行试验结果,仔鼠逃避潜伏期和总游泳距离随着LaCl暴露剂量的增加而增加;空间探索试验结果,仔鼠穿越目标平台次数和目标象限停留时间随着LaCl暴露剂量的增加而降低。2.随着LaCl剂量的增加,仔鼠海马神经元形态改变,核膜皱缩,线粒体肿胀、空泡化。3.随着LaCl剂量的增加,仔鼠海马铁蛋白增多。4.随着LaCl剂量的增加,仔鼠海马组织ROS、MDA和Fe水平增加,GSH/GSSG水平降低。5.随着LaCl剂量的增加,仔鼠海马CA1区、CA3区和DG区中ATF3表达增多。***暴露可上调仔鼠海马PERK、GRP78、ATF4、ATF3和NOX4蛋白表达水平,下调仔鼠海马SLC7A11、GPX4、NDUFB8、SDHB、UQCRC2和ATP5A蛋白表达水平。***处理上调了原代培养神经元的MDA和Fe水平。***处理可上调原代培养神经元PERK、GRP78、ATF4、ATF3和NOX4蛋白表达水平,下调原代培养神经元SLC7A11、GPX4、NDUFB8、SDHB、UQCRC2和ATP5A蛋白表达水平。结论:1.雌鼠孕哺期LaCl暴露造成仔鼠海马神经元铁死亡,造成仔鼠学习和记忆能力损害。***暴露导致神经元铁死亡可能是与内质网应激和ATF3信号通路表达上调有关。

关键词： Lung carcinoma   Zika virus infection  

摘要： The 2015 Zika virus (ZIKV) outbreak amassed international concern due to its association with the high incidence of devastating neurological conditions- microcephaly and Guillain-Barré Syndrome. Reportedly affecting over 5,000 fetal newborns, imaging studies have unveiled the pernicious effects of ZIKV infection on the fetal brain, which include cortical malformations, micrencephaly, hydrocephalus, and focal inflammation. In a collaborated effort to elucidate the mechanisms underlying how ZIKV infection incites these neurological anomalies, various studies have been employed describing its impact on global transcription in neuronal cells. ZIKV induces endoplasmic reticulum stress, and this cellular disturbance elicits the unfolded protein response (UPR). RNA-seq analyses implicate a crucial transcription factor involved in the UPR known as Activating Transcription Factor 3(ATF3) as a modulator of ZIKV infection. However, the signaling pathway that culminates in ATF3’s activation during viral infection remains unestablished. This research is aimed at investigating the effect of ZIKV on the transcriptional profile of a human lung carcinoma cell line, A549, and on evaluating NF-kappa B as an effector of ATF3 during ZIKV infection. In this study, I analyze RNA-seq data of A549 cells following infection with two isolates of ZIKV, Puerto Rican and Ugandan, using DESeq2. With immunoblot and RT-qPCR analysis, I examine ATF3 protein and RNA expression post-infection and inhibition of the NF-kappa B signaling pathway. Furthermore, I evaluate if the NF-kappa B pathway is activated during ZIKV infection by observing luciferase activity in an NF-kappa B/Nano-luciferase reporter vector. Finally, I investigate whether ZIKV infection and other stress stimuli induce alternatively spliced variants of ATF3 in A549 cells.

关键词： PERIVASCULAR ADIPOSE-TISSUE   CELLS   GENE   DYSFUNCTION   FAT   EXPRESSION   NETWORK   TARGETS   TONE  

摘要： Thoracic aortic perivascular adipose tissue (PVAT) is an adipose organ exhibiting similarities to brown adipose tissue (BAT), including cellular morphology and thermogenic gene expression. However, whether the PVAT phenotype is indistinguishable from the BAT phenotype in physiological vasculature remains unclear. We demonstrated that PVAT is distinguishable from classical BAT, given its specific vessel-tone-controlling function. Activating transcription factor 3 (ATF3) is a key factor in hypertension. Compared with wild-type mice, ATF3-deficient (ATF3(-/-)) mice fed a high-fat diet exhibited elevated mean arterial pressure, increased monocyte chemoattractant protein-1 expression and hypertrophy, plus abnormal fatty tissue accumulation in the thoracic aortic PVAT, and enhanced vascular wall tension and vasoconstrictive responses of potassium chloride, U46619, and norepinephrine in isolated aortic rings, which were restored after administration of adeno-associated ATF3 vector. We suggest that PVAT, not BAT, modulates obesity-related vascular dysfunction. ATF3 within PVAT could provide new insights into the pathophysiology of obesity-related cardiovascular diseases.

关键词： Breast cancer   ATF-3   miRNAs   TGF-?1   Bone metastases   BONE METASTASIS   TGF-BETA   RUNX2   GENE   ATF3   MICRORNAS   TARGET  

摘要： TGF-81 (transforming growth factor-beta1), a secreted polypeptide cytokine, stimulates ATF-3 (activating transcription factor-3) expression in a sustained and prolonged manner in human breast cancer cells (MDA-MB231), but not in normal human mammary epithelial cells (MCF-10A). Cyclin A (cell proliferation gene), Runx2 (metastasis gene), and MMP-13 (matrix metalloproteinase-13; invasive gene) were identified as ATF-3 target genes in these cells. Because ATF-3 has very few druggable sites, its direct targeting is difficult. Recent evidence has indicated that microRNAs (miRNAs) are key players in the post-transcriptional modulation of gene expression under several conditions. Bioinformatic analysis suggested a list of putative miRNAs that target ATF-3. Therefore, we hypothesized that TGF-81 downregulates the miRNAs that target ATF-3, resulting in the acti-vation of genes that participate in breast cancer progression and skeletal metastasis. Our findings indicate that TGF-81 downregulated the expression of miR-4638-3p in MDA-MB231 cells. At the molecular level, forced expression of miR-4638-3p reduced the expression of ATF-3 and its downstream targets, Runx2 and MMP-13, in these cells. At the cellular level, overexpression of miR-4638-3p reduced proliferation, invasion, and migration, and induced G0/G1 cell cycle arrest and apoptosis in MDA-MB231 cells. Overall, this study highlights the possibility of utilizing miR-4638-3p as a therapeutic molecule to curb skeletal metastasis of breast cancer cells.

关键词： Glioblastoma   Drug target   SPHK1   Inflammation   NF-κB/IL-6/STAT3 signal pathway   ATF3   PXT3  

摘要： Glioblastoma(GBM)is the most challenging malignant tumor of the central nervous system because of its high morbidity,mortality,and recurrence ***,mechanisms of GBM are still unclear and there is no effective drug for GBM in the ***,it is urgent to identify new drug targets and corresponding drugs for *** this study,in silico analyses and experimental data show that sphingosine kinase 1(SPHK1)is up-regulated in GBM patients,and is strongly correlated with poor prognosis and reduced overall *** of SPHK1 promoted the proliferation,invasion,metastasis,and clonogenicity of GBM cells,while silencing SPHK1 had the opposite ***1 promoted inflammation through the NF-κB/IL-6/STAT3 signaling pathway and led to the phosphorylation of JNK,activating the JNK-JUN and JNK-ATF3 pathways and promoting inflammation and proliferation of GBM cells by transcriptional activation of ***1 interacted with PTX3 and formed a positive feedback loop to reciprocally increase expression,promote inflammation and GBM *** of SPHK1 by the inhibitor,PF543,also decreased tumorigenesis in the U87-MG and U251-MG SPHK1 orthotopic mouse *** summary,we have characterized the role and molecular mechanisms by which SPHK1 promotes GBM,which may provide opportunities for SPHK1-targeted therapy.

关键词： RAN-GTP   MMP2   ATF3   CXCR3   Cell migration   Breast cancer   Patient survival   cMet   cMyc and Ki67   ACTIVATING TRANSCRIPTION FACTOR-3   PROGNOSTIC-SIGNIFICANCE   CELLS   EXPRESSION   PROMOTES   PROTEIN   ATF3   PROGRESSION   PHENOTYPE   EFFECTOR  

摘要： RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/ Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent bio-logical effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohisto-chemical staining for RAN and MMP2 are positively associated with each other in the same tumour and sepa-rately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient *** summary: Ran is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs

摘要： Excessive cardiac fibrosis is central to adverse cardiac remodeling and dysfunction leading to heart failure in many cardiac diseases. Histone methylation plays a crucial role in various pathophysiological events. However, the role of histone methylation modification enzymes in pathological cardiac fibrosis needs to be fully elucidated. Here, we identified lysine demethylase 5B (KDM5B), a histone H3K4me2/me3 demethylase, as a key epigenetic mediator of pathological cardiac fibrosis. KDM5B expression was upregulated in cardiac fibroblasts and myocardial tissues in response to pathological stress. KDM5B deficiency markedly ameliorated cardiac fibrosis, improved cardiac function, and prevented adverse cardiac remodeling following myocardial infarction (MI) or pressure overload. KDM5B knockout or inhibitor treatment constrained the transition of cardiac fibroblasts to profibrogenic myofibroblasts and suppressed fibrotic responses. KDM5B deficiency also facilitated the transformation of cardiac fibroblasts to endothelial-like cells and promoted angiogenesis in response to myocardial injury. Mechanistically, KDM5B bound to the promoter of activating transcription factor 3 (Atf3), an antifibrotic regulator of cardiac fibrosis, and inhibited ATF3 expression by demethylating the activated H3K4me2/3 modification, leading to the enhanced activation of TGF-beta signaling and excessive expression of profibrotic genes. Our study indicates that KDM5B drives pathological cardiac fibrosis and represents a candidate target for intervention in cardiac dysfunction and heart failure.