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： 胶质母细胞瘤   胶质瘤干细胞   circRPPH1   ATF3  

摘要： 目的:胶质母细胞瘤(glioblastoma,GBM)是最致命的成人颅内恶性肿瘤,因其弥漫性浸润生长的特性和背后复杂多变的未知分子机制,使得患者预后极差。非编码环状RNA(circRNA)因为其环状结构的性质,在体内的存在较为稳定,并且可以通过结合蛋白质以保持蛋白稳定,并最终促进肿瘤的发生发展。本研究从恶性胶质母细胞瘤中的胶质瘤干细胞出发,着重探究circRPPH1在胶质瘤干细胞(glioma stem cells)中的表达情况,并深入研究其内在的调控机制。本研究涉及胶质瘤干细胞和动物模型两个层面,着重探究circRPPH1对胶质瘤干细胞增殖、侵袭、自我更新能力的影响,考察其下游circRPPH1-ATF3信号及其转录调控作用,以及对TGF-β1/Smad信号通路的激活作用,以期发现新的治疗靶点,为靶向治疗提供依据。研究方法:选取胶质母细胞瘤患者来源的新鲜肿瘤组织,从中提取出胶质瘤干细胞细胞系GSC38和GSC35分别进行circRPPH1与ATF3体外敲低和过表达实验研究。通过转染技术敲低或过表达circRNA后,采用MTS增殖实验、Transwell侵袭实验、有限稀释法实验检测circRPPH1敲低与过表达后GSC的增殖、侵袭与自我更新能力的改变。采用RNA-pull down技术验证circRPPH1与ATF3的相互作用。采用RT-qPCR、Western Blot检测转染后GSC中干性标志物的表达情况。采用基因富集GSEA、双荧光素酶报告基因、RT-qPCR、ELISA、Western Blot检测ATF3的转录调控作用以及其下游TGF-β1/Smad信号通路的转导。最后,我们通过裸鼠颅内成瘤实验,在体内观察了敲低以及过表达circRPPH1对胶质母细胞瘤增殖特性的改变。结果:组织RT-qPCR结果显示circRPPH1在胶质母细胞瘤中呈现高表达。MTS实验、Transwell实验、有限稀释实验结果表明敲低circRPPH1和ATF3可以抑制GSC的增殖、侵袭与自我更新,而基因过表达能够促进这一作用。RNApulldown、Western Blot实验证明了circRPPH1能够结合ATF3蛋白,并能够提升ATF3的稳定性。双荧光素酶报告基因实验表明了ATF3可以转录调控Nestin表达。Western Blot实验证明了ATF3对下游TGF-β1/Smad信号的促进作用,同时它能够促进干性标志物表达,从而使肿瘤接受生长信号,不断增殖浸润,增加恶性程度。体内实验证明敲低circRPPH1表达能够抑制胶质瘤的增殖,而过表达circRPPH1则会促进肿瘤生长。结论:circRPPH1在胶质瘤干细胞中呈现高表达,且会导致患者的不良预后。Circ RPPH1可以通过结合ATF3,通过维持其稳定性,促使ATF3转录调控干性标志物Nestin表达,并激活下游TGF-β1/Smad信号持续表达,促进胶质母细胞瘤的增殖、侵袭与自我更新。这表明,circRPPH1-ATF3-TGF-β1/Smad信号在胶质瘤干细胞的增殖中发挥了重要调控作用。靶向circRPPH1可能是一种胶质母细胞瘤治疗的新靶点。

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： Cardiovascular medicine   Molecular biology  

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： ATF3   NF-kappa B   Ca Ski   HPV16   Cervical cancer  

摘要： Background: As a novel tumor suppressor mediator, activating transcription factor 3 (ATF3) has recently aroused an interest in its possible therapeutic applications in various cancers. In this study, we evaluated the effect of ATF3 overexpression on the cellular level of nuclear factor kappa B (NF-kappa B) in human papillomavirus (HPV)-infected Ca Ski cells. Further, we examined whether ATF3 could mediate cell cycle arrest and alter the apoptosis level of Ca Ski cells. Methods: The biological behavior of Ca Ski cells was evaluated prior and subsequent to the overexpression of ATF3 by MTT assay, fluorescence microscopy, cell cycle and annexin V/PI flow cytometric analysis. The effect of ectopic ATF3 expression on the cellular level of NF-kappa B in HPV-positive cells was evaluated by western blotting assay. Results: The overexpression of ATF3 in Ca Ski cells led to significant apoptosis and cell cycle arrest in the G1 phase. Western blotting assay revealed a discernible reduction of NF-kappa B p65 level in cervical cancer cells. Conclusion: ATF3 acts as a tumor suppressor factor in HPV16-infected Ca Ski cells and exerts anti-cancer effects on HPV16-related cervical cancer cells potentially by hindering cell growth and inducing cell cycle arrest through the down-regulation of NF-kappa B. Our results suggest that ATF3 induction or NF-kappa B suppression may be useful targets for HPV16-related cervical cancer prevention and treatment.

关键词： ER stress   integrated stress response   somatotroph axis   IGF-1   SIRT7   nonalcoholic fatty liver disease   nonalcoholic steatohepatitis   ATF3   aging   sirtuin  

摘要： Nonalcoholic fatty liver disease (NAFLD) can be ameliorated by calorie restriction, which leads to the sup-pressed somatotroph axis. Paradoxically, the suppressed somatotroph axis is associated with patients with NAFLD and is correlated with the severity of fibrosis. How the somatotroph axis becomes dysregulated and whether the repressed somatotroph axis impacts liver damage during the progression of NAFLD are unclear. Here, we identify a regulatory branch of the hepatic integrated stress response (ISR), which re-presses the somatotroph axis in hepatocytes through ATF3, resulting in enhanced cell survival and reduced cell proliferation. In mouse models of NAFLD, the ISR represses the somatotroph axis, leading to reduced apoptosis and inflammation but decreased hepatocyte proliferation and exacerbated fibrosis in the liver. NAD+ repletion reduces the ISR, rescues the dysregulated somatotroph axis, and alleviates NAFLD. These results establish that the hepatic ISR suppresses the somatotroph axis to control cell fate decisions and liver damage in NAFLD.

关键词： RAN-GTP   MMP2   ATF3   CXCR3   Cell migration   Breast cancer   Patient survival   cMet   cMyc and Ki67  

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

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： Cardiomyopathies   Epigenetics   Experimental models of disease   Histone analysis  

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.