关键词： ATF3   HPV18   HeLa cells   p53   E6  

摘要： Activating transcription factor 3 (ATF3) is the first p53 stability regulator that interferes with the ubiquitination of p53. However, the E6 oncoprotein of high-risk human papillomaviruses (HPVs) binds to and induces proteasome-dependent degradation of the host p53 protein. Herein, we investigate the effects of ATF3 overexpression on cell cycle progression and apoptosis in HPV-18-infected HeLa cells, and further examine whether ATF3 could alter the apoptosis level of HeLa cells through the inhibition of E6-mediated p53 degradation. Cytological function of HeLa cells prior and subsequent to the overexpression of ATF3 was assessed using cell cycle and annexin V/PI flow cytometry analysis. Western blotting assays revealed no significant effect of ATF3 on the levels of p53 and E6 in HeLa cells. However, annexin V staining demonstrated increases in apoptosis. ATF3 acts as a tumor suppressor factor in HPV18-related cervical cancer which mediates apoptotic functions through a p53-independent pathway.

关键词： ATF3   CCL2   cerebral ischemia   microglia   TLR4/NF-kappa B signaling  

摘要： Background: The participation of activating transcription factor 3 (ATF3) in transient middle cerebral artery occlusion and reperfusion injury has been reported. However, the precise mechanism of ATF3 in cerebral ischemia is little known so far. Thus, the study examines the mechanism of action underlying the protective role of ATF3 following middle cerebral artery occlusion (MCAO) in rats. Methods and results: The MCAO rats exhibited reduced body weight and motor ability, while increased neurological deficits and brain infarct volume. Gene ontology (GO) enrichment and KEGG pathway analyses revealed that differentially expressed genes were mainly enriched in the TLR4/NF-kappa B signaling. Moreover, ATF3 was the most differentially expressed gene in brain tissues of MCAO rats versus sham-operated rats, which could bind to CCL2. ATF3 was reduced in MCAO rats, and ATF3 inhibited CCL2 expression to mediate the TLR4/NF-kappa B signaling. Functionally, ATF3 inhibited neuronal apoptosis, microglia activation, and pro-inflammatory cytokine production to alleviate brain injury in rats. By contrast, CCL2 was overexpressed in neurons and microglia, and CCL2 mitigated the effects of ATF3 to exacerbate brain injury in rats. Conclusion: Our findings suggested that ATF3 repressed neuronal apoptosis and microglia activation caused by cerebral ischemia via targeting CCL2 and mediating the TLR4/NF-kappa B signaling.

关键词： IL-6R alpha   ATF3   HCC   Sorafenib   Regorafenib  

摘要： Sorafenib and its derivative regorafenib are the first- and second-line targeted drugs for advanced HCC, respectively. Although both drugs improve overall survival, drug resistance remains the major barrier to their full efficacy. Thus, strategies to enhance sorafenib and regorafenib efficacy against HCC are solely needed. Interleukin-6 receptor alpha (IL-6R alpha) is the receptor of IL-6, a multi-functional cytokine, which plays key roles in liver-regeneration, inflammation and development of hepatocellular carcinoma (HCC). Here we show the expression of IL-6R alpha was induced in response to sorafenib. Depletion of IL-6R alpha abolished IL-6 induced STAT3 phosphorylation at 705th tyrosine and tumor growth of HCC cells under sorafenib treatment. Mechanistically, activating transcription factor 3 (ATF3) was induced in response to sorafenib and subsequently bound to the promoter of IL-6R alpha, leading to its transcriptional activation. Depletion of ATF3 or its upstream transcription factor, ATF4, attenuated IL-6R alpha induction and IL-6 mediated sorafenib resistance. The ATF4-ATF3-IL-6R alpha cascade is also activated by regorafenib. Furthermore, blockade of IL-6R alpha with the FDA approved IL-6R alpha antibody drug, Sarilumab, drastically attenuated both sorafenib and regorafenib resistance in patient-derived xenograft (PDX) tumors, where human IL-6 could be detected by a novel in situ hybridization technique, named RNAscope. Together, our data reveal that ATF3-mediated IL-6R alpha up-regulation promotes both sorafenib and regorafenib resistance in HCC, and targeting IL-6R alpha represents a novel therapeutic strategy to enhance sorafenib/regorafenib efficacy for advanced HCC treatment.

关键词： Heparanase 2   ER stress   Hypoxia   Cisplatin   Gene expression   ATF3  

摘要： Activity of heparanase, endoglycosidase that cleaves heparan sulfate side chains in heparan sulfate proteo-glycans, is highly implicated in tumor progression and metastasis. Heparanase inhibitors are therefore being evaluated clinically as anti-cancer therapeutics. Heparanase 2 (Hpa2) is a close homolog of heparanase that lacks HS-degrading activity and functions as an endogenous inhibitor of heparanase. As a result, Hpa2 appears to attenuate tumor growth but mechanisms that regulate Hpa2 expression and determine the ratio between heparanase and Hpa2 are largely unknown. We have recently reported that the expression of Hpa2 is induced by endoplasmic reticulum (ER) and proteotoxic stresses, but the mechanism(s) underlying Hpa2 gene regulation was obscure. Here we expand the notion that Hpa2 is regulated by conditions of stress. We report that while ER and hypoxia, each alone, resulted in a 3-7 fold increase in Hpa2 expression, combining ER stress and hypoxia resulted in a noticeable, over 40-fold increase in Hpa2 expression. A prominent induc-tion of Hpa2 expression was also quantified in cells exposed to heat shock, proteotoxic stress, lysosomal stress, and chemotherapy (cisplatin), strongly implying that Hpa2 is regulated by conditions of stress. Further-more, analyses of the Hpa2 gene promoter led to the identification of activating-transcription-factor 3 (ATF3) as a transcription factor that mediates Hpa2 induction by stress, thus revealing, for the first time, a molecular mechanism that underlies Hpa2 gene regulation. Induction of Hpa2 and ATF3 by conditions of stress that often accompany the rapid expansion of tumors is likely translated to improved survival of cancer patients. (c) 2021 Elsevier B.V. All rights reserved.

关键词： activating transcription factor 3 inducer   anti-obesity   adipocyte   adipogenesis   lipolysis   browning  

摘要： In this study, we investigated whether the activating transcription factor 3 (ATF3) inducer ST32db, a synthetic compound with a chemical structure similar to that of native Danshen compounds, exerts an anti-obesity effect in 3T3-L1 white preadipocytes, D16 beige cells, and mice with obesity induced by a high-fat diet (HFD). The results showed that ST32db inhibited 3T3-L1 preadipocyte differentiation by inhibiting adipogenesis/lipogenesis-related gene (and protein levels) and enhancing lipolysis-related gene (and protein levels) via the activation of beta 3-adrenoceptor (beta 3-AR)/PKA/p38, AMPK, and ERK pathways. Furthermore, ST32db inhibited triacylglycerol accumulation in D16 adipocytes by suppressing adipogenesis/lipogenesis-related gene (and protein levels) and upregulating browning gene expression by suppressing the beta 3-AR/PKA/p38, and AMPK pathways. Intraperitoneally injected ST32db (1 mg kg(-1) twice weekly) inhibited body weight gain and reduced the weight of inguinal white adipose tissue (iWAT), epididymal WAT (eWAT), and mesenteric WAT, with no effects on food intake by the obese mice. The adipocyte diameter and area of iWAT and eWAT were decreased in obese mice injected with ST32db compared with those administered only HFD. In addition, ST32db significantly suppressed adipogenesis and activated lipolysis, browning, mitochondrial oxidative phosphorylation, and beta-oxidation-related pathways by suppressing the p38 pathway in the iWAT of the obese mice. These results indicated that the ATF3 inducer ST32db has therapeutic potential for reducing obesity.

关键词： 结直肠癌   真菌   白色念珠菌   进展   Enolase   巨噬细胞   ACOD1   ATF3   T细胞  

摘要： 第一部分白色念珠菌在结直肠癌中的丰度及其对结直肠癌的影响 目的:本研究旨在研究白色念珠菌在结直肠癌中的丰度及其对结直肠癌的影响。 方法:收集结直肠癌患者、结肠息肉患者及健康人三组共粪便标本,进行18S r RNA基因和内转录间隔(ITS)区进行初步分析。筛选出在结直肠癌丰度最高且差异明显的真菌-白色念珠菌。建立DSS+AOM诱导的小鼠结直肠癌和小鼠结直肠癌皮下瘤模型,给予白色念珠菌灌胃,统计各组肿瘤生长速度和肿瘤体积等指标。 结果:(1)结直肠癌患者肠道中的白色念珠菌水平显著增加。(2)白色念珠菌促进结直肠癌进展。 结论:白色念珠菌在结直肠癌患者肠道中增多,且可以促进结直肠癌的进展。 第二部分白色念珠菌分泌的Enolase调控巨噬细胞促进结直肠癌进展的机制 目的:本研究旨在进一步探索白色念珠菌分泌的Enolase调控巨噬细胞促进结直肠癌进展的机制。 方法:1)使用原位杂交技术检测正常肠道组织和结直肠癌组织中白色念珠菌的菌丝相比例。(2)检测Enolase对结直肠癌细胞和巨噬细胞增殖、细胞因子分泌、蛋白质组学及有机酸代谢的影响。检测敲低ACOD1和ATF3对巨噬细胞功能及代谢的影响。运用Ch IP-q PCR一步探究转录因子ATF3下游靶基因。(3)研究Enolase中和抗体对白色念珠菌作用于结直肠癌的治疗作用及对肿瘤微环境中T细胞的影响。 结果:(1)白色念珠菌分泌的Enolase在结直肠癌患者结直肠癌组织中富集。(2)肿瘤细胞分泌的聚胺促进白色念珠菌由酵母态向菌丝态转变,诱导白色念珠菌分泌更多的Enolase。(3)Enolase特异性地影响巨噬细胞而不是结肠直肠癌细胞。(4)Enolase影响RAW264.7巨噬细胞代谢相关蛋白水平的变化。(5)Enolase通过Dectin-1/SYK级联激活巨噬细胞中的ACOD1表达。(6)ATF3作为Dectin-1/SYK/ACOD1轴的下游靶点调节巨噬细胞中PGE2的转录。(7)巨噬细胞RAW264.7体外产生的PGE2抑制CD8+CTLs的比例及功能。(8)体内实验,Enolase中和抗体对白色念珠菌作用于结直肠癌具有治疗作用,能增加结直肠癌微环境中CD8+CTLs的比例及功能。(9)组织芯片结果显示,ATF3的表达与结直肠癌的生存期成负相关。 结论:(1)结直肠癌患者肠道白色念珠菌增多,肿瘤细胞分泌的聚胺诱导白色念珠菌分泌更多的Enolase。(2)Enolase刺激结直肠癌微环境的肿瘤相关巨噬细胞乌头酸脱羧酶1(ACOD1)的表达增高,进而促进衣康酸的生成,衣康酸影响巨噬细胞三羧酸循环并影响ACOD1/ATF3信号通路。(3)转录因子ATF3下游因子PEG2通过影响肿瘤微环境中CD8+细胞毒性T细胞,进而促进结直肠癌进展。

关键词： 神经病理性疼痛   DRG   mRNA测序   CB1受体   CB2受体   ATF3   NPY  

摘要： 研究背景和目的:神经病理性疼痛（Neuropathic pain,NP）是由躯体感觉神经系统的损伤或疾病直接导致的疼痛,其临床表现为自发性疼痛、痛觉过敏、痛觉超敏和感觉异常等。内源性大麻素在痛觉调节中发挥重要作用,其镇痛作用的发挥主要是通过大麻素受体实现的。大麻素1型受体（Type 1 cannabinoid receptors,CB1受体）是一种Gi/o蛋白偶联受体,主要位于神经元的突触前膜,参与内源性大麻素介导的镇痛作用。CB1受体激动剂能否缓解周围神经损伤引起的神经病理性疼痛目前仍有争议,同时其产生的中枢神经系统副作用也限制了它的临床应用。因此将CB1受体作为神经病理性疼痛的镇痛靶点并不理想。本研究旨在寻找背根神经节（Dorsal root ganglion,DRG）中CB1受体调控的下游致痛基因,探究CB1受体及相关分子在神经病理性疼痛中的作用机制,为神经病理性疼痛的临床治疗提供潜在靶点和理论证据。方法:（1）建立神经病理性疼痛动物模型——坐骨神经慢性缩窄性损伤模型（Chronic constriction injury,CCI）,并进行痛行为学检测。对CCI模型大鼠L4L6 DRG进行RNA测序（RNA sequencing,RNA-seq）,采用q PCR和免疫荧光技术（Immunofluorescence,IF）验证差异基因（Differentially expressed genes,DEGs）。（2）腹腔注射CB1受体激动剂或拮抗剂,观察激活或拮抗CB1受体对正常小鼠及CCI模型小鼠痛行为学的影响。利用Advillin-Cre ERT2小鼠和CB1fl/fl小鼠杂交产生CB1 advillin-Cre ERT2小鼠（即初级感觉神经元中CB1受体特异性敲除小鼠,以下简称CB1 c KO小鼠）。检测CB1受体敲除前后及CCI造模后CB1 c KO小鼠的痛行为学变化。对TM诱导14天、28天以及CCI造模后的CB1 c KO小鼠的DRG组织进行m RNA测序,采用q PCR和IF对CB1受体相关分子如大麻素2型受体（Type 2 cannabinoid receptors,CB2受体）、激活转录因子3（Activating transcription factor 3,ATF3）和神经肽Y（Neuropeptide Y,NPY）等进行验证。（3）应用CB2受体拮抗剂AM630、NPY sh RNA和ATF3 sh RNA分别对DRG上CB2受体、NPY、ATF3进行干预,检测其对痛行为学的影响。采用q PCR和IF技术检测DRG中CB2受体、GFAP、S100a8、S100a9、Csf1r、NPY和ATF3 m RNA及蛋白水平的变化。结果:（1）CCI造模可诱导SD大鼠和C57/BL6小鼠产生长期持续的神经病理性疼痛。RNA测序、q PCR及IF证实DRG中CB1受体在CCI造模后显著下调。（2）拮抗CB1受体可诱发正常小鼠的生理性痛觉过敏,并进一步加重CCI诱导的神经病理性疼痛,而激活CB1受体不能改善神经病理性疼痛。同时,DRG初级感觉神经元中CB1受体敲除会诱发生理性痛觉过敏并加重神经病理性疼痛。结合大、小鼠的测序结果,最终筛选出3个与CB1受体相关的分子靶点:CB2受体、NPY和ATF3。（3）DRG中的CB2受体在野生小鼠和CB1 c KO小鼠CCI造模后显著升高。拮抗CB2受体显著缓解CB1 c KO小鼠CCI造模前后的机械痛觉超敏和热痛过敏,并抑制CCI造模引起的DRG中GFAP、Csf1r、S100a8和S100a9 m RNA的上调。拮抗CB2受体也可以改善野生CCI模型小鼠的神经病理性疼痛,但激活CB2受体无镇痛作用。（4）NPY和ATF3的表达在CB1 c KO以及CCI造模后均显著升高。CCI造模后,ATF3在DRG神经元的细胞核中显著增多,并与NPY存在共定位。沉默ATF3和NPY均能挽救CB1 c KO以及野生型小鼠CCI造模导致的机械痛觉超敏。细胞实验及在体实验均显示NPY受到ATF3的正向调控。结论:（1）初级感觉神经元中CB1受体参与生理条件下基础痛阈的维持,并在神经病理性疼痛中发挥镇痛作用。（2）CB2受体、ATF3和NPY是CB1受体调控的致痛靶点。（3）在神经病理痛的维持期,CB2受体的激活促进疼痛的发展,拮抗CB2受体可缓解疼痛。（4）初级感觉神经元中CB1受体通过靶向抑制ATF3/NPY通路发挥镇痛作用。

关键词： 鼻咽癌   PLUNC   DDX17   β-catenin   ATF3   PD-L1  

摘要： 目的:探讨PLUNC在鼻咽癌中如何通过调控DDX17/β-catenin相互作用进而来调控ATF3和PD-L1的表达。方法:用5-8F,HNE2细胞构建过表达PLUNC的稳转细胞株。用HNE2,S18细胞构建过表达β-catenin或DDX17的稳转细胞株。向5-8F,HNE2细胞转染PLUNC干扰质粒或DDX17干扰质粒。向5-8F和HONE1转染β-catenin干扰质粒。WB检测鼻咽癌细胞过表达或干扰PLUNC、DDX17、β-catenin表达后β-catenin通路相关蛋白质(β-catenin、p-GSK3β,TCF4),ATF3 和 PD-L1 的表达水平。细胞免疫荧光实验检测鼻咽癌细胞过表达或干扰PLUNC,过表达或干扰β-catenin后ATF3、PD-L1在鼻咽癌细胞中的表达以及定位情况。通过Co-IP和细胞免疫荧光实验来研究PLUNC、DDX17、β-catenin的相互作用关系。结果:WB结果显示在5-8F和HNE2细胞中,PLUNC抑制β-catenin 通路相关蛋白质(β-catenin、p-GSK3β),ATF3 和 PD-L1的表达。细胞免疫荧光实验结果显示,过表达PLUNC后,ATF3在细胞核的表达减弱,PD-L1在细胞核、细胞膜、细胞浆的表达减弱。向5-8F和HNE2细胞转染干扰PLUNC质粒后,β-catenin通路相关蛋白质(β-catenin、p-GSK3β、TCF4),ATF3 和 PD-L1 表达上调。在5-8F和HNE2过表达PLUNC稳转细胞株进行Co-IP和细胞免疫荧光实验,结果显示PLUNC与DDX17存在相互作用,在细胞浆和细胞核中存在共定位。WB显示在5-8F和HNE2细胞中PLUNC抑制DDX17的表达以及干扰DDX17后,ATF3和PD-L1的表达下调。用过表达β-catenin或DDX17的HNE2和S18稳转细胞株进行Co-IP和免疫荧光实验,结果显示DDX17与β-catenin存在相互作用,在细胞核中存在共定位。用过表达β-catenin的HNE2和S18细胞进行WB和细胞免疫荧光实验,干扰5-8F和HONE1的β-catenin表达后,进行WB和细胞免疫荧光实验,结果表明β-catenin上调ATF3和PD-L1的表达。结论:PLUNC可通过抑制DDX17/β-catenin相互作用下调ATF3和PD-L1在鼻咽癌中的表达。图7幅,表11个,参考文献70篇

关键词： 肝细胞癌   长链非编码RNA   Linc01612   预后   Linc01612   miR-494   ceRNA   ATF3   p53信号通路   Linc01612   RNA结合蛋白   YBX1   NEDD4L   泛素-蛋白酶体途径降解  

摘要： 第一部分:长链非编码RNA Linc01612在肝细胞癌中发挥抑癌作用并与患者预后相关 目的:本部分旨在探究长链非编码RNA Linc01612在肝细胞癌(HCC)中的表达情况,明确Linc01612对肝癌细胞肿瘤生物学行为(增殖,侵袭,迁移与凋亡)的影响,并评估肝细胞癌组织中Linc01612的表达与患者临床预后的关系。 方法:在TCGA数据库及GEO数据库中检索并分析长链非编码RNA Linc01612(别名RP11-789C1.1)在HCC中的表达差异;运用RT-qPCR技术、原位杂交(ISH)技术检测Linc01612在配对的肝细胞癌与癌旁组织中的表达,并进行统计学差异分析;运用RT-qPCR技术,荧光原位杂交(FISH)技术检测Linc01612在肝癌细胞中的表达情况及Linc01612在肝癌细胞中的亚细胞定位;结合肝细胞癌患者的临床资料,分析Linc01612的表达与肝细胞癌患者临床数据的相关性。运用基因过表达与沉默技术升高或抑制Linc01612在肝癌细胞中的表达,并通过CCK8实验、EdU实验及克隆形成实验检测肝癌细胞增殖能力的改变;通过流式细胞分析技术检测肝癌细胞的凋亡情况;通过Transwell实验、Wound healing实验观察肝癌细胞侵袭与迁移能力的改变;运用裸鼠皮下成瘤技术与尾静脉注射构建肺转移模型技术,检测Linc01612对于肝癌细胞体内成瘤和肺转移能力的影响。 结果:TCGA数据库(LIHC数据集)及GEO数据库(GSE138178数据集)分析显示Linc01612在肝细胞癌组织中显著低表达;本中心临床样本检测同样发现Linc01612在肝细胞癌组织中的表达显著低于配对癌旁组织;临床数据分析表明Linc01612的表达水平与肝细胞癌患者的肿瘤数目以及病理分化程度密切相关;生存分析表明,Linc01612高表达患者组5年总生存率明显高于低表达患者组,但无复发生存率无统计学意义。FISH实验及核质分离实验证实Linc01612在肝癌细胞以及正常永生化肝细胞中主要定位于细胞质内。细胞功能实验发现,过表达Linc01612可以显著抑制肝癌细胞的增殖、侵袭与迁移能力,并促进肝癌细胞的凋亡,而敲低Linc01612则得到相反的结果;动物实验表明,过表达Linc01612可以抑制肝癌细胞的皮下成瘤能力和肺转移能力。 结论:长链非编码RNA Linc01612在肝细胞癌中表达下调,且其低表达提示患者预后不良;Linc01612可以抑制肝癌细胞的肿瘤生物学行为。 第二部分:长链非编码RNA Linc01612通过竞争性结合miR-494促进ATF3的表达并激活p53信号通路 目的:本部分旨在探究Linc01612调控肝细胞癌进展的具体下游分子和信号通路。 方法:通过转录组测序及生物信息学分析明确Linc01612过表达后基因的差异表达及差异基因富集的具体信号通路;运用RT-qPCR、Western blotting实验进一步验证Linc01612过表达后的差异基因(ATF3)表达;运用Western blotting、免疫组化(IHC)实验验证富集信号通路(p53信号通路)相关分子(p53、p-p53、p21、Bc1-2)的表达情况。利用生物信息学软件(LncBase Predicted和TargentScanHuman)预测 Linc01612 和 ATF3 3'UTR 可以共同结合的 miRNA(miR-494);运用双荧光素酶报告实验验证Linc01612、ATF3 3’UTR与miR-494的调控关系;通过MS2-RIP实验,进一步证实Linc01612靶向结合miR-494。蛋白半衰期实验测定Linc01612对于p53蛋白稳定性的影响;蛋白泛素化实验检测Linc01612对于p53蛋白泛素化的影响;最后,运用CCK8以及流式细胞分析技术进行细胞功能回复实验。 结果:转录组测序结果表明,Linc01612过表达后差异基因富集到了 p53信号通路,并且与p53稳定性密切相关的ATF3基因显著上调。Linc01612的481nt-499nt及ATF3 3'UTR的1082nt-1088nt均是miR-494的潜在结合位点;过表达miR-494可同时抑制Linc01612与ATF3的表达;在肝细胞癌组织中,miR-494与ATF3的表达呈显著负相关关系,Linc01612与ATF3的表达呈显著正相关关系;Linc01612可以扮演内源竞争性RNA(ceRNA)角色,通过靶向结合miR-494从而解除miR-494对靶基因ATF3的抑制作用,提高ATF3的表达水平。蛋白半衰期及蛋白泛素化水平检测表明,过表达Linc01612在提高ATF3表达的同时,减缓了由E3泛素连接酶MDM2介导的p53