关键词： PM2.5   凋亡   炎症   卵巢颗粒细胞   ATF3  

摘要： 目的:PM2.5伴随着呼吸进入血液循环,到达全身各个系统,对人体有着潜在的危害。根据调查显示PM2.5对女性生殖系统可造成损伤,尤其是对卵巢功能造成一定损伤。然而,其潜在的影响机制还不明确。本实验是通过气管滴入PM2.5对大鼠进行造模,研究其卵巢损害;观察不同浓度的PM2.5与人卵巢颗粒细胞系(KGN)共培养后细胞活性变化,对细胞周期与凋亡的影响;提取细胞总RNA进行测序寻找靶标基因,并进行机制研究。初步讨论不同浓度的PM2.5处理KGN细胞可引起细胞活性下降,周期和细胞凋亡率的改变。方法:1.采用30只3周龄大小的雌性SD大鼠,随机分为3个组,对照组、低剂量组、高剂量组,每组10只,每隔3天进行一次气管滴注PM2.5,连续滴注5个月,对照组滴注PBS,低剂量组浓度为35 μg/m3,高剂量组浓度为150 μg/m3,5月末取大鼠卵巢,进行HE染色,在显微镜下进行阅片;2.将不同浓度的PM2.5与KGN共培养后,在不同时间点采用Cell Titer-Glo试剂进行细胞活性测定;***暴露于不同浓度的PM2.5后,分别收取24 h和36h的细胞,使用AnnexinV-FITC染料进行细胞凋亡检测;4.使用不同浓度的PM2.5处理KGN,收取细胞,使用PI染料进行细胞周期检测;5.将不同浓度的PM2.5与KGN共培养后,分别收取24 h和36 h的细胞,使用DCFH-DA染料进行细胞活性氧ROS的检测;***暴露于100 μg/mL的PM2.5后,收集24 h后的对照组与实验组总RNA进行RNA-seq测序,所有样本均在Novogene(HiSeq 4000)平台上测序;7.使用不同浓度的PM2.5处理KGN,收集不同时间点的细胞上清液,用于ELIAS试剂盒检测IL-6和TNFα细胞因子的浓度。结果:1.慢性暴露于PM2.5中可引起SD大鼠卵巢炎症改变和卵泡闭锁,影响卵巢储备功能。***2.5可抑制卵巢颗粒细胞活性,呈浓度与时间依赖性改变;***2.5可诱导卵巢颗粒细胞周期阻滞于S期,且浓度越大,S期阻滞越明显。***2.5可促进卵巢颗粒细胞发生凋亡,且浓度越大,凋亡率越明显。***2.5可增加卵巢颗粒细胞炎症因子分泌,IL-6和TNFα呈浓度依赖性改变。***-seq测序数据分析发现343个差异表达基因,其中发现181个基因表达上调,162个基因部分表达被抑制,KEGG通路富集于Apotosis、NF-κB、TNF等信号通路上。***3抑制PM2.5对卵巢颗粒细胞的活性毒性、凋亡发生、炎症的改变及氧化应激水平的作用。结论:1.经气管滴注PM2.5可诱导SD大鼠卵巢发生炎症反应,卵泡闭锁增多。2.暴露于PM2.5的卵巢颗粒细胞系(KGN)可引起细胞活力下降,细胞凋亡增加,细胞周期阻滞于S期,并触发炎症与氧化应激反应。***2.5通过激活转录因子ATF3,在体外抑制卵巢颗粒细胞细胞凋亡水平,减轻炎症和氧化应激反应。

关键词： 生物信息学   WGCNA   糖尿病肾病   ATF3   蛋白尿  

摘要： 目的:探讨糖尿病肾病(Diabetic kidney disease,DKD)患者血清关键基因转录激活因子3(Activating transcription factor 3,ATF3)水平变化及其与蛋白尿的相关性,为进一步寻找早期识别DKD的生物标志物提供理论依据。方法:首先在GEO数据库中根据纳入排除标准选择合适的基因数据集,之后对其进行差异基因和加权基因共表达网络分析(WGCNA),最后将二者结果取交集获得DKD相关基因。将其导入Cytoscape软件,根据Degree算法,进行筛选获得排名靠前的5个关键基因。之后选取2020年12月-2021年11月在我院肾内科门诊或住院部明确诊断为DKD患者,根据纳入及排除标准,筛选符合条件的DKD患者。测定其随机尿白蛋白/肌酐(UACR),根据测定的UACR值将最终纳入的病例分为:正常白蛋白尿组(UACR<30mg/g)、微量白蛋白尿组(UACR:30-300mg/g)、大量白蛋白尿组(UACR>300mg/g)。收集其一般资料及生化指标,运用酶联免疫吸附法(ELISA)对患者血清ATF3浓度进行测定;三组间一般资料及各项临床指标差异采用方差分析或非参数检验进行比较;血清ATF3与各项临床指标的相关性采用Pearson或Spearman进行分析;采用多重线性逐步回归法对血清ATF3的影响因素进行分析。结果:1.选择GSE142153数据集作为研究,进行差异基因及WGCNA分析并取交集后,共获得48个DKD相关基因,进一步筛选后获得前5个关键基因,分别为IL10、IL1A、ATF3、CCL2、CXCR3。2.最终纳入88例DKD患者,正常白蛋白尿组26例,微量白蛋白尿组30例,大量白蛋白尿组32例。三组患者间年龄、性别比、BMI均未见明显差异。与正常白蛋白尿组相比,微量白蛋白尿组与大量白蛋白尿组Hb、ALB显著降低(P<0.05),Scr、BUN、UACR及24小时尿蛋白定量显著升高(P<0.05),差异具有统计学意义。3.与正常白蛋白尿组相比,其他两组血清ATF3浓度明显升高,大量白蛋白尿组血清ATF3浓度最高,差异具有统计学意义(P<0.05)。4.在对血清ATF3浓度与各临床指标及蛋白尿进行相关性分析后发现,血清ATF3浓度与Hb、ALB、GFR呈负相关(r=-0.525,-0.751,-0.406,P均<0.05),与BUN、Scr、UACR及24小时尿蛋白定量呈正相关(r=0.497,0.426,0.992,0.946,P<0.05);5.将上述有意义的指标与血清ATF3浓度进一步行多重线性逐步回归分析后发现,Hb、UACR、24小时尿蛋白定量均为ATF3的影响因素,且24小时尿蛋白定量对其影响较大。结论:DKD患者血清ATF3水平明显升高,与蛋白尿的严重程度存在正相关,有望成为监测DKD的临床指标。

关键词： 椎间盘退变   髓核细胞   激活转录因子3   铁死亡   凋亡  

摘要： 目的 腰痛是世界范围内最普遍的肌肉骨骼疾病之一,造成巨大的社会经济负担,而椎间盘退变(Intervertebral disc degeneration,IDD)是导致腰痛的主要原因。研究表明髓核细胞(Nucleus pulposus cells,NPC)功能异常,尤其是细胞死亡、炎症反应和细胞外基质(Extracellular matrix,ECM)降解增加是引起IDD的关键发病机制。铁死亡是一种铁依赖性的非凋亡和非焦亡程序性细胞死亡,伴随活性氧升高,与IDD的病理机制有关。文献报道激活转录因子3(Activation transcription factor 3,ATF3)表达及功能失调与炎症反应、细胞凋亡、铁死亡、氧化应激和ECM代谢失衡有关,但其在IDD中的作用和潜在调控机制尚未确定。本研究拟通过叔丁基过氧化氢(Tert-butylhydroperoxide,TBHP)体外诱导NPC退变,验证过表达和沉默ATF3对NPC功能的调控作用并分析相关机制。此外,构建大鼠IDD模型,评估沉默ATF3对大鼠退变椎间盘的治疗作用。对理解IDD的病理机制提供了新的见解,并为IDD疾病的治疗提供新方法和新策略。 方法 1.从Ferrdb数据库下载铁死亡相关的基因,并在ChEA3网站进行转录因子富集分析,筛选与铁死亡相关的关键转录因子。 2.通过Western blot和免疫组织化学染色检测关键转录因子及其下游关键蛋白在正常和退变髓核组织的表达差异;通过实时定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,qRT-PCR)实验检测关键转录因子在不同浓度TBHP诱导的NPCs中的表达。 3.通过Western blot、流式细胞学、酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)验证关键转录因子对NPCs铁死亡、凋亡、ECM代谢以及炎症反应的影响;通过拯救实验验证敲低关键转录因子可逆转TBHP对NPC功能的影响。 4.31G细针穿刺大鼠尾椎间盘Co8/9,构建大鼠IDD模型;在术后第4周和第8周,将含有转录因子的腺病毒注射到穿刺的IVD中。术后12周,对鼠尾行影像学及组织学评估,验证关键转录因子对IDD的影响。 结果 1.共下载248个铁死亡基因,发现转录因子ATF3在GTEx共表达库中排名第一,在综合Mean Rank TF rank的加权库贡献中排名第二;Go富集分析表明,ATF3参与调控骨骼肌组织发育,预测ATF3是与铁死亡相关的关键的转录因子。 ***3在IDD患者和TBHP处理的NPC中显著高表达;ATF3和IL-1β水平与IDD严重程度呈正相关,而COL2A1和SLC7A11水平与IDD严重程度呈负相关。 3.功能获得和功能缺失表明敲低ATF3抑制NPC凋亡、铁死亡、ECM分解和IL-1β分泌,而过表达ATF3具有相反的作用。拯救实验表明敲低ATF3可逆转TBHP对NPC功能的影响。 4.成功构建大鼠IDD模型。敲低ATF3显著降低Pfirrmann分级和组织学评分,而过表达ATF3具有相反的作用,表明敲低ATF3可抑制大鼠IDD。 结论 ATF3在IDD患者和TBHP处理的NPC中显著上调。在TBHP的诱导下,ATF3可能发生核转位,进一步增加ATF3在细胞核中的水平,从而通过促进ATF3靶基因和下游基因的调控,介导IDD的病理过程。在功能上,ATF3不仅可能通过抑制SLC7A11和SOD2来促进TBHP诱导的NPC的凋亡和铁死亡,还可能促进ECM降解和IL-1β的分泌。此外,在大鼠模型中,敲低ATF3可以减轻IDD。我们的研究结果为了解IDD的病理提供了新的见解,这可能揭示治疗IDD的新方法。

关键词： 心肌缺血再灌注损伤   ATF3   铁死亡   FANCD2  

摘要： 研究背景目前全球心血管疾病的发病率和死亡率连续多年高居首位,在我国也是呈逐年攀升趋势,其中急性心肌梗死是最为凶险的类型之一,及时的再灌注治疗恢复血流通畅,是治疗心肌梗死最为行之有效的救治手段。然而,心肌血流再通引起的缺血再灌注(Ischemia/Reperfusion,I/R)损伤是影响心梗患者预后的重要因素之一。目前临床上对心肌I/R损伤的防治仍处于探索阶段,效果不稳定,防治I/R损伤依然面临瓶颈。因此,寻找新的防治靶点和策略显得尤为重要。铁死亡(Ferroptosis)是近年来被证实的一种新型的程序性细胞死亡方式,主要与铁过载和脂质过氧化等有关,且具有区别于其他程序性死亡的形态特征。研究表明,铁死亡参与了众多心血管疾病的发生和发展,抑制心肌细胞铁死亡可显著减轻心肌I/R损伤,减轻心功能障碍。但在铁死亡纵向分子机制研究等方面仍较欠缺。本研究中,我们通过高通量测序发现在心肌I/R损伤中,转录激活因子3(activating transcription factor 3,ATF3)可能是调控心肌细胞死亡的关键因子之一。ATF3在很多心血管疾病中有相关报道,但其作用也颇具争议性。作为转录因子家族成员之一的ATF3,参与了包括自噬、凋亡、坏死等多种细胞程序性死亡。而关于ATF3在心肌I/R损伤中对铁死亡作用如何,目前尚未阐明。研究目的1.明确ATF3在心肌缺血再灌注中的表达水平;2.阐明ATF3对心肌I/R诱导的铁死亡作用如何;3.并探究ATF3调控心肌铁死亡的可能机制。研究方法在C57BL/6小鼠心肌I/R模型和C57BL/6小鼠乳鼠原代心肌细胞H/R模型的基础上,采用转录组测序(RNA-seq)分析比较不同再灌注时间下小鼠心肌组织中差异表达的基因,筛选出ATF3进行下一步研究。运用基因敲除、回复、过表达等技术,结合RNA-seq、染色质免疫共沉淀(ChIP)等分子生物学手段分析得出铁死亡负调控分子FANCD2可能是ATF3下游直接调控的基因,并进一步阐明ATF3对心肌I/R损伤中心肌细胞及铁死亡的影响。研究结果***3在心肌缺血再灌注早期表达显著上调,且向细胞核转入增加;2.敲除ATF3可显著加重小鼠心肌I/R损伤,回复ATF3表达可减轻I/R损伤;***3可直接结合铁死亡抑制基因FANCD2转录起始位点并促进其转录;***3可抑制心肌缺血再灌注/缺氧复氧诱导的铁死亡;***3可抑制Erastin和RSL3诱导的铁死亡,增加心肌细胞活力;***3对野生型小鼠心肌I/R损伤具有显著的治疗性作用。结论ATF3减轻心肌I/R诱导的铁死亡,保护心肌,可能与直接结合铁死亡抑制基因FANCD2转录起始位点并促进其转录有关。

关键词： 结直肠癌干细胞   活化转录因子3   自我更新   上皮间质转化   免疫抑制因子  

摘要： 目的研究敲减活化转录因子3(ATF3)对结直肠癌肿瘤干细胞(CRC-CSCs)自我更新能力与上皮间质转化的影响,并检测免疫抑制因子分泌的变化。方法培养人结直肠癌细胞系HCT116,通过流式细胞术分选CD133+表达的CRC-CSCs细胞,转染sh-ATF3慢病毒载体至CRC-CSCs细胞作为实验组(sh-ATF3组),以转染sh-NC慢病毒载体作为阴性对照组(sh-NC组),正常培养的CRC-CSCs细胞作为对照组。实时荧光定量PCR(qRT-PCR)检测ATF3相对表达量,CCK-8法检测细胞活性变化,成球实验检测细胞生长更新能力,平板克隆形成实验检测细胞克隆形成能力,免疫荧光染色和蛋白质免疫印迹(Western Blot)法检测E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)的表达,酶联免疫吸附法(ELISA法)检测细胞分泌免疫抑制因子TGF-β1、VEGF、IL-6、IL-10的含量变化,Western Blot检测自杀相关因子(Fas)、Fas配体(FasL)蛋白表达水平。结果对照组比较,sh-ATF3组CRC-CSCs细胞中ATF3 mRNA相对表达量下降(P<0.05),转染后48 h和72 h细胞活性降低(P<0.05),细胞成球能力与克隆形成能力均下降(P<0.01),细胞中E-cadherin蛋白表达较高而Vimentin蛋白表达较低(P<0.05),同时,细胞上清液中TGF-β1、VEGF、IL-10的含量均明显下降(P<0.01),Fas蛋白表达升高,FasL蛋白表达下降(P<0.05)。结论敲减ATF3可抑制结直肠癌肿瘤干细胞自我更新与上皮间质转化,下调免疫抑制因子TGF-β1、VEGF、IL-10表达,并且上调Fas蛋白,下调FasL蛋白的表达。

关键词： ATF3   TGF-81   NFATC2  

摘要： Activating transcription factor 3 (ATF3), an inducible stress gene, is stimulated by transforming growth factor-beta1 (TGF-81) in a protracted and relentless manner in human mammary cancer cells (hBC cells;MDA-MB231). The molecular mechanism behind this stable expression of ATF3 via TGF-81 in MDA-MB231 cells is unknown. This study found that TGF-81 stimulated the expression of the nuclear factor of activated T Cells 2 (NFATC2) in MDA-MB231 cells and provided evidence of its interaction with ATF3. The functional characterization of NFATC2 in association with ATF3 was determined by silencing of NFATC2 using siRNA. Knock-down of NFATC2 decreased the expression of both ATF3 and its target gene MMP13 (matrix metalloproteinase 13, a critical invasive gene) in hBC cells. Chromatin immunoprecipitation revealed that TGF-81 promoted NFATC2 binding and NFATC2-ATF3 complex binding at the MMP13 promoter region, whereas silencing of NFATC2 decreased their binding in hBC cells. Thus, we uncovered the mechanism of interaction between NFATC2 and ATF3 regulated by TGF-81, and NFATC2 acted as a pivotal factor in providing ATF3 stability and further drove MMP13 transcription. Targeting NFATC2 and blocking its association with ATF3 could therefore help to slow the pro-gression of breast cancer.

关键词： 胶质母细胞瘤   替莫唑胺   自噬   转录激活因子3   14-3-3ζ  

摘要： 目的:探讨在替莫唑胺(temozolomide,TMZ)治疗敏感与耐药的胶质母细胞瘤中转录激活因子3 (activatingt ranscription factor3,ATF3)、14-3-3ζ和beclin-1的表达水平变化及其与TMZ耐药产生的相关意义。方法:采用RT-PCR及免疫组织化学法检测并比较同一患者TMZ治疗前及耐药时胶质母细胞瘤组织内ATF3、14-3-3ζ和beclin-1基因及蛋白质表达水平。取30μg/m LTMZ培养的U87细胞为实验组,普通溶剂培养的U87细胞为对照组,培养72h,MTT及annexin V-别藻蓝素(allophycoc yanin,APC)单染法分别检测并比较肿瘤细胞的生长抑制率、凋亡率,RT-PCR及蛋白质印迹检测并比较细胞内ATF3、14-3-3ζ和beclin-1基因及蛋白质表达水平。结果:TMZ耐药时胶质母细胞瘤组织中ATF3、14-3-3ζ和beclin-1表达水平均高于TMZ治疗前(P<0.05);TMZ敏感的U87细胞培养72 h肿瘤细胞生长抑制率和凋亡率均增加(P<0.05),细胞内ATF3、14-3-3ζ和beclin-1表达水平均低于对照组(P<0.05);三者呈同向表达。结论:在TMZ用药过程中,胶质母细胞瘤内ATF3激活可能上调14-3-3ζ的表达,进而启动并使自噬增强,导致TMZ耐药产生。

关键词： Pelvic organ prolapse   Vaginal fibroblasts   Oxidation stress   Extracellular matrix   MMPs/TIMPs   p38/Nrf2 pathway  

摘要： Pelvic organ prolapse (POP) is a common disorder in women, and it is characterized by weakening of pelvic supportive structure with extracellular matrix (ECM) degradation. Activating transcription factor 3 (ATF3) was upregulated in anterior vaginal wall tissues of POP patients. We hypothesized that upregulation of ATF3 might contribute to POP development. This study aims to unveil the role of ATF3 in the pathogenesis of POP using a H2O2-induced in vitro model. Vaginal fibroblasts were isolated from woman with POP-Q stage greater than II and asymptomatic women with normal pelvic floor support. Knockdown of ATF3 enhanced cell viability and decreased cell apoptosis. Flow cytometry and immunnofluorescence showed that ATF3 deficiency inhibited H2O2-induced ROS production and the expression of 8 OHdG and 4-HNE. Western blot and Real-time PCR analysis revealed that ATF3 deficiency attenuated ECM component degradation (increasing collagen I, collagen III and elastin) and MMPs/TIMPs imbalance (decreasing MMP2 and MMP9 and increasing TIMP2). Moreover, knockdown of ATF3 induced the activation of p38/Nrf2/HO-1 signaling pathway. Further treatment with p38 inhibitor SB203580 abolished the protection of ATF3 deficiency against H2O2-induced cell damage, which was reverted by Nrf2 activator TBHQ. Thus, ATF3 likely contributes to POP progression by inducing cell apoptosis, oxidative stress and ECM degradation via regulating p38/Nrf2 pathway, which provides a potential therapeutic target for POP.

关键词： Gastric cancer   ATF3   Cisplatin resistance   Ferroptosis   Nrf2   Keap1 signaling  

摘要： Background Currently, resistance against cisplatin (DDP) is a frequent problem for the success of advanced gastric carcinoma (GC) chemotherapy. Here, we sought to investigate the function of activating transcription factor 3 (ATF3) n GC chemoresistance. Methods Expression of ATF3 was determined in GC cell lines (MNK45, SGC7901, and BGC823) and cisplatin (DDP)-resistant cells (SGC7901/DDP and BGC823/DDP). Biological informatics was performed to analyze ATF3 expression and prognosis in GC patients. Cisplatin resistance was evaluated. Ferroptosis was detected after ATF3 transfection of cells. The underlying molecular mechanism was also investigated. Results Transcripts of ATF3 were decreased in GC cells and GC tissues. Kaplan-Meier plotter analysis revealed that ATF3 expression was positively related to the overall survival of GC patients. In particular, lower levels of ATF3 were observed in cisplatin-resistant SGC7901/DDP and BGC823/DDP relative to their parental cells. Notably, ATF3 elevation sensitized cisplatin-resistant cells to cisplatin. Mechanically, compared with parental cells, SGC7901/DDP and BGC823/DDP cells exhibited lower ferroptosis evident by lower ROS, MDA and lipid peroxidation and higher intracellular GSH levels. However, ATF3 elevated ferroptosis in SGC7901/DDP and BGC823/DDP cells. Intriguingly, ATF3 overexpression together with ferroptosis activator erastin or RSL3 treatment further enhanced ferroptosis and cisplatin resistance;however, the ferroptosis suppressor liproxstatin-1 reversed the function of ATF3 in ferroptosis and cisplatin resistance. Additionally, cisplatin-resistant cells exhibited stronger activation of Nrf2/Keap1/xCT signaling relative to parental cells, which was restrained by ATF3 up-regulation. Importantly, restoring Nrf2 signaling overturned ATF3-mediated ferroptosis and cisplatin resistance. Conclusion ATF3 may sensitize GC cells to cisplatin by induction of ferroptosis via blocking Nrf2/Keap1/xCT signaling, supporting a prom

摘要： Gastric cancer: Possible treatment targets identified New treatments for gastric cancer could involve controlling the activity of a regulatory gene and associated signaling pathway. Over-activation of the Wnt signaling pathway, which regulates many cellular functions, occurs in around half of gastric cancers. Further, the activating transcription factor 3 gene (ATF3) is thought to influence tumorigenesis, although its role in gastric cancer is unclear. Guohua Xie and co-workers at Shanghai Jiao Tong University, China, explored the function of ATF3 in human gastric cancer tissues. Patients with low ATF3 expression had poorer prognosis and shorter life expectancy. The team discovered that reduced expression of ATF3 triggered the increased expression of two of its target genes, which then altered Wnt signaling. Reduced ATF3 expression also boosted the invasiveness of gastric cancer cells. Initial results suggest that overexpression of ATF3 could suppress gastric cancer progression. ATF3 has been reported to be dysregulated in various cancers and involved in various steps of tumorigenesis. However, the mechanisms underlying the abnormal expression of ATF3 and its biological function in gastric cancer (GC) have not been well investigated. Here, we report ATF3 as one of the key regulators of GC development and progression. Patients with low ATF3 expression had shorter survival and a poorer prognosis. In vitro and in vivo assays investigating ATF3 alterations revealed a complex integrated phenotype that affects cell growth and migration. Strikingly, high-throughput sequencing and microarray analysis of cells with ATF3 silencing or of ATF3-low GC tissues indicated alterations in the Wnt signaling pathway, focal adhesions and adherens junctions. Mechanistically, the expression of beta-catenin and cell migration inducing hyaluronidase 1 (CEMIP) was significantly upregulated in GC cells with downregulated ATF3, which was synergistically repressed by the beta-catenin/TCF3 signa