关键词： ATF3   sFlt-1   二甲双胍   滋养细胞   子痫前期  

摘要： 研究目的: 本研究以子痫前期(preeclampsia,PE)患者和HTR-8/SVneo细胞为研究对象,探讨激活转录因子3(activation of transcription factor 3,ATF3)在氧化应激损伤滋养细胞中的作用,研究二甲双胍(metformin,MET)对滋养细胞氧化应激损伤的保护机制。 研究方法: 1、胎盘组织来自吉林大学第二医院住院并终止妊娠的产妇,自2023年3月至2024年3月共21例,对照组为7例无任何合并症的正常妊娠孕妇,实验组包括7例早发型子痫前期孕妇(发病孕周<34周)及7例晚发型子痫前期孕妇(发病孕周≥34周),具体的诊断标准参照《妊娠期高血压疾病诊治指南(2020版)》。归纳统计参与研究孕妇的一般资料并于分娩时收集其胎盘组织,通过蛋白免疫印记实验(Western blot,WB)检测各组ATF3的蛋白表达水平。 2、用HTR-8/SVneo滋养细胞进行PE模型制作:缺氧24/48小时。设置不同浓度梯度的二甲双胍处理滋养细胞组(100、500、1000、1500、2000、2500、3000μmol/L),在常氧或缺氧条件下处理24/48小时,利用细胞计数试剂(Cell Counting Kit-8,CCK8)对滋养细胞进行活力检测,选择最适二甲双胍浓度及最适处理时间作为后续实验条件。 3、实验分为4组:正常对照组(NC)、正常培养+二甲双胍处理组(NC+MET)、缺氧处理组(H)、缺氧+二甲双胍处理组(H+MET)。通过WB技术检测各处理组及对照组ATF3的蛋白表达水平。 4、通过酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测正常对照组(NC)、正常培养+二甲双胍处理组(NC+MET)、缺氧处理组(H)、缺氧+二甲双胍处理组(H+MET)细胞培养上清中sFlt-1(soluble fms-like tyrosine kinase 1,sFlt-1)的蛋白表达水平。 研究结果: 1、WB结果显示,不同组间ATF3与β-actin相对比值的总体均数存在着统计学差异(F=4.278,P=0.026)。晚发型子痫前期组(LOPE)胎盘中的ATF3表达水平较正常妊娠组(NC)和早发型子痫前期组(EOPE)均明显升高(P=0.009,P<0.001);早发型子痫前期组(EOPE)胎盘中的ATF3表达水平较正常妊娠组(NC)下降(P=0.028)。 2、CCK8检测显示缺氧24小时对滋养细胞活力没有明显影响,二甲双胍最适浓度为2000μmol/L。 3、WB结果显示,不同组间ATF3与β-actin相对比值的总体均数存在着统计学差异(F=5.087,P=0.004)。缺氧处理组(H)中的ATF3表达水平较正常对照组(NC)和正常培养+二甲双胍组(NC+MET)均明显升高(P<0.001,P=0.017),加入二甲双胍处理后,缺氧+二甲双胍处理组(NC+MET)中的ATF3表达水平较缺氧处理组(H)明显下降(P=0.033),正常培养+二甲双胍处理组(NC+MET)和缺氧+二甲双胍处理组(H+MET)中的ATF3表达水平较正常对照组(NC)无明显差别(P=0.171,P=0.102)。 4、ELISA结果显示,各组间细胞培养上清中sFlt-1的浓度存在着统计差异(F=6.156,P=0.018)。正常培养组(NC)、正常培养+二甲双胍组(NC+MET)、缺氧组(H)、缺氧+二甲双胍组(H+MET)培养24h后细胞上清液中sFlt-1的浓度分别为:595.67±331.92 ng/L、321.58±91.88 ng/L、1006.05±167.45 ng/L、420.20±177.06 ng/L。缺氧处理组(H)细胞培养液中的sFlt-1表达水平较正常对照组(NC)明显升高(P=0.044),加入二甲双胍处理后,正常培养+二甲双胍处理组(NC+MET)以及缺氧+二甲双胍处理组(H+MET)细胞培养液中的sFlt-1表达水平较缺氧处理组(H)明显下降(P=0.004、P=0.009),正常培养+二甲双胍处理组(NC+MET)以及缺氧+二甲双胍处理组(H+MET)细胞培养液中的sFlt-1表达水平较正常对照组(NC)无明显差别(P=0.150、P=0.338),正常培养+二甲双胍处理组(NC+MET)较缺氧+二甲双胍处理组(H+MET)细胞培养液中的sFlt-1表达水平无明显差别(P=0.583)。 研究结论: 1、胎盘中ATF3的表达水平与子痫前期疾病的严重程度有关,在晚发型子痫前期中滋养细胞损伤较轻,ATF3代偿性上升;在早发型子痫前期中滋养细胞损伤较重,ATF3表达下降。 2、缺氧应激可使滋养细胞ATF3和sF

关键词： osteoarthritis   ferroptosis   synovitis   bioinformatics   JUN   ATF3   CDKN1A   GPX4  

摘要： Purpose: Osteoarthritis (OA) is the most common joint disease worldwide and is the primary cause of disability and chronic pain in older *** is a type of programmed cell death characterized by aberrant iron metabolism and reactive oxygen species accumulation;however, its role in OA is not known. Methods: To identify ferroptosis markers co -expressed in articular cartilage and synovium samples from patients with OA, in silico analysis was *** genes were analyzed and the results were evaluated using a ROC curve prediction *** biological function, correlation between Signature genes, immune cell infiltration, and ceRNA network analyses were performed. Signature genes and ferroptosis phenotypes were verified through in vivo animal experiments and clinical samples. The expression levels of non -coding RNAs in samples from patients with OA were determined using qRT-PCR. ceRNA network analysis results were confirmed using dual-luciferase assays. Results: JUN, ATF3, and CDKN1A were identified as OA- and ferroptosis-associated signature genes. GSEA analysis demonstrated an enrichment of these genes in immune and inflammatory responses, and amino acid metabolism. The CIBERSORT algorithm showed a negative correlation between T cells and these signature genes in the cartilage, and a positive correlation in the synovium. Moreover, RP5-894D12.5 and FAM95B1 regulated the expression of JUN, ATF3, and CDKN1A by competitively binding to miR-1972, miR-665, and miR-181a-2-3p. In vivo, GPX4 was downregulated in both OA cartilage and synovium;however, GPX4 and GSH were downregulated, while ferrous ions were upregulated in patient OA cartilage and synovium samples, indicating that ferroptosis was involved in the pathogenesis of OA. Furthermore, JUN, ATF3, and CDKN1A expression was downregulated in both mouse and human OA synovial and cartilage tissues. qRT-PCR demonstrated that miR-1972, RP5-894D12.5, and FAM95B1 were differentially expressed in OA tis

关键词： 七氟烷   铁死亡   内质网应激   ATF3   过氧化氢   神经发育毒性  

摘要： 研究背景:随着麻醉和外科手术技术的快速进步和发展,越来越多的婴幼儿及孕产妇接受麻醉和手术治疗。人类的神经系统在胎儿妊娠末期至婴幼儿出生后2～3年内是发育的重要时期,发育期大脑极易受到包括全麻药物在内的各种因素的干扰和影响,可能产生发育脑神经毒性损伤。大量动物研究和临床数据表明,在生命发育的早期阶段暴露吸入麻醉药七氟烷、异氟烷能够引起婴幼个体未成熟大脑产生广泛的神经病理性改变,导致长期的行为学异常和神经认知功能损害。因此,探讨吸入麻醉药的神经发育毒性作用及其机制成为麻醉学乃至神经科学急需解决的问题。研究表明导致神经元死亡是吸入麻醉药诱导发育脑神经毒性的主要特征,但具体机制不清。铁死亡（ferroptosis）是细胞程序性死亡的一种形式,其特征是细胞内亚铁离子(Fe2+)增加,积聚的Fe2+能够与过氧化氢（H2O2）通过芬顿反应生成羟基自由基,进而对多不饱和脂肪酸（PUFA）产生脂质过氧化损伤。研究证实,铁死亡不仅介导心脏、肝脏和肾脏等器官损伤的细胞死亡过程,还与多种神经疾病的细胞死亡相关。内质网不仅是细胞内蛋白质合成、修饰、折叠加工的场所,也是驱动脂质过氧化的中枢枢纽。细胞感受内外环境的伤害刺激后,可引起内质网应激,其中PERK/ATF4通路活化与铁死亡的发生密切相关。作为一种应激反应因子,激活转录因子3（ATF3）在细胞受到多种伤害性刺激时迅速表达,进而调控相关靶基因转录激活或抑制。在内质网应激情况下,PERK/ATF4通路活化能够进一步激活ATF3,在多种形式的细胞死亡,包括细胞凋亡、自噬、铁死亡中起重要作用。目前,七氟烷能否诱导发育脑神经元铁死亡仍未明确。内质网应激介导的ATF3活化在七氟烷诱导的发育脑神经元铁死亡过程中作用如何,其调控细胞内铁离子稳态失衡的机制是什么,以及脂质过氧化损伤级联通过什么途径被激活尚不清楚。研究目的:（1）探究七氟烷能否诱导发育脑神经元铁死亡。（2）探究内质网应激介导的ATF3活化在七氟烷诱导发育脑铁死亡过程中可能的作用机制,为吸入麻醉药的神经发育毒性作用提供一种潜在的防治策略。研究方法:（1）HT22小鼠海马神经元细胞和大鼠原代海马神经元分别给予2%、4%、8%七氟烷处理6 h、12 h、24 h后,采用MTT法检测细胞的活力,LDH释放实验检测细胞的死亡率,应用铁离子和丙二醛（MDA）检测试剂盒检测神经元细胞内Fe2+浓度和MDA含量,应用光学显微镜观察神经元细胞的形态变化,应用透射电镜观察神经元细胞线粒体形态的变化,应用蛋白印迹法检测神经元细胞内铁离子代谢相关蛋白包括转铁蛋白受体（TFR）、转铁蛋白（TF）和铁转运蛋白（FPN）的表达。神经元细胞分别给予铁螯合剂去铁胺（DFO）(100μmol/L),抗氧化剂Fer-1（50μmol/L）、Lip-1（10μmol/L）、Vit E（100μmol/L）、GSH（2.5 mmol/L）或等量生理盐水预处理1 h,然后分别给予4%、8%七氟烷处理24 h后,检测细胞的生存率和死亡率及神经元细胞内Fe2+浓度和MDA含量。（2）HT22小鼠海马神经元细胞和大鼠原代海马神经元分别给予4%、8%七氟烷处理6～24 h,应用H2O2试剂盒检测神经元细胞内H2O2含量。神经元细胞分别给予GSH（2.5 mmol/L）或生理盐水预处理1 h,然后给予七氟烷处理,应用铁离子和过氧化氢试剂盒检测神经元细胞内Fe2+浓度和H2O2含量,应用蛋白印迹法检测神经元细胞内TFR、TF和FPN的表达。神经元细胞给予H2O2（500μmol/L）处理24 h后,检测神经元细胞内死亡率、神经元细胞内Fe2+浓度和MDA含量及TFR、TF和FPN的表达。（3）HT22小鼠海马神经元细胞和大鼠原代海马神经元给予4%、8%七氟烷处理6～24 h后,应用蛋白印迹法检测神经元细胞内ATF3的表达,应用共聚焦显微镜检测神经元细胞核内ATF3的聚集。神经元细胞应用si RNA沉默ATF3基因,再给予8%七氟烷处理24 h后,检测细胞的死亡率,应用铁离子、过氧化氢和MDA试剂盒检测神经元细胞内Fe2+、H2O2和MDA含量,应用蛋白印迹法检测神经元细胞内TFR、TF和FPN的表达。（4）HT22小鼠海马神经元细胞和大鼠原代海马神经元分别给予4%、8%七氟烷处理6～24 h后,应用蛋白印迹法检测神经元细胞内质网应激相关蛋白GRP78、PERK和ATF4的表达。神经元细胞给予内质网应激抑制剂4-PBA（250μmol/L）、PERK抑制剂GSK2606414（1μmol/L）或生理盐水预处理1 h,然后给予8%七氟烷处理24 h后,检测细胞的死亡率,应用铁离子、过氧化氢和MDA试剂盒检测神经元细胞内Fe2+、H2O2和MDA含量,应用蛋白印迹法检测神经元细胞GRP78、PERK、ATF4、A

关键词： Aristolochic acid nephropathy   ATF3   c-JUN   Transcription factor  

摘要： Background: Aristolochic acid nephropathy (AAN) is a rapidly progressive interstitial nephropathy caused by Aristolochic acid (AA). AAN is associated with the development of nephropathy and urothelial carcinoma. It is estimated that more than 100 million people worldwide are at risk of developing AAN. However, the underlying mechanisms driving renal deterioration in AAN remain poorly understood, and the treatment options are limited. Methods: We obtained GSE27168 and GSE136276 series matrix data from the Gene Expression Omnibus (GEO) related to AAN. Using the R Studio environment, we applied the limma package and WGCNA package to identify co-differently expressed genes (co-DEGs). By GO/KEGG/GSVA analysis, we revealed common biological pathways. Subsequently, co-DEGs were subjected to the String database to construct a protein-protein interaction (PPI) network. The MCC algorithms implemented in the Cytohubba plugin were employed to identify hub genes. The hub genes were cross-referenced with the transcription factor (TF) database to identify hub TFs. Immune infiltration analysis was performed to identify key immune cell groups by utilizing CIBERSORT. The expressions of AAN-associated hub TFs were verified in vivo and in vitro. Finally, siRNA intervention was performed on the two TFs to verify their Results: Our analysis identified 88 co-DEGs through the "limma" and "WGCNA" R packages. A PPI network comprising 53 nodes and 34 edges was constructed with a confidence level >0.4. ATF3 and c-JUN were identified as hub TFs potentially linked to AAN. Additionally, expressions of ATF3 and c-JUN positively correlated with monocytes, basophils, and vessels, and negatively correlated with eosinophils and endothelial cells. We observed a significant increase in protein and mRNA levels of these two hub TFs. Furthermore, it was found that siRNA intervention targeting ATF3, but not c-JUN, alleviated cell damage induced by AA. The knockdown of ATF3 protects against oxidative stress

关键词： IFN gamma signaling   immunotherapy   NSCLC   RCC   RGS1  

摘要： Background: Non-responsiveness is a major barrier in current cancer immune checkpoint blockade therapies, and the mechanism has not been elucidated yet. Therefore, it is necessary to discover the mechanism and biomarkers of tumor immunotherapeutic ***: Bioinformatics analysis was performed based on CD8(+) T cell infiltration in multiple tumor databases to screen out genes related to anti-tumor immunity. Associations between Regulator of G-protein signaling 1 (RGS1) and IFN gamma-STAT1 signaling, and MHCI antigen presentation pathway were examined by RT-qPCR, western blotting, and flow cytometry. The modulatory mechanisms of RGS1 were investigated via CHIP-qPCR and dual-luciferase assay. The clinical and therapeutic implications of RGS1 were comprehensively investigated using tumor cell lines, mouse models, and clinical samples receiving ***: RGS1 was identified as the highest gene positively correlated with immunogenicity among RGS family. Inhibition of RGS1 in neoplastic cells dampened anti-tumor immune response and elicited resistance to immunotherapy in both renal and lung murine subcutaneous tumors. Mechanistically, RGS1 enhanced the binding of activating transcription factor 3 (ATF3) to the promoter of interferon gamma receptor 1 (IFNGR1), activated STAT1 and the subsequent expression of IFN gamma-inducible genes, especially CXCL9 and MHC class I (MHCI), thereby influenced CD8(+) T cell infiltration and antigen presentation and processing. Clinically, lower expression level of RGS1 was associated with resistance of PD1 inhibition therapy and shortened progression-free survival among 21 NSCLC patients receiving ***: Together, these findings uncover a novel mechanism that elicits immunotherapy resistance and highlight the function of tumor-intrinsic RGS1, which brings new insights for future strategies to sensitize anti-PD1 immunotherapy.

关键词： triphenyl phosphate   diphenyl phosphate and 4-hydroxyphenyl phosphate (diphenyl) ester   apoptosis   endoplasmic reticulum stress   eIF2 alpha-ATF4/ATF3-CHOP pathway  

摘要： Purpose: Triphenyl phosphate (TPHP) is a widely used organophosphate flame retardant, which can be transformed in vivo into diphenyl phosphate (DPHP) and 4-hydroxyphenyl phosphate (diphenyl) ester (OH-TPHP) through biotransformation process. Accumulation of TPHP and its derivatives in biological tissues makes it necessary to investigate their toxicity and molecular ***: The present study evaluated the cellular effects of TPHP, DPHP, and OH-TPHP on cell survival, cell membrane damage, oxidative damage, and cell apoptosis using HeLa cells as in vitro model. RNA sequencing and bioinformatics analysis were conducted to monitor the differently expressed genes, and then RT-qPCR and Western bolt were used to identify potential molecular mechanisms and key hub ***: Results showed that OH-TPHP had the most significant cytotoxic effect in HeLa cells, followed by TPHP;and no significant cytotoxic effects were observed for DPHP exposure within the experimental concentrations. Biological function enrichment analysis suggested that TPHP and OH-TPHP exposure may induce endoplasmic reticulum stress (ERS) and cell apoptosis. The nodes filtering revealed that ERS and apoptosis related genes were involved in biological effects induced by TPHP and OH-TPHP, which may be mediated through the eukaryotic translation initiation factor 2 alpha/activating transcription factor 4 (ATF4)/ATF3- CCAAT/ enhancer-binding protein homologous protein (CHOP) cascade pathway and death receptor 5 (DR5) /P53 signaling ***: Above all, these findings indicated that ERS-mediated apoptosis might be one of potential mechanisms for cytotoxicity of TPHP and OH-TPHP.

关键词： ATF3   DNA methylation   transcription factor   thyroid cancer  

摘要： Background: Aberrant expression of oncogenes and/or tumor suppressor genes (TSGs) drives the tumorigenesis and development of thyroid cancer. We investigated the expression and function of a member of the activating transcription factor (ATF)/cAMP-responsive element-binding protein (CREB) transcription factor (TF) family, ATF3, in thyroid ***: Data from 80 patients with papillary thyroid cancer (PTC) in the First Affiliated Hospital of Sun Yat-sen University and 510 PTC samples in The Cancer Genome Atlas thyroid cancer database were utilized for gene expression and prognosis analyses. The survival data were analyzed by Kaplan-Meier curves and Cox regression with adjustment for age, sex, multilocality, extrathyroidal extension, lymph metastases, and history of neoadjuvant treatment. DNA methylation was analyzed by methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing PCR. TFs binding to ATF3 promoter were identified by DNA pull-down combined with mass spectrum assay, and confirmed by quantitative PCR (qPCR), luciferase reporter assay, and chromatin immunoprecipitation (ChIP)-qPCR. We conducted functional assays in vitro and in a xenograft mouse model to evaluate the function of ATF3 in thyroid cancer. Integrated analyses based on RNA sequencing, ChIP-seq, and CUT&Tag assays were performed to explore the mechanisms underlying the function of ***: ATF3 was significantly downregulated in PTC and patients with low ATF3 expression had reduced progression-free survival (adjusted hazard ratio = 0.50 [CI 0.26-0.98], p = 0.043). DNA hypermethylation in ATF3 promoter disrupted the binding of SP1 and MYC-MAX, leading to inactivation of the gene. ATF3 functioned as a TSG by inhibiting the proliferation and mobility of thyroid cancer cells. And ATF3 regulated the expression of a number of genes by binding to the regulatory elements of them, particularly for genes in MAPK and PI3K/AKT pathways. Among these target genes, filamin C was pos

关键词： ATF3/CFS1 axis   M2 macrophages   miR-27a-3p   NSCLC   TAM  

摘要： Non-small cell lung cancer (NSCLC) imposes a significant economic burden on patients and society due to its low overall cure and survival rates. Tumour-associated macrophages (TAM) affect tumour development and may be a novel therapeutic target for cancer. We collected NSCLC and tumour-adjacent tissue samples. Compared with the tumour-adjacent tissues, the Activation Transcription Factor 3 (ATF3) and Colony Stimulating Factor 1 (CSF-1) were increased in NSCLC tissues. Levels of ATF3 and CSF-1 were identified in different cell lines (HBE, A549, SPC-A-1, NCI-H1299 and NCI-H1795). Overexpression of ATF3 in A549 cells increased the expression of CD68, CD206 and CSF-1. Moreover, levels of CD206, CD163, IL-10 and TGF-beta increased when A549 cells were co-cultured with M0 macrophages under the stimulation of CSF-1. Using the starbase online software prediction and dual-luciferase assays, we identified the targeting between miR-27a-3p and ATF3. Levels of ATF3, CSF-1, CD206, CD163, IL-10 and TGF-beta decreased in the miR-27a mimics, and the tumour growth was slowed in the miR-27a mimics compared with the mimics NC group. Overall, the study suggested that miR-27a-3p might inhibit the ATF3/CFS1 axis, regulate the M2 polarization of macrophages and ultimately hinder the progress of NSCLC. This research might provide a new therapeutic strategy for NSCLC.