Early Growth Response Gene-1 Deficiency Interrupts TGFβ1 Signaling Activation and Aggravates Neurodegeneration in Experimental Autoimmune Encephalomyelitis Mice

Early growth response protein 1(Egr-1)triggers the transcription of many genes involved in cell growth,differentiation,synaptic plasticity,and neurogenesis.However,its mechanism in neuronal survival and degeneration is still poorly understood.This study demonstrated that Egr-1 was down-regulated at mRNA and protein levels in the central nervous system(CNS)of experimental autoimmune encephalomyelitis(EAE)mice.Egr-1 knockout exacerbated EAE progression in mice,as shown by increased disease severity and incidence;it also aggravated neuronal apoptosis,which was associated with weakened activation of the BDNF/TGFβ1/MAPK/Akt signaling pathways in the CNS of EAE mice.Consistently,Egr-1 siRNA promoted apoptosis but mitigated the activation of BDNF/TGFβ1/MAPK/Akt signaling in SH-SY5Y cells.Our results revealed that Egr-1 is a crucial regulator of neuronal survival in EAE by regulating TGFβ1-mediated signaling activation,implicating the important role of Egr-1 in the pathogenesis of multiple sclerosis as a potential novel therapy target.

Expression of insulin-like growth factor-1 mRNA and protein level of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkeys

BACKGROUND： Insulin-like growth factor-I（IGF-1）, as one of the important members of growth factor family, participants in the regulation of many physiological functions and behaviors, having very strong neuroprotective effect. However, the expression of IGF-1 following cerebral ischemia/reperfusion is still disputed. OBJECTIVE： To observe the expression of IGF-1 and protein of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkey. DESIGN ： A completely randomized grouping design, controlled animal experiment SETTING ： Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University MATERIALS： ① Totally 17 rhesus monkeys , of either gender, aged 4 to 5 years, were enrolled . Seven rhesus monkeys observed with gene chip were randomly divided into 2 groups： sham operation group （n=3） and ischemia/reperfusion group 〈n=4〉. Ten rhesus monkeys observed with in situ hybridization and immunohistochemistry method were randomly divided into 2 groups： sham operation group 〈n=3 〉and ischemia/reperfusion group （n=7）. Rhesus monkeys observed under microscope were divided into 2 groups： sham operation group （n=6） and ischamia/reperfusion group （n=-11）.②Materials used in the experiment： cresyl violet （Sigma Company, America）; immunohistochemical reagent kit （ Huamei Bio-engineering Company）; In situ hybridization reagent kit （Boshide Bio-engineering Co.Ltd, Wuhan）; 12 800 dots chip （Boxing Company, Shanghai）. METHODS ： This experiment was carried out at the Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University from January 2001 to December 2003.① The onset area of middle cerebral artery was blocked for 2 hours, middle cerebral artery ischemia/reperfusion models were created.② After ischemia/reperfusion for 24 hours, cerebral tissue sections of rhesus monkeys were prepared and stained with cresyl violet. Image analysis was performed with 5001W image analysis software. Morphological change of corpora striata of operative side was observed in the rhesus monkeys between two groups. Total RNA was extracted from cerebral tissue. ③ Detection of gene chip： Cy3-duTP and Cy5-duTP were used to respectively perform reverse transcription labeling. The sample was reversely transcribed into cDNA, then hybridized with cDNA of cerebral tissue. Genes with the separate absolute value of cy3 and cy5〉800, cY3/cy5 〉 2（high expression） or 〈 0.5 （low expression） were found out. Those were genes with differential expression. ④ The expressions of IGF-1 mRNA and protein level of corpora striata in ischemic side of rhe- sus monkeys were detected between sham operation group and ischemia/reperfusion group at 9 and 24 hours after ischemia/reperfusion with in situ hybridization method and immunohistochemical method. Brown granules were IGF-1 protein positive cells. ⑤ Analysis of variance was used in the difference comparison of measurement data among groups. MAIN OUTCOME MEASURES ： ① Change of morphological structure of corpora striata at ischemic side in rhesus monkeys. ② Change of cerebral gene expression profiles at ischemia/reperfusion in rhesus monkeys between two groups.③ Expression of IGF-1 mRNA and protein level of corpora striata at ischemia/reperfu- sion in rhesus monkeys between two groups. RESULTS ： ① Pathological change ： Obvious pathological change of cerebral infarction appeared in the ischemia and reperfusion group, while there was no such pathological change in the sham operation group.② Change of gene expression profile ： There were 4480 genes with difference expression in the ischemia/reperfusion group and sham-operation group, in which, 260 genes had high expression and their absolute value was over 800, and 63 genes had low expression, cy3/cy5 of IGF-1 was 0.379, being relative low ex- pression. ③ IGF-1 mRNA and protein positive cell counts in corpora striata at cerebral ischemic side[IGF-1 mRNA： 〈9.72±1.18）,（9.11 ±0.76）,（14.77±0.60） counts/field：lGF-1 protein： （15.11 ±1.83）,（15.39±0.78）, （34.62±0.97）counts/field, P 〈 0.05-0.01]. CONCLUSION： IGF-1 mRNA and protein are lowly expressed in middle cerebral artery of rhesus monkeys at ischemia/reperfusion.

EGR-1和MMP-2在早期自然流产患者中的表达及临床意义

目的探讨早期生长反应因子(EGR-1)和基质金属蛋白酶-2(MMP-2)在早期自然流产患者绒毛中的表达及临床意义。方法采用RT-PCR和Westernblot检测20例早期自然流产患者(流产组)和20例正常早孕人工流产(对照组)绒毛组织中EGR-1和MMP-2的表达情况。结果流产组中EGR-1、MMP-2 mRNA和蛋白水平明显低于对照组,差异有统计学意义(P<0.05)。结论EGR-1和MMP-2在早孕绒毛组织中表达异常可能参与早期妊娠失败的发生。

三阴性乳腺癌组织中EGR1、AR、H3K4me3表达与其临床病理特征及预后的相关性

目的探讨三阴性乳腺癌组织中早期生长反应基因1(EGR1)、雄激素受体(AR)、组蛋白H3第4位赖氨酸三甲基化(H3K4me3)表达与其临床病理特征及预后的相关性。方法回顾性分析89例三阴性乳腺癌患者的临床资料,所有患者均行手术治疗,采用免疫组化染色法测定EGR1、AR、H3K4me3表达,比较肿瘤组织及癌旁正常组织内EGR1、AR、H3K4me3表达差异,分析EGR1、AR、H3K4me3表达与其临床病理的关系,分析EGR1、AR、H3K4me3表达与其预后的关系。结果肿瘤组织内EGR1阳性表达率、AR阳性表达率低于对照组,H3K4me3阳性表达率高于对照组,差异有统计学意义(P<0.05);EGR1阳性表达、AR阳性表达患者临床分期Ⅰ~Ⅱ期、无淋巴结转移、中高分化占比高于EGR1阴性表达、AR阴性表达患者,H3K4me3阳性表达患者临床分期Ⅲ期、淋巴结转移、低分化占比高于H3K4me3阴性表达患者,差异有统计学意义(P<0.05);随访1年,89例患者共36例出现复发转移,发生率为40.45%(36/89);复发转移组EGR1阳性表达、AR阳性表达率低于未复发转移组,H3K4me3阳性表达率高于未复发转移组,差异有统计学意义(P<0.05)。结论EGR1、AR在三阴性乳腺癌中多呈阴性表达,H3K4me3则以阳性表达为主,三者表达均与临床分期、淋巴结转移及分化程度存在密切关系,或可作为临床治疗的新靶点。

EGR3通过抑制PRMT1/p-STAT3通路减轻肥胖相关性肾病足细胞炎症损伤

目的:肥胖会导致肥胖相关性肾病(obesity related glomerulopathy,ORG),但其发病机制并不明确。本研究拟检测早期生长反应蛋白3(early growth response protein 3,EGR3)在ORG患者和高脂饮食诱导的肥胖小鼠肾皮质组织中的表达,并探讨EGR3抑制棕榈酸(palmitic acid,PA)诱导的人足细胞炎症损伤的分子机制。方法:收集排除其他疾病导致的肾损害并经组织病理学证实的ORG患者(n=6)和高脂饮食诱导的肥胖小鼠的肾皮质组织(n=10)。使用150μmol/L PA干预人和小鼠足细胞48 h;人足细胞中分别过表达或沉默EGR3。采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测白细胞介素(interleukin,IL)-6和IL-1β的含量;real-time RT-PCR检测EGR3、足细胞分子标志NPHS1(nephrosis1)、NPHS2(nephrosis2)、足糖萼蛋白(podocalyxin,PODXL)、平足蛋白(podoplanin,PDPN)mRNA的表达;RNA-seq检测人足细胞过表达EGR3并150μmol/L PA干预后与对照组的差异表达基因(differentially expressed genes,DEGs);免疫共沉淀(co-immunoprecipitation,Co-IP)+液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS)检测EGR3可能的相互作用蛋白质,并与RNA-seq的结果取交集;Co-IP验证EGR3与蛋白精氨酸甲基转移酶1(protein arginine methyltransferases 1,PRMT1)的相互作用;沉默EGR3和PRMT1抑制剂干预后检测PA诱导的足细胞培养液中IL-6和IL-1β的含量;蛋白质印迹法检测分别过表达或沉默EGR3后磷酸化信号转导及转录激活蛋白3(phosphorylated signal transducer and activator of transcription 3,p-STAT3)的蛋白质表达。结果:EGR3在ORG患者和高脂饮食诱导的肥胖小鼠肾皮质组织中的表达均显著上调(均P<0.01),150μmol/L PA干预人和小鼠足细胞48 h后显著上调2种细胞EGR3的表达(均P<0.05)。人足细胞过表达或沉默EGR3分别抑制或促进PA干预后细胞培养液中IL-6和IL-1β的分泌,并分别上调或下调NPHS1、PODXL、NPHS2及PDPN的表达(均P<0.05)。RNA-seq结果显示共有988个DEGs,Co-IP+LC-MS共发现238个可能与EGR3相互作用的蛋白质,且Co-IP证实PRMT1为EGR3的相互作用蛋白质。PRMT1抑制剂能部分减少人足细胞沉默EGR3后PA诱导的IL-6及IL-1β的分泌(均P<0.05);此外,过表达或沉默EGR3负调控PRMT1及p-STAT3的表达。结论:EGR3可能通过抑制PRMT1/p-STAT3通路减轻ORG足细胞炎症损伤。

微RNA-196a-1-3p靶向Ras响应元件结合蛋白调控胆管癌细胞增殖的机制研究

目的探讨转化生长因子β(TGF-β)调控人胆管癌细胞系RBE细胞增殖的关键微RNA(miRNA)及其潜在的机制。方法该研究起止时间为2020年1月至2022年1月。磷酸盐缓冲液(PBS)处理为对照组,TGF-β处理为TGF-β组,TGF-β抗体处理为抗体组。检测三组RBE细胞的增殖水平。miRNA高通量测序检测三组RBE细胞的miRNA调控变化,并进行miRNA模拟物过表达筛选鉴定受TGF-β调控的影响RBE细胞增殖水平的关键miRNA。miRNA数据库(miRDB)在线分析miRNA的潜在底物,并通过小干扰RNA(siRNA)敲低筛选鉴定影响RBE细胞增殖水平的关键底物。结果相比于对照组,TGF-β组RBE细胞的增殖水平上升(1.62±0.07比2.35±0.09,P<0.05),抗体组RBE细胞的增殖水平下降(1.62±0.07比1.11±0.08,P<0.05)。过表达微RNA-196a-1-3p(miR-196a-1-3p)时,RBE细胞的增殖水平下降(P<0.05)。敲低Ras响应元件结合蛋白(RREB1)时,RBE细胞的增殖水平下降(P<0.05)。过表达miR-196a-1-3p后,RBE细胞中RREB1的信使RNA(mRNA)和蛋白水平下降(P<0.05)。敲低miR-196a-1-3p后,RBE细胞中RREB1与SMAD家族蛋白3(SMAD3)的相互作用增加。敲低SMAD3后,RBE细胞的增殖水平下降(P<0.05)。与仅敲低SMAD3相比,敲低SMAD3的同时过表达RREB1的RBE细胞的增殖水平无显著变化,并且同时敲低SMAD3和miR-196a-1-3p的RBE细胞的增殖水平无显著变化。结论TGF-β能够通过miR-196a-1-3p/RREB1/SMAD3轴促进RBE细胞增殖;miR-196a-1-3p和RREB1可作为潜在的治疗胆管癌的靶标,为针对该靶标的新药研发奠定了基础。

Insulin-like growth factor-binding protein-3 inhibits IGF-1-induced proliferation of human hepatocellular carcinoma cells

OBJECTIVE Basic fibroblast growth factor(b FGF)and platelet-derived growth factor(PDGF)produced by hepatocellular carcinoma(HCC)cells are responsible for the cell growth.Accumulating evidence shows that insulin-like growth factor-binding protein-3(IGFBP-3)suppresses HCC cell proliferation in both IGF-dependent and independent manners.The present study is to investigate whether treatment with exogenous IGFBP-3 inhibits bF GF and PDGF production and the cell proliferation of HCC cells.METHODS Cell Counting Kit 8 assay were designed to detect HCC cell proliferation,transcription factor early growth response-1(EGR1)involving in IGFBP-3 regulation of b FGF and PDGF were detected by RT-PCR and Western blot assays.Western blot assay was adopted to detect the IGFBP-3 regulating insulin-like growth factor 1 receptor(IGF-1R)signaling pathway.RESULTS The present study demonstrates that IGFBP-3 suppressed IGF-1-induced b FGF and PDGF expression while it does not affect their expression in the absence of IGF-1.To delineate the underlying mechanism,Western-blot and RT-PCR assays confirmed that the transcription factor early growth response protein 1(EGR1)is involved in IGFBP-3 regulation of b FGF and PDGF.IGFBP-3 inhibition of type 1 insulin-like growth factor receptor(IGF1R),ERK and AKT activation is IGF-1-dependent.Furthermore,transient transfection with constitutively activated AKT or MEK partially blocks the IGFBP-3 inhibition of EGR1,b FGF and PDGF expression.CONCLUSION In conclusion,these findings suggest that IGFBP-3suppresses transcription of EGR1 and its target genes b FGF and PDGF through inhibiting IGF-1-dependent ERK and AKT activation.It demonstrates the importance of IGFBP-3 in the regulation of HCC cell proliferation,suggesting that IGFBP-3 could be a target for the treatment of HCC.

转化生长因子-β_(1)相关血清微小核糖核酸对糖尿病肾病早期诊断的临床价值

目的探讨转化生长因子β_(1)(TGF-β_(1))相关血清微小核糖核酸(miRNA)对糖尿病肾病(DKD)早期诊断的临床价值。方法收集2019年1月至2021年12月在缙云县壶镇镇中心卫生院及缙云县人民医院就诊的2型糖尿病(T2DM)患者260例作为研究对象。根据尿白蛋白排泄率(UAER),研究对象分为单纯T2DM组135例、早期DKD组82例、临床DKD组43例。另选取50名健康志愿者作为对照组。检测各组血清miR-27a、miR-27b、miR-29c、miR-200c水平和TGF-β_(1)水平,并进行指标间的相关关系分析。利用受试者工作特征(ROC)曲线分析血清miRNAs对DKD的早期诊断价值。结果单纯T2DM组、早期DKD组和临床DKD组患者血清miR-27a、miR-27b、miR-200c水平及TGF-β_(1)水平均高于对照组,miR-29c水平低于对照组(F=235.624、70.351、108.127、672.304,P均<0.001)。TGF-β_(1)与血清miR-27a、miR-27b、miR-200c呈正相关,与miR-29c呈负相关(r=0.668、0.429、0.618、-0.502,P均<0.001)。血清miR-27a、miR-27b、miR-29c、miR-200c对T2DM患者发生DKD的诊断曲线下面积(AUC)为0.913、0.775、0.812、0.894,miR-27a和miR-200c的敏感度和特异度均>0.7。验证队列中血清miR-27a、miR-27b、miR-29c、miR-200c对T2DM患者发生DKD的诊断AUC分别为0.901、0.787、0.802、0.896,差异有统计学意义(Z=-9.012,P<0.01)。结论血清miR-27a、miR-27b、miR-29c、miR-200c对于早期DKD都有一定的诊断价值。

Nrf2、LTBP2、PECAM1与NSCLC患者EGFR靶向治疗反应性关系及意义

目的:探讨血清核因子E2相关因子2(Nrf2)、转化生长因子结合蛋白2(LTBP2)、血小板内皮细胞黏附分子1(PECAM1)与非小细胞肺癌(NSCLC)患者表皮生长因子受体(EGFR)靶向治疗反应性关系及意义。方法:选取我院2021年1月—2023年1月收治的95例NSCLC患者为研究对象,根据EGFR靶向治疗3个月后的治疗反应性分为有效组(54例)、无效组(41例)。比较2组治疗前及治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平,分析治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平与NSCLC患者EGFR靶向治疗反应性相关性,偏回归分析治疗无效的影响因素,受试者工作特征曲线(ROC)分析治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平联合检测对NSCLC患者EGFR靶向治疗效果的预测价值。结果:治疗1个月、2个月后无效组血清Nrf2、LTBP2、PECAM1水平均高于有效组(P<0.05);相关性分析发现,治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平与治疗反应性均呈负相关(P<0.05);Logistic回归分析发现,治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平为NSCLC患者EGFR靶向治疗效果的影响因素(P<0.05);ROC曲线分析发现,治疗1个月、2个月后血清Nrf2、LTBP2、PECAM1水平联合预测治疗无效的曲线下面积(AUC)分别为0.761、0.816,最佳预测敏感度、特异度分别为(85.37%、66.67%)、(92.68%、70.37%),高于单一指标预测(P<0.05)。结论:血清Nrf2、LTBP2、PECAM1水平与NSCLC患者EGFR靶向治疗反应性密切相关,并在预测治疗效果方面具有较高效能。

老年缺血性脑卒中患者血清Egr-1、Neuregulin 1表达水平对血管性痴呆的预测效能

目的分析老年缺血性脑卒中患者血清早期生长反应因子1(Egr-1)、神经调节蛋白1(NRG1)表达水平对血管性痴呆(VD)的预测效能。方法将2019年5月至2022年3月该院收治的122例老年缺血性脑卒中患者作为研究对象,随后根据治疗后6个月内是否发生血管性痴呆分为痴呆组(n=55)、非痴呆组(n=67),根据简易精神状态评价量表(MMSE)评分将痴呆组患者分为轻度组、中度组、重度组。采用酶联免疫吸附试验检测血清Egr-1、NRG1表达水平,比较各组血清Egr-1、NRG1表达水平;采用受试者工作特征(ROC)曲线分析血清Egr-1、NRG1对老年缺血性脑卒中患者血管性痴呆的预测效能,采用多因素Logistic回归分析影响缺血性脑卒中患者发生血管性痴呆的危险因素。结果痴呆组患者血清Egr-1表达水平明显高于非痴呆组,NRG1表达水平明显低于非痴呆组,差异有统计学意义(P<0.05)。血管性痴呆患者重度组血清Egr-1表达水平明显高于中度组,中度组高于轻度组,差异有统计学意义(P<0.05),NRG1表达水平明显低于中度组、轻度组,中度组低于轻度组,差异有统计学意义(P<0.05)。ROC曲线结果显示,血清Egr-1联合NRG1预测老年缺血性脑卒中患者发生血管性痴呆的曲线下面积(AUC)为0.925,高于血清Egr-1(AUC=0.874)、NRG1(AUC=0.785)单独预测。多因素Logistic回归分析显示,血清Egr-1≥1.63 pg/mL(OR=3.404,95%CI:1.906~6.081)、NRG1<12.66 pg/mL(OR=2.795,95%CI:1.709~4.572)为影响老年缺血性脑卒中患者血管性痴呆发生的危险因素(P<0.05)。结论血清Egr-1表达水平在老年缺血性脑卒中血管性痴呆患者中异常升高,而血清NRG1异常降低,且二者表达水平与患者血管性痴呆严重程度密切相关,同时对缺血性脑卒中患者血管性痴呆的发生有良好的预测效能。

急性缺血性脑卒中患者血清miR-124-3p和EGR1的表达水平及其预测并发吞咽障碍的价值

目的:探讨血清微RNA-124-3p(microRNA-124-3p,miR-124-3p)、早期生长反应因子1(early growth response 1,EGR1)在急性缺血性脑卒中(acute ischemic stroke,AIS)患者合并吞咽障碍中的预测价值。方法:回顾性选取2020年5月至2023年3月期间郑州大学第五附属医院神经内科收治住院的181例AIS患者为研究对象,依据吞咽功能测试分为障碍组(62例)和无障碍组(119例)。采用酶联免疫吸附法检测血清EGR1表达水平,real-time RT-PCR检测血清miR-124-3p表达水平;AIS患者血清miR-124-3p、EGR1表达水平与基线资料的相关性采用Spearman及Pearson分析;血清miR-124-3p、EGR1表达水平对AIS患者并发吞咽障碍的预测价值采用受试者操作特征(receiver operating characteristic,ROC)曲线分析;AIS患者并发吞咽障碍的影响因素采用logistic回归分析。结果:障碍组血清EGR1表达水平、年龄、美国国立卫生研究院卒中量表(National Institutes of Health Stroke Scale,NIHSS)评分均高于无障碍组(均P<0.05);血清miR-124-3p表达水平、Barthel指数、肌力分级>3级患者比例均低于无障碍组(均P<0.05)。AIS并发吞咽障碍患者血清miR-124-3p表达水平与年龄、NIHSS评分呈负相关(r=−0.514、−0.520,均P<0.05),与Barthel指数、肌力分级呈正相关(r=0.509、0.492,均P<0.05);EGR1表达水平与年龄、NIHSS评分呈正相关(r=0.498、0.516,均P<0.05),与Barthel指数、肌力分级呈负相关(r=−0.527、−0.494,均P<0.05)。血清miR-124-3p、EGR1表达水平单独及联合预测AIS患者并发吞咽障碍的曲线下面积(area under curve,AUC)分别为0.795、0.811、0.880。EGR1是AIS患者并发吞咽障碍的独立危险因素,miR-124-3p是AIS患者并发吞咽障碍的保护因素(均P<0.05)。结论:并发吞咽障碍的AIS患者血清miR-124-3p呈低表达,EGR1呈高表达,临床可根据二者的联合检测对吞咽障碍进行早期评估,以改善预后。

血清早期生长应答因子1与2型糖尿病视网膜病变的相关性

目的探讨血清早期生长应答因子1(Egr-1)水平与2型糖尿病(T2DM)视网膜病变(DR)的相关性。方法选取医院2020年1月至2022年6月收治的T2DM患者84例,按是否合并DR分为A组(合并DR,40例)和B组(不合并DR,44例),另选取同期健康体检者48例作为对照组(C组)。常规检测3组患者的血糖、肾功能等指标水平,比较血清Egr-1水平。结果B组、C组超敏C反应蛋白(hs-CRP)、空腹血糖(FBG)、糖化血红蛋白(HbA1C)水平明显高于A组,且C组明显高于B组(P<0.05);C组尿素氮(BUN)、Egr-1明显高于A组和B组(P<0.05),且B组与A组相当(P>0.05);C组24 h尿微量白蛋白水平明显高于B组(P<0.05),胰岛素抵抗指数(HOMA-IR)与B组相当(P>0.05)。Egr-1与24 h尿微量白蛋白,BUN,FBG,HbA1C,hs-CRP水平及DR分期均呈正相关性(r=0.639,0.567,0.688,0.601,0.939,0.712;P<0.05)。随着DR分期的增加,血清Egr-1水平逐渐增加,而Ⅰ、Ⅱ期DR患者Egr-1水平相当(P>0.05)。结论血清Egr-1可能作为DR的一种新型炎性生物学标志物用于临床诊断。

2型糖尿病患者血清miR-140-3p和EGR1水平与肠道菌群的关系研究

目的 探究2型糖尿病(T2DM)患者血清微小RNA-140-3p(miR-140-3p)和早期生长反应因子1(EGR1)水平与肠道菌群的关系。方法 回顾性选取2021年3月至2022年12月在青岛大学附属青岛市中心医院就诊治疗的125例T2DM患者作为T2DM组,另选取同期体检健康者100名作为对照组。采用实时荧光定量PCR测定血清miR-140-3p、EGR1 mRNA水平,比较对照组与T2DM组的一般资料以及血清miR-140-3p、EGR1 mRNA水平;采用Pearson分析T2DM患者血清miR-140-3p、EGR1 mRNA水平与肠道菌水平的相关性。结果 与对照组比较,T2DM组的体重指数、腰围、空腹血糖、空腹胰岛素、胰岛素抵抗指数、甘油三酯、总胆固醇均显著上调,差异均有统计学意义(P<0.05)。T2DM组血清miR-140-3p较对照组均显著下调,血清EGR1 mRNA水平显著上调,差异均有统计学意义(P<0.05)。与对照组相比,T2DM组乳酸杆菌、拟杆菌水平均显著减少,T2DM组双歧杆菌、肠杆菌、肠球菌、酵母菌水平均显著增加,差异均有统计学意义(P<0.05)。Pearson相关分析显示,T2DM患者血清miR-140-3p水平与乳酸杆菌、拟杆菌水平均呈正相关(P<0.05),与双歧杆菌、肠杆菌、肠球菌、酵母菌水平均呈负相关(P<0.05);T2DM患者血清EGR1 mRNA水平与乳酸杆菌、拟杆菌水平均呈负相关(P<0.05),与双歧杆菌、肠杆菌、肠球菌、酵母菌水平均呈正相关(P<0.05)。结论 T2DM患者血清miR-140-3p表达水平显著下调,EGR1表达水平显著上调,且与患者肠道菌水平具有一定相关性。

盐诱导激酶抑制剂HG-9-91-01对小鼠脓毒症相关认知功能障碍的保护作用及其机制

目的:脓毒症相关认知功能障碍是脓毒症患者常见的并发症,目前病理机制不明,缺乏有效的防治手段。盐诱导激酶(salt-induced kinase,SIK)是调节代谢、免疫、炎症反应等的重要分子,与多种神经系统疾病的发生和发展相关。本研究旨在探讨SIK在脓毒症小鼠海马中的表达,以及SIK抑制剂HG-9-91-01在脓毒症相关认知功能障碍中的作用及其机制。方法:首先,将C57BL/6小鼠随机分为对照组(Con组)和脓毒症模型组[脂多糖(lipopolysaccharide,LPS)组]。LPS组小鼠以8 mg/kg的剂量腹腔注射LPS,Con组小鼠予注射等体积生理盐水。在注射后1、3、6 d取小鼠海马组织,分别采用实时荧光定量聚合酶链反应(quantitative PCR,qPCR)和蛋白质印迹法检测SIK1、SIK2、SIK3的mRNA和蛋白质的表达水平。然后,将小鼠随机分为Con组、LPS组、SIK抑制剂组(HG组)。LPS组和HG组注射LPS构建脓毒症模型,HG组在注射LPS后的第3~6天以10 mg/kg的剂量腹腔注射HG-9-91-01,LPS组注射等体积溶媒。在注射LPS后的第7~11天进行Morris水迷宫实验(Morris water maze,MWM)以评估3组小鼠的认知功能。行为学检测后取3组小鼠的海马组织,采用qPCR检测炎症因子和小胶质细胞标志分子的mRNA表达水平,蛋白质印迹法检测诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、CD68、离子钙结合衔接分子1(ionized calcium binding adaptor molecule 1,Iba-1)、N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体(NMDA receptor,NR)亚型、cAMP反应元件结合蛋白(cAMPresponse element-binding protein,CREB)调控的转录共激活因子1(CREB-regulated transcription coactivator 1,CRTC1)、胰岛素样生长因子1(insulin-like growth factor 1,IGF-1)的蛋白质表达水平,免疫组织化学(immunohistochemistry,IHC)检测海马CA1区、CA3区、齿状回(dentate gyrus,DG)Iba-1阳性细胞的表达,并进行Sholl分析。结果:与Con组比较,LPS组小鼠海马组织SIK1、SIK2、SIK3的mRNA和蛋白质表达水平均上调(均P<0.05)。与Con组比较,LPS组小鼠的逃避潜伏期明显延长,目标象限停留时间百分比降低,运动速度下降(均P<0.05);与LPS组相比,HG组小鼠的逃避潜伏期明显缩短,目标象限停留时间百分比增加(均P<0.05)。与Con组比较,LPS组海马组织炎症因子[肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)、白细胞介素6(interleukin-6,IL-6)]和I型小胶质细胞标志分子iNOS、CD68的mRNA表达均上调,而II型小胶质细胞标志分子CD206和精氨酸酶-1(arginase-1,Arg-1)的mRNA表达均下调;与LPS组比较,HG组TNF-α、IL-1β、IL-6、iNOS的mRNA表达均下调,而CD206和Arg-1的mRNA表达均上调(均P<0.05)。与Con组比较,LPS组海马组织iNOS、CD68、Iba-1的蛋白质表达均上调;与LPS组比较,HG组iNOS、CD68、Iba-1的蛋白质表达均下调(均P<0.05)。与Con组比较,LPS组海马组织CA1、CA3、DG区Iba-1阳性细胞数量均增加;与LPS组比较,HG组CA1、CA3、DG区Iba-1阳性细胞数量均减少(均P<0.05)。Sholl分析结果显示:距离小胶质细胞胞体8~38μm半径范围内,LPS组小胶质细胞突起与同心圆的交点较Con组明显减少(均P<0.05);在距离小胶质细胞胞体14~20μm半径范围内,HG组小胶质细胞突起与同心圆的交点较LPS组明显增多(均P<0.05)。与Con组比较,LPS组小鼠海马突触相关蛋白NR亚型NR1、NR2A、NR2B及IGF-1的蛋白质表达下调,磷酸化的CRTC1(phosphorylatedCRTC1,p-CRTC1)的蛋白质表达上调;与LPS组比较,HG组小鼠海马突触相关蛋白NR1、NR2A、NR2B及IGF-1的蛋白质表达上调,p-CRTC1的蛋白质表达下调(均P<0.05)。结论:脓毒症小鼠海马组织SIK表达上调,SIK抑制剂HG-9-91-01可改善小鼠脓毒症相关认知功能障碍,其机制可能与激活CRTC1/IGF-1通路,抑制神经炎症,增强突触可塑性有关。

Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines

AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. Egr-1 gene of transfected HHCC and ECa109 cells showed obvious suppression of the cell growth and malignant phenotypes, but no suppression in SMMC7721 (HCC cell line) cells.

甲状腺乳头状癌患者血清EGR1/2 mRNA水平检测在临床早期实验诊断中的价值研究

目的 探讨血清早期生长反应因子1(early growth response factor 1,EGR1)mRNA,早期生长反应因子2(early growth response factor 2,EGR2)mRNA表达水平在甲状腺乳头状癌(papillary thyroid carcinoma,PTC)患者早期诊断中的价值。方法 选择2019年1月~2022年2月在苏州市立医院确诊治疗的78例PTC患者(PTC组)和46例甲状腺良性肿瘤患者(良性肿瘤组)为研究对象,另以同期到该院体检的38例健康者为对照组,收集整理所有PCT患者的临床基本资料。采用实时荧光定量PCR法检测各组血清EGR1 mRNA和EGR2 mRNA表达水平,比较PTC患者、良性肿瘤患者和对照组血清EGR1 mRNA和EGR2 mRNA表达水平差异,分析PTC患者血清EGR1 mRNA和EGR2 mRNA水平与患者临床病理特征的关系,采用Pearson法分析PTC患者血清EGR1 mRNA和EGR2 mRNA表达相关性,采用ROC曲线分析血清EGR1 mRNA和EGR2 mRNA水平对PTC患者早期诊断的价值。结果 对照组、良性肿瘤组和PTC组血清EGR1 mRNA(1.03±0.14,0.81±0.10,0.74±0.08),EGR2 mRNA(0.98±0.12,0.76±0.09,0.67±0.06)水平依次显著降低,差异具有统计学意义(F=103.402,166.508,均P<0.001)。肿瘤分期为Ⅲ~Ⅳ(0.71±0.13)、有淋巴结转移(0.69±0.12)、浸润程度超过包膜(0.67±0.17)的PTC患者血清EGR1 mRNA表达水平低于肿瘤分期为Ⅰ~Ⅱ、无淋巴结转移、浸润程度未浸润包膜的患者(0.78±0.15,0.77±0.16,0.79±0.18),差异有统计学意义(t=2.206,2.415,2.945,均P<0.05);肿瘤分期为Ⅲ~Ⅳ(0.63±0.08)、有淋巴结转移(0.63±0.11)、浸润程度超过包膜(0.58±0.12)的PTC患者血清EGR2 mRNA表达水平低于肿瘤分期为Ⅰ~Ⅱ、无淋巴结转移、浸润程度未浸润包膜的患者(0.71±0.12,0.70±0.14,0.73±0.16),差异有统计学意义(t=3.481,2.382,4.455,均P<0.05);Pearson相关性分析显示,PTC患者血清中EGR1 mRNA与EGR2 mRNA表达呈正相关(r=0.216,P<0.05);血清EGR1 mRNA和EGR2 mRNA水平联合检测早期诊断PTC的ROC曲线下面积为0.829,敏感度、特异度分别为85.90%,73.91%。结论 PTC患者血清EGR1 mRNA,EGR2 mRNA水平下调,其水平变化与PTC病情发展密切相关,二者联合检测对PTC早期诊断具有重要意义。

Lipocalin-2-Mediated Insufficient Oligodendrocyte Progenitor Cell Remyelination for White Matter Injury After Subarachnoid Hemorrhage via SCL22A17 Receptor/Early Growth Response Protein 1 Signaling

Insufficient remyelination due to impaired oligodendrocyte precursor cell(OPC)differentiation and maturation is strongly associated with irreversible white matter injury(WMI)and neurological deficits.We analyzed whole transcriptome expression to elucidate the potential role and underlying mechanism of action of lipocalin-2(LCN2)in OPC differentiation and WMI and identified the receptor SCL22A17 and downstream transcription factor early growth response protein 1(EGR1)as the key signals contributing to LCN2-mediated insufficient OPC remyelination.In LCN-knockdown and OPC EGR1 conditional-knockout mice,we discovered enhanced OPC differentiation in developing and injured white matter(WM);consistent with this,the specific inactivation of LCN2/SCl22A17/EGR1 signaling promoted remyelination and neurological recovery in both atypical,acute WMI due to subarachnoid hemorrhage and typical,chronic WMI due to multiple sclerosis.This potentially represents a novel strategy to enhance differentiation and remyelination in patients with white matter injury.

miR-181b-5p调节EGR1/BIM通路对ALL细胞增殖、凋亡和迁移的影响

目的 探讨miR-181b-5p在急性淋巴细胞白血病(ALL)中的作用及其潜在的分子机制。方法 采用qRT-PCR检测四川大学华西医院2019年1月—2021年1月80例ALL患者miR-181b-5p和早期生长反应因子1(EGR1) mRNA的表达水平。分别使用miR-181b-5p模拟物、miR-181b-5p抑制物或(和)EGR1过表达质粒转染人ALL细胞系NALM-6细胞。采用MTT、流式细胞仪和划痕愈合实验检测miR-181b-5p对细胞增殖、凋亡和迁移的影响。通过双荧光素酶验证miR-181b-5p和EGR1的相互作用关系。Western blot检测细胞中EGR1蛋白和细胞增殖、凋亡和迁移相关蛋白的表达水平。结果 miR-181b-5p在ALL中表达显著上调,EGR1表达下调(P<0.05)。抑制miR-181b-5p不仅抑制细胞增殖、迁移,诱导细胞凋亡,而且上调EGR1和BIM蛋白表达(P<0.05);上调miR-181b-5p则具有与之相反的效果(P<0.05)。EGR1是ALL细胞中miR-181b-5p的靶基因(P<0.05)。miR-181b-5p过表达可明显削弱EGR1过表达对ALL细胞增殖、凋亡和迁移的影响(P<0.05)。结论 抑制miR-181b-5p可通过靶向上调EGR1和BIM表达在ALL中抑制细胞增殖和迁移,促进细胞凋亡。

三阴性乳腺癌组织中EGR1表达与临床病理特征预后的相关性

目的:探讨早期生长反应基因1(EGR1)在三阴性乳腺癌(TNBC)组织中的表达与临床病理特征、预后的相关性。方法:选取2016年1月至2018年3月期间于我院收治的50例TNBC患者、53例非TNBC的乳腺癌患者为研究对象,比较TNBC癌组织、非TNBC癌组织和癌旁正常组织EGR1的表达水平,并分析TNBC癌组织EGR1表达与临床病理特征、预后的相关性。结果:TNBC癌组织EGR1的高表达率显著低于非TNBC癌组织、癌旁正常组织,且非TNBC癌组织EGR1的高表达率显著低于癌旁正常组织,差异有统计学意义(P<0.05)。肿瘤直径≥3cm、组织学分级为G3级、有淋巴结转移、TNM分期为Ⅲ期、Ki-67指数≥50%的TNBC患者癌组织EGR1高表达率显著低于<3cm、G2与G1级、无淋巴结转移、Ⅰ期、<50%者,且G2级、Ⅱ期的TNBC患者癌组织EGR1高表达率显著低于G1级、Ⅰ期者,差异有统计学意义(P<0.05)。癌组织EGR1高表达患者的中位生存时间显著高于低表达者,差异有统计学意义(P<0.05)。多因素Cox比例风险回归模型分析结果表明,淋巴结转移是TNBC患者预后不良的独立危险因素,癌组织EGR1高表达是其独立保护因素(P<0.05)。结论:TNBC癌组织EGR1呈低表达,癌组织EGR1高表达是患者预后不良的独立保护因素。

七氟醚-N2O麻醉对青年小鼠空间学习和记忆功能及pCREB和Egr1表达的影响

目的研究七氟醚-N2O麻醉后青年小鼠的空间学习和记忆能力以及海马磷酸化环腺苷酸应答元件结合蛋白(phosphorylated cyclic AMP response element-binding protein,pCREB)和早期生长反应因子1(early growth response factor 1,Egr1)的表达,并与老年小鼠进行比较。方法12只3月龄和12只18月龄小鼠随机分为对照组和实验组,每组各6只。48 h后进行为期6天的获得训练。获得训练结束后24 h进行空间探索实验,实验结束后15 min收集两组小鼠的海马组织,通过Western blot和实时定量PCR方法评估pCREB和Egr1的表达水平。结果未见麻醉损害青年小鼠的空间学习和记忆功能以及海马中pCREB和Egr1的表达水平,麻醉组与对照组比较差异均无统计学意义;而老年小鼠Sev+N2O组在麻醉后第4、5、6天逃逸潜伏期显著长于对照组(P<0.05),两组小鼠游泳速度差异无统计学意义;在空间探索实验中,Sev+N2O组目标象限路程百分比和时间百分比显著低于对照组(P<0.01);海马中pCREB和Egr1的表达水平均较对照组显著下降(P<0.05)。结论七氟醚-N2O麻醉对青年小鼠的空间学习记忆能力没有损害,也不影响海马中pCREB和Egr1的表达,但会对老年小鼠造成损害。