背景:阿尔茨海默病的免疫相关致病机制尚不明确,通过生物信息学探讨阿尔茨海默病患者NK细胞与铜死亡机制的相关性,可为研究阿尔茨海默病的发生发展提供新方向.目的:使用生物信息学分析方法筛选阿尔茨海默病患者外周血中NK细胞与铜死亡相关的关键基因,并在临床标本水平加以验证.方法:利用GEO在线数据库筛选阿尔茨海默病患者外周血转录组差异表达基因和NK细胞相关基因,与已报告的铜死亡因子取交集,获取差异表达的铜死亡相关基因;接着采用RT-qPCR技术对基因相对表达量进行实验验证,实验样本来自于安徽省立医院神经内科2021-2023年住院患者的外周血,按照纳排标准纳入疾病组30例及对照组20例.后续进一步使用在线GeneMANIA网站构建蛋白质-蛋白质互作网络;利用R语言进行免疫浸润分析;基于ENCODE数据库进行转录因子预测.结果与结论:①利用GSE63060数据集中阿尔茨海默病患者外周血转录组差异表达基因、GSE168522数据集中NK细胞相关基因、已报告的铜死亡基因,应用在线韦恩图工具、LASSO算法及随机森林机器学习方法筛选获取4个差异表达的铜死亡相关基因:铁氧还原蛋白1(ferredoxin 1,FDX1)、铜离子转运ATP酶a肽(ATPase Cu2+transporting alpha polypeptide,ATP7A)、丙酮酸脱氢酶β(pyruvate dehydrogenase El subunit beta,PDHB)和二氢硫辛酰胺琥珀酰转移酶(dihydrolipoamide succinyltransferase,DLST).②临床样本实验验证,FDX1和ATP7A在阿尔茨海默病患者外周血中表达上调(P<0.001),且在载脂蛋白E4不同基因型中呈差异性表达(P<0.01,P<0.001);PDHB和DLST在阿尔茨海默病患者外周血中表达下调(P<0.001),在载脂蛋白E4不同基因型中表达无显著性意义(P>0.05).③蛋白质-蛋白质互作网络发现20种功能蛋白与关键基因相关联,免疫浸润分析结果验证了关键基因与12个免疫细胞具有显著相关性(以P<0.05为具有相关性).④生物信息学分析与实验验证结果提示,FDX1,ATP7A,PDHB和DLST基因在阿尔茨海默病中存在差异性表达,这4个基因在阿尔茨海默病外周血的NK细胞中可能通过铜死亡机制参与了疾病的发生发展,文章结果为阿尔茨海默病的诊断及治疗提供了潜在的靶点.
Cuproptosis-related genes in natural killer cells of Alzheimer's disease
BACKGROUND:The immune-related pathogenesis of Alzheimer's disease is still unclear.Exploring the correlation between natural killer cells and cuproptosis mechanism in Alzheimer's disease patients through bioinformatics can provide a new direction for the study of the occurrence and development of Alzheimer's disease.OBJECTIVE:To screen the key genes related to cuproptosis of natural killer cells in peripheral blood of patients with Alzheimer's disease by bioinformatics analysis and verify them in clinical specimens.METHODS:The GEO online database was used to screen the transcriptome differentially expressed genes and natural killer cell related genes in the peripheral blood of patients with Alzheimer's disease,and intersected with the reported cuproptosis factors.Differentially expressed cuproptosis-related genes were obtained.Then RT-qPCR technology was used to verify the relative gene expression levels.The experimental samples were all from peripheral blood of hospitalized patients in the Department of Neurology of Anhui Provincial Hospital from 2021 to 2023,and 30 patients in the disease group and 20 in the control group were included according to the inclusion and exclusion criteria.The protein-protein interaction network was further constructed using the online GeneMANIA website.R language was used for immune infiltration analysis.Transcription factor prediction was conducted based on ENCODE database.RESULTS AND CONCLUSION:(1)The differential expression genes of peripheral blood transcriptome of Alzheimer's disease patients in GSE63060 data set,natural killer cell related genes in GSE168522 data set,and reported cuproptosis genes were used to screen and obtain four differentially expressed cuproptosis-related genes by using online Venn diagram tool,LASSO algorithm,and random forest machine learning methods:ferredoxin 1(FDX1),ATPase Cu2+transporting alpha polypeptide(ATP7A),pyruvate dehydrogenase El subunit beta(PDHB),and dihydrolipoamide succinyltransferase(DLST).(2)Clinical sample experiments showed that FDX1 and ATP7A were up-regulated in peripheral blood of patients with Alzheimer's disease(P<0.001),and were differentially expressed in different genotypes of apolipoprotein E4(P<0.01,P<0.001).The expression of PDHB and DLST in peripheral blood of patients with Alzheimer's disease was down-regulated(P<0.001),and there was no difference in apolipoprotein E4 genotypes(P>0.05).(3)Protein-protein interaction network found that 20 functional proteins were associated with key genes,and immunoinfiltration analysis showed that key genes were significantly associated with 12 immune cells(P<0.05 was considered to be relevant).(4)Bioinformatics analysis and experimental verification results suggest that FDX1,ATP7A,PDHB,and DLST are differentially expressed in Alzheimer's disease,may participate in the occurrence and development of Alzheimer's disease through the cuproptosis mechanism in peripheral blood natural killer cells,and also provide potential targets for the diagnosis and treatment of Alzheimer's disease.
万方数据
维普
