系统性红斑狼疮外周血单个核细胞中铁死亡相关关键基因筛选分析

Screening and analysis of ferroptosis related genes in peripheral blood mononuclear cells of patients with systemic lupus erythematosus

目的通过生物信息学的方法分析系统性红斑狼疮(SLE)患者PBMCs的差异表达基因,筛选并分析铁死亡相关关键基因,从转录水平探索铁死亡参与SLE发病的可能机制。方法从美国国家生物技术信息中心(NCBI)的子数据库基因芯片公共数据库(GEO)检索出符合筛选标准的健康对照组(HC组)和SLE患者(SLE组)的数据集和样本信息,利用GEO2R、R语言及相关软件包进行分析获得差异表达基因、基因本体论(GO)富集分析和京都基因和基因组百科全书(KEGG)信号通路富集分析结果。利用STRING、Cytoscape等工具分析差异基因的蛋白交互作用网络(PPI),探索关键基因与通路,寻找潜在作用靶点。此外,通过实时荧光定量反转录PCR(RT-qPCR)验证关键基因在SLE中的表达。采用Mann-Whitney U秩和检验比较2组PBMCs中关键基因的表达差异;采用Spearman秩相关分析探索其与SLE疾病活动度的关系。结果研究纳入了6个数据集,SLE组和HC组的差异基因中与铁死亡相关的基因合计166个。差异基因主要在肺泡巨噬细胞、中性粒细胞、CD49+细胞和CD31+细胞等免疫细胞中特异性表达。GO富集分析和KEGG通路富集分析提示差异基因主要参与了细胞的氧化应激反应、感染和TNF信号通路等多个与SLE密切相关的信号通路。不同算法筛选出的Hub基因均提示RELA基因为关键基因,且RT-qPCR证实,相较于HC组RELA基因表达水平[0.75(0.37,1.13)],SLE组中表达水平[2.02(1.19,4.06)]出现上升,差异具有统计学意义(Z=-3.08,P=0.002),并与SLE样本对应的SLEDAI呈正相关(r=0.52,P=0.019)。结论多种免疫细胞,包括肺泡巨噬细胞和CD49+NK细胞等的铁死亡过程参与SLE的发病机制,其中RELA基因可能通过NF-κB通路参与SLE患者PBMCs铁死亡过程。

Objective To analyze the differentially expressed genes in PBMCs of patients with systemic lupus erythematosus(SLE)by bioinformatics methods screening and analyzing the key genes related to ferroptosis,and explore the possible mechanism of ferroptosis involved in the pathogenesis of SLE at the transcription level.Methods The data sets and samples of healthy people(HC)and SLE patients who met the screening criteria were retrieved from the Gene Expression Omnibus(GEO),a sub-database of the National Center for Biotechnology Information(NCBI).The differentially expressed genes,GO enrichment analysis and KEGG pathway enrichment analysis were analyzed by GEO2R,R language and related software packages.The protein interaction network(PPI)of differential genes was analyzed by STRING,Cytoscape and other tools to explore the key genes and pathways.In addition,real-time quantitative reverse transcription PCR(RT-qPCR)was used to verify the expression of key genes.Mann-Whitney U test was used to compare the expression of key genes in PBMCs between the two groups.Spearman rank correlation analysis was used to explore the relationship between SLE disease activity and the level of key genes.Results Six data sets were included in this study.A total of 166 genes related to ferroptosis were differentially expressed between SLE and HC groups.The differential genes were specifically expressed in alveolar macrophages,neutrophils,CD49+cells and CD31+cells.GO enrichment analysis and KEGG pathway enrichment analysis showed that the differentially expressed genes were mainly involved in multiple signaling pathways closely related to SLE,such as oxidative stress response,infection and TNF signaling pathway.Hub genes screened by different algorithms all suggested RELA as a key gene,and RT-qPCR confirmed that compared with the RELA gene expression level in the HC group[0.75(0.37,1.13)],the expression level in SLE group[2.02(1.19,4.06)]was increased,the difference was statistically significant(Z=-3.08,P=0.002),and was positively correlated with the corresponding SLEDAI score of SLE samples(r=0.52,P=0.019).Conclusion The ferroptosis of many immune cells,including alveolar macrophages and CD49+NK cells,is involved in the pathogenesis of SLE.RELA may be involved in the ferroptosis of PBMCs in SLE through the NF-κB pathway.