基于生物信息学筛选铁死亡参与多发性硬化症机制的潜在关键靶点及初步验证

Bioinformatics-based screening of potential key targets of ferroptosis involved in multiple sclerosis mechanisms and preliminary validation

目的利用生物信息学技术从铁死亡角度筛选多发性硬化症(MS)的潜在关键基因,并通过动物实验初步验证,探索铁死亡在MS中的作用机制。方法从GEO数据库中下载MS数据集GSE21942和GSE43591;从FerrDb数据库及相关文献获得铁死亡相关基因。应用R软件对数据集GSE21942进行差异分析和WGCNA分析。将两种分析结果和铁死亡相关基因取交集,得到MS铁死亡相关差异表达基因(FRDEGs),并对FRDEGs进行GO和KEGG富集分析。应用LASSO和SVM-RFE两种机器学习算法进一步筛选关键基因,并利用数据集GSE43591进行验证,绘制ROC曲线评估关键基因的诊断价值。应用ssGESA算法探索关键基因与免疫细胞的相关性。采用qPCR和Western blot方法进一步验证关键基因的表达。结果共筛选出20个FRDEGs,GO和KEGG富集分析结果表明,它们主要涉及对氧化应激的反应、蛋白质磷酸化的负调控、NOD样受体信号通路、MAPK信号通路、Th17细胞分化等过程。通过机器学习和外部数据集验证筛选出2个与铁死亡相关的关键基因(CIRBP和TNFAIP3),ROC结果提示CIRBP和TNFAIP3均具有较高的诊断价值。免疫浸润分析结果表明CIRBP和TNFAIP3与活化CD4 T细胞、活化B细胞等显著相关。qPCR和Western blot结果显示,与正常组相比,EAE组小鼠中CIRBP和TNFAIP3的mRNA、蛋白表达水平显著升高(P<0.05)。结论CIRBP和TNFAIP3是MS中与铁死亡相关的潜在关键基因,铁死亡参与MS的发病机制可能与CIRBP和TNFAIP3的高表达有关。

Objective To screen the potential key genes in multiple sclerosis(MS)from the perspective of ferroptosis by bioinformatics technology,and to explore the mechanism of ferroptosis in MS by preliminary verification in animal experiment.Methods We downloaded MS datasets GSE21942 and GSE43591 from the GEO database and obtained ferroptosis-related genes from the FerrDb database and related literature.The dataset GSE21942 was analyzed using R software for differential analysis and WGCNA analysis.The intersection of the results from both analyses and the ferroptosis-related genes yielded the MS ferroptosis-related differentially expressed genes(FRDEGs).Subsequently,GO and KEGG enrichment analyses were performed on the FRDEGs.Two machine learning algorithms,LASSO and SVM-RFE,were employed to further screen the key genes,which were validated using the dataset GSE43591,and the diagnostic value of the key genes was assessed by plotting ROC curves.The ssGESA algorithm was used to explore the correlation between key genes and immune cells.Finally,the expression of key genes was further validated using qPCR and Western blot methods.Results A total of 20 FRDEGs were screened.The results of GO and KEGG enrichment analysis indicated that they were mainly involved in response to oxidative stress,negative regulation of protein phosphorylation,participation in the NOD-like receptor signaling pathway,MAPK signaling pathway,Th17 cell differentiation,etc.Machine learning and validation with external datasets screened two key genes(CIRBP and TNFAIP3)related to ferroptosis.The ROC results indicated that both CIRBP and TNFAIP3 had high diagnostic values.The results of immune infiltration analysis showed that CIRBP and TNFAIP3 were significantly associated with activated CD4 T cells and activated B cells.qPCR and Western blot assays showed that the mRNA and protein expression levels of CIRBP and TNFAIP3 were significantly increased in mice of the EAE group compared with those of the normal group(P<0.05).Conclusion CIRBP and TNFAIP3 are potential key genes associated with ferroptosis in MS,and the involvement of ferroptosis in the pathogenesis of MS may be related to the high expression of CIRBP and TNFAIP3.