基于生物信息学方法对抑郁症中铁死亡特征 基因的筛选与实验验证

Identification and Experimental Validation of Ferroptosis-Related Signature Genes in Depression Based on Bioinformatics Analysis

摘要: 目的：基于生物信息学方法筛选出抑郁症中铁死亡相关特征基因并通过体内实验验证，探讨铁死亡在抑郁症中的作用及价值。方法：以美国国家生物技术信息中心(NCBI)基因表达综合数据库(GEO)中的微阵列芯片数据集GSE20388为源，采用R语言limma包，以P < 0.05，|log2FC| > 0.1为标准，进行差异分析，得到差异基因；再与从Ferrdb数据库选出的铁死亡相关基因取交集，得到差异表达的铁死亡相关基因。对GSE20388数据集进行加权基因共表达网络分析(Weighted Gene Co-Expression Network Analysis, WGCNA)，筛选出与抑郁症最相关的模块基因；利用最小绝对收缩与选择算子回归模型(Least Absolute Shrinkage and Selection Operator, LASSO)模型和蛋白质相互作用网络分析(Protein-Protein Interaction Network Analysis, PPI)进一步筛选出关键基因，对关键基因进行富集分析。最后，通过慢性不可预见轻度应激(CUMS)的方法建立抑郁样小鼠模型，收集海马组织标本采用实时荧光定量PCR (Quantitative Real-Time PCR, RT-qPCR)方法验证关键基因信使RNA (Messenger RNA, mRNA)的表达水平。结果：我们分析并确定了3个关键基因：OTUB1，GOT1，CYLD；GO富集分析表明这些基因参与了细胞的代谢，氨基酸的代谢以及蛋白的泛素化/去泛素化；KEGG富集分析显示与氨基酸代谢，碳代谢以及TNF信号通路密切相关。免疫浸润分析结果显示，调节性T细胞和未成熟树突状细胞在抑郁模型中高表达而单核细胞在抑郁症模型中低表达且差异显著(P < 0.001)。qPCR结果显示：CYLD与GOT1均在抑郁模型中显著高表达，而GPX4与OTUB1在抑郁模型中显著低表达。MDA结果显示：与对照组相比，抑郁小鼠模型中MDA水平显著高表达。结论：CYLD、GOT1、OTUB1是抑郁症与铁死亡的关键枢纽基因，有着深远的治疗价值。

Abstract: Objective: To identify ferroptosisrelated characteristic genes in depression using bioinformatics approaches and validate them through in vivo experiments, thereby exploring the role and potential value of ferroptosis in depression. Methods: Based on the microarray dataset GSE20388 from the NCBI GEO database, differential expression analysis was performed using the R package “limma” with thresholds of P < 0.05 and |log2FC| > 0.1 to obtain differentially expressed genes (DEGs). These DEGs were intersected with ferroptosisrelated genes retrieved from the FerrDb database to identify ferroptosisassociated DEGs. Weighted gene coexpression network analysis (WGCNA) was applied to GSE20388 to identify modules most correlated with depression. The least absolute shrinkage and selection operator (LASSO) regression and proteinprotein interaction (PPI) network analyses were then used to further screen for hub genes, followed by functional enrichment analysis of these genes. Finally, a depressionlike mouse model was established using the chronic unpredictable mild stress (CUMS) protocol. Hippocampal tissues were collected, and mRNA expression levels of the hub genes were validated by quantitative realtime PCR (RTqPCR). Results: Three hub genes—OTUB1, GOT1, and CYLD—were identified. GO enrichment analysis indicated their involvement in cellular metabolism, amino acid metabolism, and protein ubiquitination/deubiquitination. KEGG enrichment analysis revealed significant associations with amino acid metabolism, carbon metabolism, and the TNF signaling pathway. Immune infiltration analysis demonstrated that regulatory T cells and immature dendritic cells were significantly upregulated, whereas monocytes were significantly downregulated in the depression model (P < 0.001). RT-qPCR results showed that CYLD and GOT1 were significantly upregulated, while GPX4 and OTUB1 were significantly downregulated in the depression model. Additionally, malondialdehyde (MDA) levels were significantly elevated in depressed mice compared with controls. Conclusion: CYLD, GOT1, and OTUB1 are key hub genes linking depression and ferroptosis, highlighting their potential therapeutic value in depression.