Table1_Bioinformatics Analysis Identifies Potential Ferroptosis Key Genes in the Pathogenesis of Pulmonary Fibrosis.xls

Objective: Ferroptosis has an important role in developing pulmonary fibrosis. The present project aimed to identify and validate the potential ferroptosis-related genes in pulmonary fibrosis by bioinformatics analyses and experiments.
Methods: First, the pulmonary fibrosis tissue sequencing data were obtained from Gene Expression Omnibus (GEO) and FerrDb databases. Bioinformatics methods were used to analyze the differentially expressed genes (DEGs) between the normal control group and the pulmonary fibrosis group and extract ferroptosis-related DEGs. Hub genes were screened by enrichment analysis, protein-protein interaction (PPI) analysis, and random forest algorithm. Finally, mouse pulmonary fibrosis model was made for performing an exercise intervention and the hub genes’ expression was verified through qRT-PCR.
Results: 13 up-regulated genes and 7 down-regulated genes were identified as ferroptosis-related DEGs by comparing 103 lung tissues with idiopathic pulmonary fibrosis (IPF) and 103 normal lung tissues. PPI results indicated the interactions among these ferroptosis-related genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway enrichment and Genome-Ontology (GO) enrichment analyses showed that these ferroptosis-related genes involved in the organic anion transport, response to hypoxia, response to decrease oxygen level, HIF-1 signaling pathway, renal cell carcinoma, and arachidonic acid metabolism signaling pathway. The confirmed genes using PPI analysis and random forest algorithm included CAV1, NOS2, GDF15, HNF4A, and CDKN2A. qRT-PCR of the fibrotic lung tissues from the mouse model showed that the mRNA levels of NOS2 and GDF15 were up-regulated, while CAV1 and CDKN2A were down-regulated. Also, treadmill training led to an increased expression of CAV1 and CDKN2A and a decrease in the expression of NOS2 and GDF15.
Conclusion: Using bioinformatics analysis, 20 potential genes were identified to be associated with ferroptosis in pulmonary fibrosis. CAV1, NOS2, GDF15, and CDKN2A were demonstrated to be influencing the development of pulmonary fibrosis by regulating ferroptosis. These findings suggested that, as an aerobic exercise treatment, treadmill training reduced ferroptosis in the pulmonary fibrosis tissues, and thus, reduces inflammation in the lungs. Aerobic exercise training initiate concomitantly with induction of pulmonary fibrosis reduces ferroptosis in lung. These results may develop our knowledge about pulmonary fibrosis and may contribute to its treatment.

研究背景与目的：铁死亡（Ferroptosis）在肺纤维化的发生发展中具有重要作用。本研究旨在通过生物信息学分析与实验验证，筛选并鉴定肺纤维化中潜在的铁死亡相关基因。
研究方法：首先，从基因表达综合数据库（Gene Expression Omnibus, GEO）及铁死亡数据库（FerrDb）获取肺纤维化组织测序数据。采用生物信息学方法分析正常对照组与肺纤维化组间的差异表达基因（differentially expressed genes, DEGs），并提取铁死亡相关差异表达基因。通过富集分析、蛋白质-蛋白质相互作用（protein-protein interaction, PPI）分析及随机森林算法筛选核心基因。最后，构建小鼠肺纤维化模型并实施运动干预，通过实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）验证核心基因的表达水平。
研究结果：通过对比103份特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）肺组织与103份正常肺组织，共鉴定出13个上调表达的铁死亡相关差异表达基因及7个下调表达的铁死亡相关差异表达基因。蛋白质-蛋白质相互作用分析结果显示，上述铁死亡相关基因间存在相互调控关系。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析与基因本体（Gene Ontology, GO）富集分析结果表明，这些铁死亡相关基因参与了有机阴离子转运、缺氧应答、氧水平降低应答、缺氧诱导因子-1（hypoxia-inducible factor-1, HIF-1）信号通路、肾细胞癌及花生四烯酸代谢信号通路。经蛋白质-蛋白质相互作用分析与随机森林算法验证的核心基因包括CAV1、NOS2、GDF15、HNF4A及CDKN2A。对小鼠模型纤维化肺组织的qRT-PCR检测结果显示，NOS2与GDF15的mRNA表达水平上调，而CAV1与CDKN2A的mRNA表达水平下调。此外，跑台训练可上调CAV1与CDKN2A的表达，并下调NOS2与GDF15的表达。
研究结论：本研究通过生物信息学分析，共鉴定出20个与肺纤维化中铁死亡相关的潜在基因。经验证，CAV1、NOS2、GDF15及CDKN2A可通过调控铁死亡影响肺纤维化的发生发展。上述研究结果表明，作为一种有氧运动治疗手段，跑台训练可减轻肺纤维化组织中的铁死亡水平，进而缓解肺部炎症。在诱导肺纤维化的同时开展有氧运动训练，可减轻肺部铁死亡程度。本研究结果可加深我们对肺纤维化的认识，并为其治疗提供新的参考依据。