鉴定和验证脂多糖诱导的急性肺损伤中的铁死亡相关基因。
Identification and validation of ferroptosis-related genes in lipopolysaccharide-induced acute lung injury.
机构信息
Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Breast Surgery, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, China.
出版信息
Cell Signal. 2023 Aug;108:110698. doi: 10.1016/j.cellsig.2023.110698. Epub 2023 May 5.
BACKGROUND
Emerging evidence reveals the important role of ferroptosis in the pathophysiological process of acute lung injury (ALI). We aimed to identify and validate the potential ferroptosis-related genes of ALI through bioinformatics analysis and experimental validation.
METHODS
Murine ALI model was established via intratracheal instillation with LPS and confirmed by H&E staining and transmission electronic microscopy (TEM). RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs) between control and ALI model mice. The potential differentially expressed ferroptosis-related genes of ALI were identified using the limma R package. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, gene set enrichment analysis (GSEA), and protein-protein interactions (PPI) were applied for the differentially expressed ferroptosis-related genes. CIBERSORT tool was used to conduct immune cell infiltration analysis. Finally, protein expressions and RNA expression of ferroptosis DEGs were validated in vivo and in vitro by western blots and RT-qPCR.
RESULTS
Among 5009 DEGs, a total of 86 differentially expressed ferroptosis-related genes (45 up-regulated genes and 41 down-regulated genes) were identified in the lungs between control and ALI. GSEA analysis showed that the genes enriched were mainly involved in response to molecule of bacterial origin and fatty acid metabolic process. The GO and KEGG enrichment analysis indicated that the top 40 ferroptosis DEGs were mainly enriched in reactive oxygen species metabolic process, HIF-1signaling pathway, lipid and atherosclerosis, and ferroptosis. The PPI results and Spearman correlation analysis suggested that these ferroptosis-related genes interacted with each other. Immune infiltration analysis confirmed that ferroptosis DEGs were closely related to immune response. Consistent with the RNA-seq data, the western blot and RT-qPCR unveiled increased mRNA expressions of Cxcl2, Il-6, Il-1β, and Tnfα, and protein expressions of FTH1, TLR4 as well as decreased ACSL3 in LPS-induced ALI. In vitro, the upregulated mRNA levels of CXCL2, IL-6, SLC2A1, FTH1, TNFAIP3, and downregulated NQO1 and CAV1 in LPS-stimulated BEAS-2B and A549 cells were verified.
CONCLUSION
We identified 86 potential ferroptosis-related genes of LPS-induced ALI through RNA-seq. Several pivotal ferroptosis-related genes involved in lipid metabolism and iron metabolism were implicated in ALI. This study may be helpful to expand our understanding of ALI and provide some potential targets to counteract ferroptosis in ALI.
背景
新出现的证据表明铁死亡在急性肺损伤（ALI）的病理生理过程中起着重要作用。我们旨在通过生物信息学分析和实验验证来鉴定和验证 ALI 的潜在铁死亡相关基因。
方法
通过气管内滴注 LPS 建立小鼠 ALI 模型，并通过 H&E 染色和透射电镜（TEM）进行确认。使用 RNA 测序（RNA-seq）筛选对照和 ALI 模型小鼠之间的差异表达基因（DEGs）。使用 limma R 包鉴定潜在的 ALI 差异表达铁死亡相关基因。进行基因本体论（GO）富集分析、京都基因与基因组百科全书（KEGG）通路富集分析、基因集富集分析（GSEA）和蛋白质-蛋白质相互作用（PPI）分析差异表达的铁死亡相关基因。使用 CIBERSORT 工具进行免疫细胞浸润分析。最后，通过 Western blot 和 RT-qPCR 在体内和体外验证铁死亡 DEGs 的蛋白表达和 RNA 表达。
结果
在 5009 个 DEGs 中，共鉴定出肺组织中 86 个差异表达的铁死亡相关基因（45 个上调基因和 41 个下调基因）。GSEA 分析表明，富集的基因主要参与对细菌来源分子的反应和脂肪酸代谢过程。GO 和 KEGG 富集分析表明，前 40 个铁死亡 DEGs 主要富集在活性氧代谢过程、HIF-1 信号通路、脂质和动脉粥样硬化以及铁死亡中。PPI 结果和 Spearman 相关性分析表明，这些铁死亡相关基因相互作用。免疫浸润分析证实，铁死亡 DEGs 与免疫反应密切相关。与 RNA-seq 数据一致，Western blot 和 RT-qPCR 揭示了 LPS 诱导的 ALI 中 Cxcl2、Il-6、Il-1β 和 Tnfα 的 mRNA 表达增加，以及 FTH1、TLR4 的蛋白表达减少，ACSL3 减少。在体外，LPS 刺激的 BEAS-2B 和 A549 细胞中验证了 CXCL2、IL-6、SLC2A1、FTH1、TNFAIP3 的上调 mRNA 水平和 NQO1 和 CAV1 的下调。
结论
通过 RNA-seq 我们鉴定出了 86 个 LPS 诱导的 ALI 的潜在铁死亡相关基因。一些涉及脂质代谢和铁代谢的关键铁死亡相关基因参与了 ALI。这项研究可能有助于我们更深入地了解 ALI，并为对抗 ALI 中的铁死亡提供一些潜在的靶点。
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