线粒体相关基因在特发性肺纤维化中的分析

doi:10.3877/cma.j.issn.1674-6902.2024.02.002

目的

通过生物信息学方法和肺纤维化(idiopathic pulmonary fibrosis, IPF)小鼠模型分析与特发性肺纤维化发生发展相关的线粒体相关基因。

方法

从基因表达综合数据库(gene expression omnibu, GEO)中获得IPF样本和正常样本间的差异表达基因；通过MitoCarta3.0数据库和所得差异基因进行匹配，以获取线粒体相关差异基因(mitochondrial-related differential expressed genes, MiRDEGs)，并对其进行基因本体论(GeneOntology, GO)分析和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes, KEGG)功能富集分析；构建MiRDEGs的PPI网络，并使用Cytohubba获得核心MiRDEGs；建立肺纤维化小鼠模型验证鉴定出的核心MiRDEGs。

结果

最终得到26个上调的MiRDEGs和24个下调的MiRDEGs，主要涉及到有氧呼吸，线粒体结构和物质代谢。其中10个MiRDEGs被cytohubba鉴定为核心MiRDEGs。结果表明，在小鼠肺纤维化模型中，FASN，PDK4，ACSL1的表达低于对照组，而ALDH18A1，MTHFD2，ALDH1L2，PC的表达则高于对照组。

结论

IPF中的线粒体相关基因及其对免疫浸润的影响与IPF的发病相关并可能为潜在的治疗靶点。

关键词: 特发性肺纤维化, 线粒体, 免疫, 生物信息学

Objective

Mitochondria-related genes associated with the development of idiopathic pulmonary fibrosis were analyzed by bioinformatics methods and the pulmonary fibrosis mouse model.

Methods

Datasets were acquired from the GEO database. Analysis of differential expressed genes (DEGs) was conducted for each dataset using the GEO2R online tool. Mitochondrial-related differential expressed genes (MiRDEGs) were identified by intersecting MitoCarta3.0 database genes with DEGs. Subsequently, functional enrichment analyses and a Protein-Protein Interaction (PPI) network construction were conducted for MiRDEGs, with hub MiRDEGs identified using CytoHubba. Mouse model of bleomycin-induced pulmonary fibrosis were used to validate hub MiRDEGs.

Results

Our study identified 26 upregulated and 24 downregulated MiRDEGs linked to mitochondrial function, structure and metabolism pathways. Cytohubba identified 10 hub MiRDEGs. Immune infiltration analysis indicated abundant M₂ macrophage, M₁ macrophage, and M₀ macrophage infiltration in IPF samples. Notably, among the hub MiRDEGs, ACSL1 exhibited the most pronounced negative correlation with M₂ macrophage infiltration, while PC showed the strongest positive correlation. Animal experiments demonstrated that lower expression of FASN, PDK4, and ACSL1, and higher expression of ALDH18A1, MTHFD2, ALDH1L2, and PC in the pulmonary fibrosis mouse model compared to the control group.

Conclusion

By comprehensively exploring the genes related to mitochondria and emphasizing their impact on immune infiltration in IPF, this investigation provides insights into the molecular mechanisms underlying IPF and potential therapeutic target.

Key words: Idiopathic fibrosis of the lung, Mitochondria, Immune, Bioinformatics

表1 核心MiRDEGs引物序列

MiRDEGs	引物序列
FASN	forward：5′- GGAGGTGGTGATAGCCGGTAT-3′
	reverse：5′-TGGGTAATCCATAGAGCCCAG-3′
PDK4	Forward：5′-AGGGAGGTCGAGCTGTTCTC-3′
	reverse: 5′- GGAGTGTTCACTAAGCGGTCA-3′
HMGCS2	forward：5′-GAAGAGAGCGATGCAGGAAAC-3′
	reverse：5′-GTCCACATATTGGGCTGGAAA-3′
ACSL1	forward：5′-TGCCAGAGCTGATTGACATTC-3′
	reverse: 5′-GGCATACCAGAAGGTGGTGAG-3′
ACADL	forward：5′-TCTTTTCCTCGGAGCATGACA-3′
	reverse：5′-GACCTCTCTACTCACTTCTCCAG-3′
ALDH18A1	forward：5′-GTCCAGCCCTCAGCTATTAGA-3′
	reverse：5′-CTTCAGCTCACTTCGGTGGG-3′
MTHFD2	forward：5′-AGTGCGAAATGAAGCCGTTG-3′
	reverse：5′-GACTGGCGGGATTGTCACC-3′
ALDH1L1	forward：5′-CAGGAGGTTTACTGCCAGCTA-3′
	reverse：5′-CACGTTGAGTTCTGCACCCA-3′
ALDH1L2	forward：5′-ACCAGCCGGGTTTATTTCAAA-3′
	reverse：5′-ACTCCCACTACTCGGTGGC-3′
PC	forward: 5′-CCACCTCTCCGAACCGTGACA-3′
	reverse- 5′-GAGAAGATTGGTAGGGGAGGC-3′

表1 核心MiRDEGs引物序列

MiRDEGs	引物序列
FASN	forward：5′- GGAGGTGGTGATAGCCGGTAT-3′
	reverse：5′-TGGGTAATCCATAGAGCCCAG-3′
PDK4	Forward：5′-AGGGAGGTCGAGCTGTTCTC-3′
	reverse: 5′- GGAGTGTTCACTAAGCGGTCA-3′
HMGCS2	forward：5′-GAAGAGAGCGATGCAGGAAAC-3′
	reverse：5′-GTCCACATATTGGGCTGGAAA-3′
ACSL1	forward：5′-TGCCAGAGCTGATTGACATTC-3′
	reverse: 5′-GGCATACCAGAAGGTGGTGAG-3′
ACADL	forward：5′-TCTTTTCCTCGGAGCATGACA-3′
	reverse：5′-GACCTCTCTACTCACTTCTCCAG-3′
ALDH18A1	forward：5′-GTCCAGCCCTCAGCTATTAGA-3′
	reverse：5′-CTTCAGCTCACTTCGGTGGG-3′
MTHFD2	forward：5′-AGTGCGAAATGAAGCCGTTG-3′
	reverse：5′-GACTGGCGGGATTGTCACC-3′
ALDH1L1	forward：5′-CAGGAGGTTTACTGCCAGCTA-3′
	reverse：5′-CACGTTGAGTTCTGCACCCA-3′
ALDH1L2	forward：5′-ACCAGCCGGGTTTATTTCAAA-3′
	reverse：5′-ACTCCCACTACTCGGTGGC-3′
PC	forward: 5′-CCACCTCTCCGAACCGTGACA-3′
	reverse- 5′-GAGAAGATTGGTAGGGGAGGC-3′

图1 IPF样本和正常样本间的差异表达基因。注：A-C: GSE24206，GSE53845，GSE92592差异表达基因火山图；D-F：GSE24206，GSE53845，GSE92592差异表达基因热图

图2 IPF相关MiRDEGs的鉴定及其GO和KEGG分析；MiRDEGs的PPI网络和核心MiRDEGs的鉴定。注：A、B：Venn图显示了mitcarta3.0数据库中线粒体定位基因与GSE24206、GSE53845和GSE92592数据集中的差异基因的交集；C、D：MiRDEGs的GO和KEGG功能富集分析；E：MiRDEGs的PPI网络；F：基于CytoHubba筛选出的十个核心MiRDEGs

图3 肺纤维化小鼠模型构建及核心MiRDEGs在肺纤维化小鼠模型中的验证。注：A、B：对照组和博来霉素组小鼠肺组织的HE和Msson染色；B：核心MiRDEGs在对照组和肺纤维化组小鼠肺组织中的表达水平差异

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Analysis of mitochondrial related genes in idiopathic pulmonary fibrosis

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Analysis of mitochondrial related genes in idiopathic pulmonary fibrosis