Table 3_Liver fibrosis in biliary atresia: identification of the key gene EDIL3 via integrated bioinformatics.xls

BackgroundBiliary atresia (BA) is one of the most destructive liver and biliary diseases in neonates and is characterized by progressive fibrous inflammatory obstruction of the intrahepatic and extrahepatic bile ducts, ultimately leading to liver fibrosis and liver failure. This study aimed to use integrated bioinformatics methods to identify differentially expressed genes (DEGs) in BA liver tissue, identify key genes, and explore their mechanisms in liver fibrosis.
MethodsWe obtained data from the gene expression omnibus (GEO) dataset GSE122340 [171 BA patients and 7 normal controls (NCs)]. DEGs were screened via the limma package, followed by gene ontology (GO)/kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis. A protein-protein interaction (PPI) network was constructed via STRING and Cytoscape, and core genes were selected via the maximum clique centrality (MCC), maximum neighborhood component (MNC), and degree algorithms from the CytoHubba plugin. Further focus was placed on the key gene EGF-like repeats and discoidin I-like domains 3 (EDIL3) through gene set enrichment analysis (GSEA), expression validation, subcellular localization analysis, and clinical tissue sample validation. To minimize batch effects, we performed ComBat correction on the combined gene expression data of GSE122340 and the validation dataset GSE46960 before interdataset comparison.
ResultsWe identified a total of 3706 DEGs, including 2774 upregulated DEGs and 932 downregulated DEGs. The functional enrichment analysis revealed that the DEGs were involved mainly in biological processes such as the cell cycle, DNA replication, and extracellular matrix organization, as well as signaling pathways such as herpes simplex virus infection, phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT), and tumor necrosis factor (TNF). Protein-protein interaction (PPI) network analysis revealed 7 core genes (BRCA1, TOP2A, BRCA2, BUB1B, HSP90AA1, PLK4, and EDIL3), with EDIL3 showing the greatest increase in BA. EDIL3 is located on chromosome 5, and its encoded protein is expressed primarily in the cell membrane and extracellular region. GSEA indicated that high EDIL3 expression was significantly associated with apoptosis and activation of the PI3K-AKT signaling pathway. Clinical sample validation revealed that EDIL3 expression was significantly elevated in BA liver tissue, and its high expression was significantly negatively correlated with the survival rate of patients’ native livers.
ConclusionDiscoidin I-like domain 3 is a novel gene with a potentially key role in BA-related liver fibrosis, possibly influencing the proliferation and apoptosis of cholangiocytes by regulating the PI3K-AKT signaling pathway, thereby participating in the occurrence and development of liver fibrosis. This study provides new insights into the molecular mechanisms and potential treatment strategies for BA.

背景：胆道闭锁（biliary atresia, BA）是新生儿中最具破坏性的肝胆疾病之一，以肝内和肝外胆管进行性纤维炎性梗阻为特征，最终可进展为肝纤维化与肝衰竭。本研究旨在通过整合生物信息学方法，筛选胆道闭锁肝组织中的差异表达基因（differentially expressed genes, DEGs），鉴定关键核心基因，并探讨其在肝纤维化发生发展中的作用机制。

方法：本研究从基因表达综合数据库（Gene Expression Omnibus, GEO）中获取数据集GSE122340的数据，该数据集包含171例胆道闭锁患者与7例正常对照（normal controls, NCs）样本。采用limma R包筛选差异表达基因，随后进行基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析。利用STRING数据库与Cytoscape软件构建蛋白质相互作用（protein-protein interaction, PPI）网络，并通过CytoHubba插件的最大团中心性（maximum clique centrality, MCC）、最大邻域分量（maximum neighborhood component, MNC）以及度（degree）三种算法筛选核心基因。进一步通过基因集富集分析（Gene Set Enrichment Analysis, GSEA）、表达验证、亚细胞定位分析以及临床组织样本验证，重点关注核心基因表皮生长因子样重复序列和凝血酶敏感蛋白I样结构域3（EGF-like repeats and discoidin I-like domains 3, EDIL3）。为最小化批次效应，在跨数据集比较前，本研究对GSE122340与验证数据集GSE46960的合并基因表达数据进行了ComBat校正。

结果：本研究共筛选得到3706个差异表达基因，其中上调基因2774个，下调基因932个。功能富集分析结果显示，差异表达基因主要富集于细胞周期、DNA复制、细胞外基质组织等生物学过程，以及单纯疱疹病毒感染、磷脂酰肌醇3-激酶（phosphoinositide 3-kinase, PI3K）-蛋白激酶B（protein kinase B, AKT）、肿瘤坏死因子（tumor necrosis factor, TNF）等信号通路。蛋白质相互作用网络分析共筛选得到7个核心基因，分别为BRCA1、TOP2A、BRCA2、BUB1B、HSP90AA1、PLK4及EDIL3，其中EDIL3在胆道闭锁组织中的表达上调幅度最为显著。EDIL3基因定位于5号染色体，其编码的蛋白主要表达于细胞膜与细胞外区域。基因集富集分析结果显示，EDIL3高表达与细胞凋亡及PI3K-AKT信号通路激活显著相关。临床组织样本验证结果表明，胆道闭锁肝组织中EDIL3的表达水平显著升高，且其高表达与患者自体肝存活率呈显著负相关。

结论：凝血酶敏感蛋白I样结构域3（EDIL3）是一个在胆道闭锁相关肝纤维化中发挥关键作用的新型基因，其可能通过调控PI3K-AKT信号通路影响胆管上皮细胞的增殖与凋亡，进而参与肝纤维化的发生与发展。本研究为阐明胆道闭锁的分子机制及开发潜在治疗策略提供了新的思路。