Screening of target genes for bile acid metabolism in Crohn’s disease and its value in disease diagnosis and therapeutic monitoring

doi:10.12307/2025.937

Abstract

Abstract: BACKGROUND: Bile acid metabolism plays a crucial role in the development and progression of Crohn’s disease. There is no research on changes in bile acid metabolism and key target genes following treatment with biological agents.
OBJECTIVE: To investigate the expression characteristics of bile acid metabolism-related genes in patients with Crohn’s disease, identify key genes associated with response to biological agents.
METHODS: Transcriptome data were obtained through the GEO database to analyze differentially expressed genes between inflammation-control groups and inflammation-treatment groups. GO, KEGG, and GSEA enrichment analyses were used to evaluate the effects of biological agent therapy on bile acid metabolism. Protein-protein interaction network and WGCNA algorithm were employed to analyze differentially expressed genes, identifying modules closely related to biological agent treatment response, which led to the determination of UGT2A3 as a key gene in bile acid metabolism. In the inflammation group of the GSE186582 dataset, samples were divided into high and low expression groups based on UGT2A3 levels to study its relationship with immune infiltration and explore the interaction between UGT2A3 and the immune microenvironment. Clinical characteristics and intestinal manifestations were compared between high and low expression groups, and correlations between UGT2A3 and clinical indicators (C-reactive protein, erythrocyte sedimentation rate, Crohn’s disease activity index, and Crohn’s disease endoscopic activity score) were investigated. The competing endogenous RNA regulatory network of UGT2A3 was constructed, and its upstream miRNA was functionally enriched to explore the molecular mechanism of UGT2A3 in bile acid metabolism. Single-cell analysis and clustering were performed using high-throughput sequencing data of GSE134809 to observe the expression of UGT2A3 in different samples and cell populations. Colon tissue samples from untreated and biologic-treated Crohn’s disease patients and healthy colon tissue samples from patients with intestinal polyps were collected, and UGT2A3 expression was detected by immunohistochemistry, qRT-PCR, and western blot assay. Fresh feces from Crohn’s disease patients and healthy controls were collected to detect bile acid levels, and the relationship between UGT2A3 and fecal bile acid levels was analyzed.
RESULTS AND CONCLUSION: A total of 11 bile acid metabolism-related genes were screened, showing significant changes in gene expression after biological agent therapy. GO and KEGG enrichment analyses revealed that intestinal nutrient absorption and metabolic processes normalized after treatment, while leukocyte chemotaxis and inflammatory response pathway activity decreased. GSEA analysis revealed significant enrichment of bile acid metabolism-related pathways after treatment. Protein-protein interaction network construction and WGCNA analysis identified UGT2A3 as a key gene closely associated with treatment response. UGT2A3 expression was significantly decreased in inflamed tissues of Crohn’s disease patients and returned to normal levels after biological agent therapy. This result was confirmed in clinical specimens. UGT2A3 expression levels showed significant negative correlations with C-reactive protein, erythrocyte sedimentation rate, Crohn’s Disease Activity Index, and Crohn’s Disease Endoscopic Index of Severity. Receiver Operating Characteristic curve analysis demonstrated that UGT2A3 has good diagnostic value (Area Under Curve AUC=0.801 0) and effectively reflects treatment outcomes. Immune infiltration analysis showed significantly increased infiltration of various immune cells in samples with low UGT2A3 expression, and its expression levels negatively correlated with immune scores, microenvironment scores, and stromal scores. Compared with the low UGT2A3 expression group, patients with high expression showed less fecal occult blood and penetrating inflammation, with milder intestinal strictures and general condition severity. Fecal bile acid analysis revealed that UGT2A3 expression strongly negatively correlated with primary bile acid content and strongly positively correlated with secondary bile acid content.

Key words: Crohn’s disease, inflammatory bowel disease, biologics, bile acid metabolism, bioinformatics, engineered tissue construction

CLC Number:

Chen Hui, Zhang Lu, Chen Jing, Wang Nanzhang, Wang Ruochun, Lu Cuihua, Ji Yifei. Screening of target genes for bile acid metabolism in Crohn’s disease and its value in disease diagnosis and therapeutic monitoring[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(32): 7028-7040.

Figures/Tables 15

果见图2。GO分析显示，152个上调基因在以下生物学过程中富集：粒细胞趋化性、粒细胞迁移、髓系白细胞迁移、白细胞趋化性、中性粒细胞迁移、中性粒细胞趋化性；KEGG分析显示，152个上调基因在以下代谢通路中富集：肌肉细胞中的细胞骨架、细胞因子-细胞因子受体相互作用、磷脂酰肌醇3-激酶-蛋白激酶B信号通路、阿米巴病、局部黏附。GO分析显示，172个下调基因在以下生物学过程中富集：有机阴离子运输、羧酸运输、异物代谢过程、有机酸运输、肠道吸收；KEGG分析显示，172个下调基因在以下代谢通路中富集：蛋白质消化和吸收、脂肪消化和吸收、胆汁分泌、视黄醇代谢、化学致癌-DNA加合物等。总的来说，经生物制剂治疗后，克罗恩病患者的肠道营养物质吸收以及胆汁酸代谢等多种物质代谢过程有一定程度恢复，白细胞趋化运动及炎症反应途径经治疗后的回落也在意料之中。对第1组和第2组差异表达基因分别进行GSEA富集分析，发现5条在对照组及治疗组显著上调的胆汁酸代谢相关信号通路(图2C，D)，包括药物诱导胆汁酸通路、胆汁酸和胆盐的循环利用、胆汁循环、胆汁酸合成和胆汁运输障碍、葡萄糖醛化。关键基因UGT2A3位于葡萄糖醛化(REACTOME_GLUCURONIDATION)途径中，这进一步验证了生物制剂治疗有益于胆汁酸代谢相关的多个通路的恢复。 2.3 筛选胆汁酸代谢相关核心差异基因基于STRING 12.0中的蛋白质互作数据，在Cytoscape中绘制蛋白质互作网络(图3)，在Centiscape2.2插件中，利用中介中心性、接近中心性、度中心性3种算法筛选出324个差异基因中评分最高的51个关键基因(表1)，其中包含胆汁酸代谢相关交集差异基因的大部分基因：ABCC2、CYP3A4、UGT2A3、ABCB1、ABCG2、SLC5A1，这从侧面证明了胆汁酸代谢相关交集差异基因的关键性。 2.4 UGT2A3为生物制剂治疗响应密切的胆汁酸代谢相关核心基因利用WCGNA识别共表达模块，当软阈值设置为8时，邻接函数能够较好地满足无尺度条件(图4A)，得到了19个与治疗前后状态相关的模块(图4B)，计算每个颜色模块的特征值与临床状态(未经生物制剂治疗与生物制剂治疗后)之间的相关系数和相关性显著性，筛选出了与生物制剂治疗响应密切的蓝色模块(-0.54，P < 0.000 1)、粉色模块(-0.58，P < 0.000 1)，见图4C，共包含481个基因。在胆汁酸代谢相关交集差异基因中，只有UGT2A3对生物制剂治疗显示出了较强的响应(图4D)，并且UGT2A3也作为蛋白质互作网络的关键基因，其在治疗后的表达恢复可能与生物制剂作用机制相偶联。 2.5 UGT2A3在炎症肠道组织中明显下调且经生物制剂治疗后表达恢复对GSE134809数据集进行单细胞分析细胞聚类，使用JackStrawPlot功能可视化确定主成分个数的结果，确定显著性最大的主成分数为20(图5A，B)，观察了UGT2A3在不同样本和细胞群间的表达情况，发现其表达主要集中在肠上皮细胞、杯状细胞，并且炎症组与对照组相比UGT2A3的表达密度明显减少(图5C-F)，这些结果在临床炎症组结肠炎症标本的免疫组化实验中得到验证(图5G-H)。在GSE186582数据集炎症组、对照组、治疗组的UGT2A3表达箱式图中，炎症组UGT2A3表达明显下调，治疗组UGT2A3表达恢复(图6A)。通过临床结肠组织样本进一步验证了UGT2A3在炎症组、治疗组、对照组中的表达，Western blot检测(图6B，C)和qRT-PCR检测(图6D)显示炎症组织中UGT2A3显著低表达，并且经生物制剂治疗后UGT2A3表达恢复。 2.6 UGT2A3表达与克罗恩病患者临床数据的相关性及诊断能力相关性"

分析表明，UGT2A3与全身炎症反应和克罗恩病的活动性密切相关(图7A-D)，UGT2A3与C-反应蛋白呈强负相关(r=-0.798 0，P < 0.000 1)，UGT2A3与血细胞沉降率呈中等程度负相关(r=-0.647 9，P < 0.05)，UGT2A3与克罗恩病活动指数呈中等程度负相关(r=-0.584 5，P < 0.000 1)，UGT2A3与克罗恩病内镜活动评分呈中等程度负相关(r=-0.584 7，P < 0.000 1)。针对UGT2A3高、低表达组临床数据对比结果显示，两组在性别、年龄、体质量指数、病程、吸烟史、伴随疾病、肛瘘史方面比较无明显差异(P > 0.05)，见表2。与UGT2A3高表达组相比，UGT2A3低表达组临床患者显示出了更多的粪隐血可能性、更严重的穿透性炎症、肠道狭窄等，见表3。粪便代谢组学分析发现，对照组初级胆汁酸水平明显低于炎症组(P < 0.000 1)，次级胆汁酸水平明显高于炎症组(P < 0.000 1)，见图7E。相关性分析显示，UGT2A3的表达与初级胆汁酸水平呈强正相关(r=0.757 2，P < 0.05)，与次级胆汁酸水平呈强负相关(r=-0.745 8，P < 0.05)，见图7F-G。炎症组与对照组、炎症组与治疗组的受试者工作特征曲线显示UGT2A3具有良好的诊断能力(曲线下面积AUC=0.801 0)，见图7H，并且能有效反映治疗带来的变化(曲线下面积AUC=0.800 8)，见图7I。 2.7 胆汁酸相关基因与免疫浸润的相关性及UGT2A3高低表达组免疫浸润分析对GSE186582数据集的炎症组样本进行免疫细胞浸润分析，得到上调的9个胆汁酸代谢相关交集差异基因与免疫细胞之间的相关性热图(图8A)，结果提示黑素细胞、内皮细胞、微血管内皮细胞、成纤维细胞、单核细胞、周细胞、淋巴管内皮细胞、前脂肪细胞与UGT2A3负相关性较强(P < 0.000 1)。UGT2A3与免疫评分的相关性分析显示，UGT2A3表达与免疫评分(ImmuneScore)中度相关(r=-0.515，P < 0.000 1)，与微环境评分(MicroenvironmentScore)高度相关(r=-0.718，P < 0.000 1)，与基质评分(StromaScore)中度相关(r=-0.649，P < 0.000 1)，见图8B-D。将炎症组分"

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