冠状动脉粥样硬化性心脏病患者心外膜脂肪组织的生物信息学分析
目的：通过生物信息学方法挖掘冠状动脉粥样硬化性心脏病（Coronary artery disease，CAD）患者心外膜脂肪组织（Epicardial adipose tissue，EAT）中的关键基因，通过富集分析、构建蛋白-蛋白互相作用网络，探讨免疫细胞浸润情况，联合CAD患者外泌体（Exosome）差异表达基因推测EAT来源外泌体差异表达基因并进行验证，从细胞及分子水平上探讨EAT在CAD疾病过程中的作用机制。方法：从GEO数据库下载GSE64554、GSE120774数据集，根据临床信息将心外膜脂肪组织的测序数据分为冠心病组和对照组，利用R语言及相关软件包进行生物信息学分析。首先获取CAD患者与对照组EAT间差异表达基因，并进行富集分析，构建蛋白-蛋白相互作用网络，以评估所选基因的生物学功能及可能参与其调控的转录因子。构建GSE64554数据集中心外膜脂肪组织的加权基因共表达网络（Weighted gene co-expression network analysis, WGCNA），获取同冠心病表型相关的基因模块，将所获EAT间差异表达基因与模块内Hub基因取交集获得关键基因，同时通过Cibersort反卷积算法对EAT组织的免疫细胞浸润情况进行分析。通过exoRbase数据库获取CAD患者与健康对照组血液外泌体间差异表达基因，CAD患者同健康对照组EAT间差异表达基因与CAD患者同健康对照组外泌体间差异表达基因区交集，将其作为CAD的诊断治疗标记物并收集临床样本通过rt-qPCR对其进行验证。对所选基因进行GO/KEGG富集分析和Metascape富集分析。结果：获得心外膜脂肪组织间差异表达基因1511个，其中表达上调的基因956个，表达下调的基因555个。构建EAT组织表达谱的加权基因共表达网络，获得同CAD表型相关的模块，将模块内Hub基因与差异表达基因取交集获得EAT在CAD发生发展中的关键基因DDX47、FEM1C、NOL11、SRP54、ABI1、PATL1、BNIP2、 C1orf159、CHCHD4。免疫细胞浸润分析显示，CAD患者EAT中幼稚CD4+ T细胞丰度升高而静息树突状细胞表达丰度减低（P ＜ 0.05）。获得CAD患者外泌体差异基因1658个，其中表达上调的基因278个，表达下调的基因1380个，将所获EAT间差异表达基因与外泌体间差异表达基因交集，共获得129个基因，选取表达丰度较高的BPI、BIRC5、CXCL12、RNASE1、F2R作为CAD患者潜在诊断治疗标记物，并同过rt-qPCR进行验证。GO/KEGG富集分析表明EAT间差异表达基因主要富集于细胞质基质、MHC蛋白复合物、RNA降解、抗原加工和呈递等，构建PPI网络，通过Cytoscape软件Cytohubba插件MCC算法获得连接度最高的基因RPS27A。Metascape富集分析差异表达基因主要富集于细胞对DNA损伤刺激反应、RNA代谢、调节细胞对压力的反应、适应性免疫系统等，TRRUST数据库预测CIITA转录因子可能参与了EAT间差异表达基因的调控。结论：（1）心外膜脂肪组织可能通过促炎和免疫途径参与CAD的发生发展，DDX47、FEM1C、NOL11、SRP54、ABI1、PATL1、BNIP2、C1orf159、CHCHD4、RPS27A可能作为关键基因并发挥重要作用。（2）CAD患者EAT中幼稚CD4+ T细胞丰度升高而静息树突状细胞表达丰度减低。（3）BPI、BIRC5、CXCL12、RNASE1、F2R可能由心外膜脂肪组织分泌并可作为CAD的诊断标记物。
Objective: This paper is mainly to identify the differential genes and possible pathogenesis of Epicardial adipose tissue (EAT) and Exosome in CAD patients at the cellular and molecular levels. Methods: We downloaded the GEO database and analyzed the genes differentially expressed in epicardial adipose tissue from healthy patients and coronary artery disease patients and established the Weighted gene co-expression network by using R language and related software packages. We also used GO/KEGG enrichment analysis and Metascape enrichment analysis to explore the protein interaction network and biological pathways of the choosen genes and analyzed key genes and pathways to find potential targets for use. Last, We interacted the dierentially expressed genes in EAT and exosomes from healthy patients and coronary artery disease patients and Q-PCR was performed the genes which are higher expression. Results: Compared with the epicardial adipose tissue with healthy individuals, the epicardial adipose tissue of CAD patients had 1511 differential genes, including 956 genes with up-regulated expression and 555 genes with down-regulated expression. According to the WGCNAthe study identified 9 module which phenotype is the most highly associated with CAD. By interacting the dierentially expressed genes and Hub genes in modules, we identified the key gene DDX47FEM1CNOL11SRP54ABI1PATL1BNIP2 C1orf159CHCHD4.A total of 129 differential genes were obtained by cross-tabbing EAT and EXO, and Q-PCR was performed to analyze the expression of BPIBIRC5CXCL12RNASE1F2R, the differences were proved to be significant after statistical analysis. GO enrichment analysis showed that differential genes were mainly involved in the cytosolMHC protein complex integral component of lumenal side of endoplasmic reticulum membranelumenal side of endoplasmic reticulum membrane MHC class II protein complex. KEGG signaling pathway enrichment analysis is mainly for RNA degradation, antigen processing, and presentation. By TRRUST datasets, we predicted that transcription factors CIITA may play a vital role in epicardial adipose tissue.Conclusion: DDX47FEM1CNOL11SRP54ABI1PATL1BNIP2 C1orf159CHCHD4 is the key gene in the pathogenesis of epicardial adipose tissue (EAT). Immunocytic infiltration analysis showed that the abundance of naive CD4+ T cells in EAT which from CAD patients was significantly increased (P <0.05). BPIBIRC5CXCL12RNASE1F2R may excrete from epicardial adipose tissue and have the potential in CAD diagnosis.
柴晏, 赵玉青, 郭旭男, 王东英, 边云飞
10.12114/j.issn.1007-9572.2022.0487
基础医学生物科学研究方法、生物科学研究技术内科学
冠心病心外膜脂肪组织外泌体生物信息学分析免疫浸润
柴晏, 赵玉青, 郭旭男, 王东英, 边云飞.冠状动脉粥样硬化性心脏病患者心外膜脂肪组织的生物信息学分析[EB/OL].(2022-07-09)[2025-08-04].https://chinaxiv.org/abs/202207.00056.点此复制
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