内质网应激相关基因在主动脉夹层疾病中的差异性表达及与免疫浸润的相关性

doi:10.12122/j.issn.1673-4254.2024.05.07

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (5): 859-866.doi: 10.12122/j.issn.1673-4254.2024.05.07

内质网应激相关基因在主动脉夹层疾病中的差异性表达及与免疫浸润的相关性

周伟¹(), 聂军¹, 胡佳², 蒋艺枝³, 张大发¹()

^1.皖南医学院第一附属医院，心胸外科，安徽芜湖 241002
^2.皖南医学院第一附属医院，神经内科，安徽芜湖 241002
^3.皖南医学院第一附属医院，血液内科，安徽芜湖 241002

收稿日期:2023-10-26 出版日期:2024-05-20 发布日期:2024-06-06
通讯作者: 张大发 E-mail:welson1987@163.com;zhangdafa2256@126.com
作者简介:周伟，博士，主治医师，E-mail: welson1987@163.com
基金资助:
国家自然科学基金(82200146);皖南医学院第一附属医院引进人才专项科研启动基金(YR20230106);皖南医学院重大疾病非编码RNA转化研究项目(RNA202009);皖南医学院校重点项目基金(WK2022ZF16)

Differential expressions of endoplasmic reticulum stress-associated genes in aortic dissection and their correlation with immune cell infiltration

Wei ZHOU¹(), Jun NIE¹, Jia HU², Yizhi JIANG³, Dafa ZHANG¹()

^1.Department of Cardiothoracic Surgery, First Affiliated Hospital of Wannan Medical College, Wuhu 241002, China
^2.Department of Neurology, First Affiliated Hospital of Wannan Medical College, Wuhu 241002, China
^3.Department of Hematology, First Affiliated Hospital of Wannan Medical College, Wuhu 241002, China

Received:2023-10-26 Online:2024-05-20 Published:2024-06-06
Contact: Dafa ZHANG E-mail:welson1987@163.com;zhangdafa2256@126.com
Supported by:
National Natural Science Foundation of China(82200146)

摘要/Abstract

摘要：

目的探讨内质网应激相关基因（ERSAGs）在主动脉夹层疾病（AD）中的差异性表达及免疫浸润相关性，为AD的治疗寻找新的靶点。方法检索GEO数据库，下载GSE190635及GSE98770两个主动脉夹层mRNA数据集，R软件分析AD患者主动脉与正常主动脉差异表达基因（DEGs），从GeneCards网站下载ERSAGs基因集。GeneMANIA数据库分析内质网应激（ERS）差异基因晚期糖基化终末产物受体（AGER）相互作用的蛋白。基于GSE98770数据集使用R语言CIBERSORT包计算AD患者主动脉壁组织内22种免疫细胞分布比例。临床收集20例主动脉壁标本，分为AD组和非AD组（n=10/组），qRT-PCR检测AGER表达量。使用TRRUST数据库和NetworkAnalyst数据库分析预测AGER上游转录因子、miRNA及调控作用化合物。结果获取到ERS差异基因AGER，与其相互作用的蛋白共有20种，主要生物功能：模式识别受体信号通路，DNA结合转录因子活性的正向调节，骨髓白细胞迁移，白细胞迁移，调节I-kappaB激酶/NF-kappaB信号传导。AD中AEGR表达水平与Treg细胞丰度间呈正相关（r=0.59，P<0.05）。qRT-PCR检测显示，AD组主动脉壁AGER表达量为1.00±0.30，非AD组表达量为1.76±0.68，差异具有统计学意义（P<0.05）。ROC曲线分析显示，AGER预测AD的AUC=0.86（95% CI：0.67~1.00，P=0.0073），cut-off值为1.335，对应的敏感性和特异性分别为80%、90%。AGER调控网络预测到3种转录因子，3种miRNAs，27种化合物。结论内质网应激相关基因AGER在主动脉夹层疾病中的低表达，其可能通过Treg细胞影响AD的发生。

关键词: 主动脉夹层, 内质网应激, 免疫浸润, 生物信息学

Abstract:

Objective To explore differentially expressed endoplasmic reticulum stress-associated genes (ERSAGs) in aortic dissection (AD) and their correlations with immune cell infiltration to identify new therapeutic targets for AD. Methods Two AD mRNA expression datasets (GSE190635 and GSE98770) were downloaded from GEO database for analysis of differentially expressed genes between the aorta of AD patients and normal aorta using R software. ERSAGs dataset was downloaded from GeneCards website, and GeneMANIA database was used to analyze the protein-protein interaction network of the differentially expressed ERSAGs and the proteins interacting with these genes. Based on GSE98770 dataset we analyzed the distributions of 22 immune cells within the aortic wall of AD patients using CIBERSORT package of R software. Surgical aortic wall specimens were obtained from 10 AD patients and 10 non-AD patients for detecting AGER mRNA expression using qRT-PCR, and the upstream transcriptional factors, miRNAs, and chemicals targeting AGER were analyzed using the TRRUST database and NetworkAnalyst database. Results Bioinformatic analysis suggested significant differential expression of AGER in AD, which interacted with 20 proteins involved in pattern recognition receptor signaling pathway, positive regulation of DNA-binding transcription factor activity, myeloid leukocyte migration, leukocyte migration, and regulation of the I-κB kinase/NF-κB signaling. In AD, AGER expression level was positively correlated with Treg cell abundance (r=0.59, P<0.05). The results of qRT-PCR demonstrated significantly lower expression of AGER mRNA in AD than in non-AD patients (1.00±0.30 vs 1.76±0.68, P<0.05). ROC curve analysis showed that at the cut-off value of 1.335, AGER had an AUC of 0.86 (95% CI: 0.67-1.00, P=0.0073) for predicting AD. Three transcriptional factors, 3 miRNAs, and 27 chemicals were predicted in the AGER regulatory network. Conclusion AGER is lowly expressed in the aorta of AD patients and may influence the occurrence of AD through Treg cells.

Key words: aortic dissection, endoplasmic reticulum stress, immune cell infiltration, bioinformatics

周伟, 聂军, 胡佳, 蒋艺枝, 张大发. 内质网应激相关基因在主动脉夹层疾病中的差异性表达及与免疫浸润的相关性[J]. 南方医科大学学报, 2024, 44(5): 859-866.

Wei ZHOU, Jun NIE, Jia HU, Yizhi JIANG, Dafa ZHANG. Differential expressions of endoplasmic reticulum stress-associated genes in aortic dissection and their correlation with immune cell infiltration[J]. Journal of Southern Medical University, 2024, 44(5): 859-866.

图/表 8

表1 qRT-PCR引物序列

Tab.1 Primer sequences for qRT-PCR

Primers

Sequence (5'-3')

β-Actin

F:AGCGAGCATCCCCCAAAGTT;

R:GGGCACGAAGGCTCATCATT

AGER

F: GTGTCCTTCCCAACGGCTC;

R: ATTGCCTGGCACCGGAAAA

图1 AD差异表达基因

Fig.1 Differentially expressed genes (DEGs) in AD. A: Volcano plot of the DEGs in GSE190635. Red points represent up-regulated genes, and blue points represent down-regulated genes; Black points represent genes without significant difference (NS). B: Heatmap of DEGs in GSE190635. C: Volcano plot for the DEGs in GSE98770. D: Heatmap of the DEGs in GSE98770.

图2 ERS差异基因分析韦恩图

Fig.2 Venn diagram of the DEGs in AD.

图3 PPI和功能分析

Fig.3 Protein-protein interaction network and functional analysis.

图4 免疫细胞浸润分析

Fig.4 Immune cell infiltration analysis in the aorta of AD patients. A: CIBERSORT algorithm-based topography of the 22 immune cells in AD samples in GSE98770 dataset. B: Correlation analysis of the 22 immune cells. C: Correlation analysis of AGER with the 22 immune cells. D: Correlation analysis between Treg cell percentage and AGER expression level. Pearson method, *P<0.05.

图5 主动脉临床标本验证

Fig.5 Validation of AGER mRNA expression in clinical aorta specimens. A: qRT-PCR detection of AGER expression in AD and NAD groups. B: ROC curve analysis. **P<0.01.

表2 AGER转录因子

Tab.2 Transcriptional factors （TF） of AGER

TF	Target	Type	PMID
NFKB1	AGER	Activation	18622638;19616578
PPARG	AGER	Repression	18855759
RELA	AGER	Activation	18622638;19616578

图6 AGER上游miRNA及调控作用化合物预测

Fig.6 Prediction of upstream miRNAs (A) and regulatory chemicals (B) of AGER.

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内质网应激相关基因在主动脉夹层疾病中的差异性表达及与免疫浸润的相关性

Differential expressions of endoplasmic reticulum stress-associated genes in aortic dissection and their correlation with immune cell infiltration

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