基于多组学分析技术探索外泌体介导的多发性肌炎/皮肌炎分子调控网络机制  

The role of molecular regulatory network in the pathogenesis of exosome-mediated polymyositis/dermatomyositis based on multi-omics analysis techniques

作　　者：徐姝玥 万佳蔚 徐蔷薇 韩志君 高明珠 Xu Shuyue;Wan Jiawei;Xu Qiangwei;Han Zhijun;Gao Mingzhu(Clinical Medical Research Center,the Second Hospital of Wuxi,Nanjing Medical University,Wuxi 214000,China;Department of Laboratory,Central Hospital of Jiangnan University,Wuxi 214000,China;Department of Rheumatology and Immunology,Central Hospital of Jiangnan University,Wuxi 214000,China;Clinical Medical Research Center,Central Hospital of Jiangnan University,Wuxi 214000,China)

机构地区：[1]南京医科大学附属无锡第二医院临床医学研究中心,无锡214000 [2]江南大学附属中心医院检验科,无锡214000 [3]江南大学附属中心医院风湿免疫科,无锡214000 [4]江南大学附属中心医院临床医学研究中心,无锡214000

出　　处：《中华风湿病学杂志》2024年第7期445-451,I0004,I0005,I0006,共10页Chinese Journal of Rheumatology

基　　金：国家自然科学基金青年项目(82001711);江苏省自然科学基金面上项目(BK20201137);无锡市卫生健康委科研项目(M202207,Q202007,BJ2020039,HB2020035,BJ2023029,HB2023023)。

摘　　要：目的通过多组学联合生物信息方式分析探索外泌体介导的多发性肌炎/皮肌炎(PM/DM)的潜在发生机制,为PM/DM提供诊断新靶标和治疗新思路。方法收集2021年1月至2023年6月来自无锡市第二人民医院的PM/DM患者及健康体检者的血清外泌体样本,基于HiSeq高通量测序技术和同位素标记相对和绝对定量(iTRAQ)定量蛋白组学技术对PM/DM组与健康对照组血清外泌体中的微RNA(miRNA)和蛋白组分开展测序分析,并使用R语言进行limma差异分析,基因本体(GO)、京都基因和基因组百科全书(KEGG)、基因富集分析(GSEA)功能分析及免疫相关性分析,筛选核心基因并建立miRNA-靶基因-转录因子共表达分子网络。最后使用实时荧光定量聚合酶链反应(qRT-PCR)对核心基因进行实验验证。统计学方法以t检验进行数据分析,并绘制受试者工作特征(ROC)曲线评价核心基因的检验效能。结果初步筛选出42个上调差异蛋白,61个下调差异蛋白,以及22个上调差异miRNA,19个下调miRNA,最终确定7个核心基因,13个关联差异miRNA以及4个转录因子。根据功能分析认为核心基因组织蛋白酶G(CTSG)、髓过氧化物酶(MPO)、组蛋白H1-5涉及的中性粒细胞胞外诱捕网的形成、NF-κB通路等炎症相关途径在外泌体介导的PM/DM发生机制中起重要作用。肥大细胞、未成熟树突状细胞、自然杀伤细胞、调节性T细胞、辅助性T细胞2等免疫细胞在疾病的血清外泌体中表达程度较高。健康对照和PM/DM组MPO[(1.08±0.47)和(2.05±0.62),t=-3.50,P=0.004]、CTSG[(1.11±0.51)和(2.27±1.10),t=-2.72,P=0.022]、H1-5[(1.03±0.25)和(1.81±0.73),t=-2.89,P=0.019]相对表达量比较差异具有统计学意义,ROC曲线中,MPO[AUC(95%CI)=0.92(0.78,1)]、CTSG[AUC(95%CI)=0.81(0.59,1)]、H1-5[AUC(95%CI)=0.84(0.64,1)]表明3个核心基因精度良好。结论本研究鉴定了PM/DM血清外泌体中的关键差异分子,并构建了miRNA-靶基因-转录因子的调控�Objective To explore the potential pathogenesis of exosome-mediated polymyositis/dermatomyositis(PM/DM)through multi-omics combined with bioinformatic analysis approach and to identify potential new targets for the diagnosis and treatment of PM/DM.Methods Collect serum exosome samples from PM/DM patients and healthy individuals who underwent physical examination in Wuxi Second People's Hospital from January 2021 to June 2023.HiSeq high-throughput sequencing technology and iTRAQ quantitative proteomics techniques were used to perform a sequencing analysis of miRNA and protein components in serum exosomes from patients with PM/DM and healthy control.R language was adapted to conduct the limma differential analysis,gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG),gene set enrichment analysis(GSEA)functional analysis,and immunological correlation analysis.Based on these analysis,we identified core genes and established a miRNA-target gene-transcription factor co-expression molecular network.Subsequently,we employed quantitative real-time PCR(qRT-PCR)to experimentally validate the core genes.Data analysis was performed using t-test statistical analysis and receiver operating characteristic(ROC)curves were plotted to evaluate the test efficacy of the core genes.Results Initially,42 up-regulated differential proteins and 61 DEP down-regulated differential proteins,as well as 22 up-regulated differential miRNAs and 19 down-regulated miRNAs were screened,and 7 core genes,13 associated differential miRNAs,and 4 transcription factors were finally identified.Based on the functional analysis we concluded that the core genes CTSG,MPO,H1-5 involved in the formation of neutrophil extracellular trap network,NF-κB pathway and other inflammation-related pathways might play an important role in exosome-mediated PM/DM pathogenesis.Immune cells such as mast cells,immature dendritic cells,natural killer cells,regulatory T cells,and helper T cells 2 were expressed to a higher extent in the disease.In the t-test,MPO[(1.08

关 键 词：皮肌炎 外泌体 蛋白质组学 基因组学 多发性肌炎 

分 类 号：R593.26[医药卫生—内科学]