Secretory phenotype in PBMCs of elderly patients with rheumatoid arthritis

Purpose: The senescence-associated secretory phenotype (SASPs) might increase risk of age-related diseases and concomitant diseases in elderly rheumatoid arthritis (ERA) patients. This study aims to investigate the SASPs in peripheral blood mononuclear cells (PBMCs) for ERA patients. Methods: We performed RNA-seq of the PBMCs from 5 aged RA (RA_A) and 4 young RA (RA_Y) patients. By comparing the differentially expressed genes (DEGs) of RA_Y and RA_A using DESeq package, we identified the senescent secretory phenotype of ERA. The Gene Ontology (GO) functional enrichment, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and GSEA analysis were performed using clusterProfiler package. The significant protein–protein interaction (PPI) networks identified by Cytoscape. The proteomics data that investigate secretory phenotype of ERA was download from proteomics database. The overlapping SASPs at the intersection of proteomic and transcriptomic were then validated using real-time PCR (RT-PCR). Results: The PBMCs of RA_A and RA_Y had heterogeneity transcriptomic feature. By comparing RA_A with RA_Y groups, 348 up-regulated and 363 down-regulated DEGs were identified. Gene functional enrichment indicated that up-regulated DEGs in RA_A, SASPs for ERA patients, enriched in PI3K-Akt signaling pathway, MAPK signaling pathway, toll-like receptor family, neutrophil degranulation and immune-related pathways and so on. GSEA analysis indicated, humoral immune response pathways were activated in RA_A. By performing RT-PCR, these five SASPs, SPTA1, SPTB, VNN1, TNXB, KRT1, in PBMCs of RA patients were finally validated. Conclusion: Our study revealed the aging phenotypes in PBMCs of RA patients, and validated five SASPs, which providing novel insights for targeting SASPs therapy. Overall design: We performed RNA-seq on peripheral blood mononuclear cells (PBMCs) from aged RA patients (RA_A) and young RA patients (RA_Y). By comparing the differentially expressed genes (DEGs) between RA_A and RA_Y, the SASPs in PBMCs of ERA patients was revealed and validated by performing RT-PCR.

研究目的：衰老相关分泌表型（senescence-associated secretory phenotype, SASP）可能增加老年类风湿关节炎（elderly rheumatoid arthritis, ERA）患者罹患年龄相关性疾病及合并症的风险。本研究旨在探究ERA患者外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中的SASP特征。研究方法：本研究对5例老年类风湿关节炎患者（RA_A）及4例青年类风湿关节炎患者（RA_Y）的PBMCs进行RNA测序（RNA-seq）。借助DESeq软件包对比RA_Y组与RA_A组的差异表达基因（differentially expressed genes, DEGs），以此鉴定ERA患者的衰老分泌表型。采用clusterProfiler软件包开展基因本体（Gene Ontology, GO）功能富集分析、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析及基因集富集分析（GSEA）。通过Cytoscape软件筛选得到显著蛋白质相互作用（protein–protein interaction, PPI）网络。从蛋白质组学数据库下载用于探究ERA患者分泌表型的蛋白质组学数据。取蛋白质组学与转录组学结果的交集SASP，通过实时荧光定量PCR（real-time PCR, RT-PCR）完成验证。研究结果：RA_A组与RA_Y组患者的PBMCs存在转录组异质性。对比两组后，共鉴定出348个上调DEGs及363个下调DEGs。基因功能富集分析显示，RA_A组中上调的DEGs（即ERA患者的SASP相关基因）显著富集于PI3K-Akt信号通路、MAPK信号通路、Toll样受体家族、中性粒细胞脱颗粒及免疫相关通路等。GSEA分析表明，RA_A组的体液免疫应答通路被激活。经RT-PCR验证，类风湿关节炎患者PBMCs中的5个SASP基因（SPTA1、SPTB、VNN1、TNXB、KRT1）的表达水平得到确认。研究结论：本研究揭示了类风湿关节炎患者PBMCs的衰老表型，并验证了5个SASP基因，为靶向SASP的治疗策略提供了全新的研究思路。整体实验设计：本研究对老年类风湿关节炎患者（RA_A）及青年类风湿关节炎患者（RA_Y）的PBMCs开展RNA测序。通过对比RA_A组与RA_Y组的DEGs，揭示并通过RT-PCR验证了ERA患者PBMCs中的SASP特征。