冠状动脉慢血流疾病转录组学研究及差异基因验证

Transcriptomics study of coronary slow flow disease and verification of differentially expressed genes

目的探讨冠状动脉慢血流(SCF)患者与健康人群的差异基因及SCF发病涉及的信号通路和蛋白相互关系。方法收集2018年住院的SCF患者和同期健康体检者各43例,记录患者一般资料,采集血液测定糖代谢、脂代谢和肾脏代谢相关指标。血液单核细胞中提取RNA,通过RNA测序获得差异基因表达谱。并进行GO功能注释、KEGG通路富集和蛋白相互作用网络分析及表型分析。ELISA检测SCF患者和对照组炎症细胞因子的水平,实时荧光定量PCR(qPCR)验证差异基因。结果SCF组男性27例(63%),女性16例(37%),年龄(54.3±8.8)岁,对照组男性29例(67%),女性14例(33%),年龄(57.2±8.3)岁。SCF组有吸烟史、空腹血糖异常、血脂异常的比例及体重指数高于对照组(P<0.05)。SCF组和对照组存在117个差异基因,其中上调基因73个,下调基因44个。GO功能注释和KEGG通路分析发现,差异基因主要与抗原处理和呈递、细胞吞噬、免疫球蛋白A的肠道免疫网络、辅助型T细胞1(Th1)、Th2和Th17细胞分化通路相关。与对照组相比,SCF组炎症细胞因子白细胞介素-6、白细胞介素-10、肿瘤坏死因子-α和干扰素-γ的表达水平升高(P<0.05)。2组差异最显著的前12个基因中,11个qPCR分析与转录组结果一致,蛋白相互作用网络分析表明甲酰化多肽受体-2(FPR2)和凝血酶致敏因子3(THBS3)蛋白位于整个蛋白作用网络的核心位置。结论SCF患者与健康人群的基因存在差异。FPR2和THBS3等基因可能通过抗原处理和呈递以及Th17细胞分化等免疫相关通路影响SCF的发生和发展。

Objective To determine the differentially expressed genes between patients with coronary slow flow(SCF)and healthy controls,as well as to define the signal pathways and protein interactions related to the pathogenesis of the disease.Methods A total of 43 hospitalized SCF patients in the year of 2018 and 43 healthy subjects,who underwent health checkup in the same year,were enrolled in this study.General data were obtained,blood samples were collected to determine the related indicators of glucose metabolism,lipid metabolism and kidney metabolism.RNA was extracted from blood monocytes,and the differential gene expression profiles were investigated by RNA-Seq.GO function annotation,KEGG pathway enrichment,protein interaction network analysis(PPI)and phenotype analysis were performed.The levels of related cytokines were detected by ELISA,and qPCR was used to verify differentially expressed genes of the two groups.Results In the SCF group,there were 27(62.79%)males and 16 females(37.21%),the average age was(54.3±8.8)years.In the control group,there were 29 males(67.44%)and 14 females(31.56%),the average age was(57.2±8.3)years.The percent of smoking history,abnormal fasting blood glucose,abnormal blood lipid levels and body mass index were significantly higher in the SCF group than in the control group(all P<0.01).There were 117 differentially expressed genes between SCF patients and healthy controls,of which 73 were up-regulated and 44 were down-regulated.Biological function analysis of the differentially expressed genes showed that these genes were mainly related to antigen processing and presentation,cell phagocytosis,immunoglobulin,intestinal immune network,Th1 and Th2 cell differentiation and Th17 cell differentiation pathways.The expression levels of inflammatory cytokines IL-6,IL-10,tumor necrosis factor-αand interferon-γwere significantly higher in SCF patients than in healthy controls(all P<0.05).Among the top 12 genes with the most significant differences between the two groups,qPCR analysis indicated consistent results with the transcriptome results in 11 out of 12 genes.PPI analysis showed that FPR2 and THBS3 proteins were at the core of the entire protein interaction network.Conclusion Genes such as FPR2 and THBS3 may play important roles in the pathogenesis of SCF through immune-related pathways such as antigen processing and presentation and Th17 cell differentiation.