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基于生信分析对脓毒症相关性脑病中神经炎症相关核心基因的筛选OA北大核心CSTPCD

Screening of core genes in neuroinflammation of sepsis associated encephalopathy based on bioinformatics analysis

中文摘要英文摘要

目的:通过生信分析筛选脓毒症相关性脑病(sepsis associated encephalopathy,SAE)中小胶质细胞介导神经炎症的核心基因,并通过体外细胞学实验验证.方法:从基因表达综合数据库(gene expression omnibus,GEO)中获取脓毒症患者外周血全转录组测序数据集GSE65682以及体外脂多糖(lipopolysaccharides,LPS)诱导小胶质细胞活化模型基因芯片数据集GSE103156.采用加权基因共表达网络分析(weighted gene co-expression network analysis,WGCNA)筛选GSE65682数据集中与脓毒症临床诊断显著相关的模块,与GSE103156数据集中LPS处理前后小胶质细胞的差异表达基因(differentially expressed gene,DEG)相交,利用基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)对DEG进行功能富集分析.采用STRING构建蛋白质-蛋白质相互作用网络、Cytoscape以及Lasso回归分析筛选核心基因.构建LPS诱导BV2小胶质细胞活化体外细胞模型,采用实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)检测基因表达;采用慢病毒载体法过表达组蛋白去乙酰化酶9(histone deacetylase 9,HDAC9),Western blot检测炎症相关分子表达.结果:WGCNA分析得到GSE65682数据集中与脓毒症临床诊断相关的9个模块、共332个基因,通过Limma分析得到GSE103156数据集中LPS刺激前后小胶质细胞的1 272个DEGs,二者取交集后得到18个交集基因,进一步采用Lasso回归分析筛选得到4个枢纽基因分别为七跨膜G蛋白偶联受体183(G protein-coupled receptor 183,GPR183)、HDAC9、烟酰胺腺嘌呤二核苷酸激酶(nicotinamide adenine dinucleotide kinase,NADK)、富含亮氨酸重复蛋白 25(leucine rich repeat containing 25,LRRC25).RT-qPCR结果证实在LPS刺激的小胶质细胞炎性活化模型中,Gpr183和Hdac9基因的mRNA表达下调、Lrrc25表达上调,而Nafk表达无明显变化;Western blot显示过表达HDAC9可促进LPS诱导小胶质细胞促炎因子白介素(interleukin,IL)-1β、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的表达、促进JAK1-STAT3磷酸化.结论:本研究利用生物信息学筛选得到SAE中小胶质细胞介导神经炎症的4个关键基因,并初步证实HDAC9对于小胶质细胞具有促炎活性,为SAE的机制研究提供了新的思路和数据.

Objective:To identity core genes of neuroinflammation mediated by microglia in sepsis-associated encephalopathy(SAE)through bioinformatics analysis and validate them through in vitro cellular experiments.Methods:Transcriptomic datasets of peripheral blood from sepsis patients(GSE65682)and in vitro lipopolysaccharide(LPS)-stimulated microglial cell activation model(GSE103156)were obtained from the Gene Expression Omnibus(GEO)database.Weighted gene co-expression network analysis(WGCNA)was used to screen the modules significantly related to clinical diagnosis of sepsis in the GSE65682 dataset.The intersection between differentially expressed genes(DEG)in LPS-treated and untreated microglial cells from the GSE 103156 dataset and the WGCNA modules was determined.Functional enrichment analysis of DEG was performed using gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).A protein-protein interaction network was constructed using STRING,and core genes were screened by Cytoscape and Lasso regression analysis.An in vitro cellular activation model of LPS-induced BV2 microglial was established,and gene expression was detected using quantitative real-time PCR(RT-qPCR).Histone deacetylase 9(HDAC9)was overexpressed in microglia using the lentiviral vector method,and Western blot was employed to detect the inflammation related molecule expression.Results:The WGCNA analysis identified nine modules associated with the clinical diagnosis of sepsis in the GSE65682 dataset,comprising 332 genes.Limma analysis identified 1 272 DEGs in LPS-stimulated microglial cells from the GSE 103156 dataset.Eighteen overlapping genes were obtained,and the Lasso regression analysis further selected four hub genes:G protein-coupled receptor 183(GPR183),HDAC9,nicotinamide adenine dinucleotide kinase(NADK),and leucine rich repeat containing 25(LRRC25).RT-qPCR confirmed downregulation of mRNA expression of Gpr183 and Hdac9 genes and upregulation of Lrrc25 expression in the inflammatory activation model of LPS-stimulated microglial cell,with no significant change in Nadk expression.Western blot showed that overexpression of HDAC9 promoted the expression of pro-inflammatory cytokines interleukin(IL)-1β and inducible nitric oxide synthase(iNOS)in LPS-induced microglial cells and enhanced JAK1-STAT3 phosphorylation.Conclusion:This study identitfies four key genes mediating neuroinflammation in SAE through bioinformatics analysis and preliminarily demonstrates that HDAC9 has pro-inflammatory activity in microglia,providing new insights and data for further mechanistic research on SAE.

苏思璇;尚彦星;段程伟;梁彩霞;张冬梅

南通大学医学院病原生物学系,江苏 南通 226001||南通大学第二附属医院临床医学研究中心,江苏省人体免疫学医学重点学科/实验室建设单位,南通市代谢免疫学与疾病微环境重点实验室,南通市分子免疫学医学重点实验室,南京医科大学康达学院南通临床医学院,江苏 南通 226001南通大学第二附属医院临床医学研究中心,江苏省人体免疫学医学重点学科/实验室建设单位,南通市代谢免疫学与疾病微环境重点实验室,南通市分子免疫学医学重点实验室,南京医科大学康达学院南通临床医学院,江苏 南通 226001

临床医学

脓毒症相关性脑病小胶质细胞生信分析差异基因核心基因

sepsis associated encephalopathymicrogliabioinformatics analysisdifferential genescore genes

《南京医科大学学报(自然科学版)》 2024 (007)

915-926 / 12

江苏省自然科学基金(BK20211108);江苏省医学重点学科/实验室建设单位(JSDW202249);南通市自然科学基金(JC2023114);南京医科大学康达学院科研创新团队项目(KD2022KYCXTD005);中国高校产学研创新基金—华通国康医学科研专项(2023HT042)

10.7655/NYDXBNSN231122

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