通过转录组测序和生物信息学方法鉴别小胶质细胞炎症激活过程中的关键通路和基因

Identification of key pathways and genes involved in microglia inflammation by bioinformatics analysis of transcriptome sequencing

目的通过转录组测序和生物信息学分析,探究小胶质细胞炎症激活的关键通路和基因。方法使用质量浓度为1μg/ml的脂多糖对小胶质细胞进行刺激,建立小胶质细胞炎症模型。使用ELISA法和RT-qPCR检测小胶质细胞炎症模型IL-6和TNF-α水平。对建立的小胶质细胞炎症模型进行转录组测序,采用生物信息学方法筛选出差异表达基因。对差异表达基因进行GO分析和KEGG分析。使用String数据库构建差异表达基因的蛋白互作网络,并使用Cytoscape软件进行蛋白互作网络的可视化。使用MCODE应用程序提取蛋白互作网络的模块,使用cytohubba应用程序提取枢纽基因。采用RT-qPCR验证枢纽基因的mRNA表达水平。对枢纽基因进行富集分析,预测其靶向miRNA,预测可能与其作用的药物。结果经ELISA和RT-qPCR检验,小胶质细胞炎症模型建立成功。通过转录组测序和生物信息学分析,筛选出434个差异表达基因。GO分析显示这些差异表达基因主要集中在细胞对细胞因子刺激的反应、炎症反应、对外界刺激反应的调节等条目。KEGG分析显示这些差异表达基因主要集中在趋化因子信号通路、TNF信号通路、IL-17信号通路等通路。构建了这些差异表达基因的蛋白互作网络图,并进行了模块分析和枢纽基因的提取,枢纽基因大部分位于模块1内,模块1的种子基因为S1pr1,枢纽基因包括S1pr1、Cxcr4、Cx3cl1、Cx3cr1、Cxcl10、Cxcl2、Ccl4、Ccl5、Ccl9、Fpr1。RT-qPCR结果显示,与培养液组相比,脂多糖组中S1pr1、Cxcr4、Cx3cl1、Cx3cr1的mRNA表达下调,Cxcl10、Cxcl2、Ccl4、Ccl5、Ccl9、Fpr1的mRNA表达上调。枢纽基因的富集分析主要集中在趋化因子介导的信号通路、视紫红质样受体、细胞趋化性等条目。预测了可能和枢纽基因作用的miRNA和药物。结论通过对小胶质细胞炎症模型进行转录组测序和生物信息学分析,筛选出了差异表达基因,提取了枢纽基因和种子基因,有助于进一步理解小胶质细胞炎症的分子机制,为相关疾病的诊治提供潜在的靶点。

Objective To explore the key pathways and genes involved in microglia inflammation through transcriptome sequencing and bioinformatics analysis.Methods BV2 cells were stimulated by lipopolysaccharide to establish microglia inflammation model.The levels of IL-6 and TNF-αwere detected by ELISA and RT-qPCR.The established microglia inflammation model was sequenced by transcriptome sequencing,and the differentially expressed genes were screened by bioinformatics method.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis of differentially expressed genes were performed.The protein-protein interaction network of differentially expressed genes was constructed by using string database,and the protein-protein interaction network was visualized by using Cytoscape software.The protein interaction network module was extracted by using MCODE app.The hub gene was extracted by using cytohubba app and was verified through RT-qPCR.We conducted enrichment analysis of hub genes,predicted their targeted miRNAs and interacting drugs.Results The microglia inflammation model was successfully established and verified by ELISA and RT-qPCR.We screened 434 differentially expressed genes by bioinformatics analysis of transcriptome sequencing results.GO analysis showed that these differentially expressed genes were mainly concentrated in cellular response to cytokine stimulus,inflammatory response,regulation of response to external stimulation.KEGG analysis showed that these differentially expressed genes were mainly concentrated in Chemokine signaling pathway,TNF signaling pathway,IL-17 signaling pathway.We constructed the protein interaction network of these differentially expressed genes,and carried out module analysis and extraction of hub genes.Most of hub genes are located in module 1,and the seed gene of module 1 is S1pr1.Hub genes include S1pr1,Cxcr4,Cx3cl1,Cx3cr1,Cxcl10,Cxcl2,Ccl4,Ccl5,Ccl9,Fpr1.RT-qPCR results showed that compared with the culture medium group,the mRNA expressions of S1pr1,Cxcr4,Cx3cl1 and Cx3cr1 were down-regulated,and the mRNA expressions of Cxcl10,Cxcl2,Ccl4,Ccl5,Ccl9 and Fpr1 were up-regulated in the LPS group.The enrichment analysis of hub genes mainly focused on chemokine-mediated signaling pathway,Class A/1(Rhodopsin-like receptors),cell chemotaxis and so on.Drugs and miRNAs that may interact with hub genes were predicted.Conclusion Through transcriptome sequencing and bioinformatics analysis of microglia inflammation model,differentially expressed genes were screened,hub genes and seed genes were extracted,which will help us further understand the molecular mechanism of microglia inflammation and provide potential targets for the treatment of related diseases.