季节性与常年性变应性鼻炎鼻黏膜上皮细胞基因表达生物信息学分析

Bioinformatics analysis of nasal epithelial cell gene expression in seasonal and perennial allergic rhinitis

目的:通过对季节性与常年性变应性鼻炎(AR)患者鼻黏膜上皮细胞的基因表达进行转录组测序及生物信息学分析,获得季节性AR与常年性AR鼻黏膜上皮细胞基因表达的差异。方法:体外培养人鼻黏膜上皮细胞系(human nasal epithelial cells,HNEpC),分别给予100μg/ml蒿(mugwort)或户尘螨(house dust mite,HDM)提取物处理24 h,提取细胞总RNA,实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qPCR)检测细胞因子白细胞介素(IL)6、IL-8、IL-33以及胸腺基质淋巴细胞生成素(thymic stromal lymphopoietin,TSLP)的表达。2019年11月至2020年11月,就诊于吉林大学中日联谊医院耳鼻咽喉头颈外科的季节性AR、常年性AR患者以及健康对照者各3例,体外培养患者原代鼻黏膜上皮细胞,给予相应过敏原处理24 h,提取细胞总RNA进行转录组测序,对结果进行生物信息学分析。以SPSS 22.0对数据进行统计学分析。结果:qPCR结果显示,蒿提取物与HDM提取物处理的HNEpC细胞因子IL-6、IL-8、IL-33以及TSLP变化趋势相同。而对季节性AR、常年性AR患者与健康对照者鼻黏膜上皮细胞转录组测序分析发现,二者受到相应过敏原处理后生物学过程及信号通路存在差异。基因本体(gene ontology,GO)富集分析显示,蒿过敏AR患者差异表达基因(differentially expressed genes,DEG)主要富集在氧化还原过程、凋亡负向调控过程、细胞黏附过程;HDM过敏AR患者DEG则主要富集在细胞黏附、细胞增殖的负向调控及药物反应的生物学过程中。京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号通路富集分析显示,蒿过敏AR患者DEG在花生四烯酸代谢、p53信号通路及转化生长因子β(TGF-beta)信号通路中存在显著富集,而HDM过敏AR患者DEG主要富集在细胞周期、Fanconi贫血通路及DNA复制信号通路。基因探针富集分析(Gene Set Enrichment Analysis,GSEA)显示,蒿过敏AR患者在炎性反应、肿瘤坏死因子/核因子κB(TNF-α/NF-κB)信号通路、IL-2/STAT5信号通路显著上调,可能促进炎性反应;HDM过敏AR患者在G2M、E2F、MYC显著下调,可能抑制细胞增殖。蛋白质相互作用网络显示,TNF与CDK1分别为蒿、HDM过敏AR患者蛋白质相互作用网络的中心,是有最多相互作用的蛋白。结论:季节性AR与常年性AR可能通过影响鼻黏膜上皮细胞的不同生物学过程及信号通路,从而导致AR的发生发展存在差异。

Objective Transcriptome sequencing and bioinformatics analysis were performed on the gene expression of nasal epithelial cells in patients with seasonal allergic rhinitis(AR)and perennial AR,so as to obtain the differences in the gene expression of nasal epithelial cells between seasonal AR and perennial AR.Methods The human nasal epithelial cell line(HNEpC)was cultured in vitro,treated with 100μg/ml mugwort or house dust mite(HDM)extracts for 24 hours.Total cell RNA was extracted,and quantitative real-time polymerase chain reaction(qPCR)was used to detect the expression of cytokines,including IL-6,IL-8,IL-33 and thymic stromal lymphopoietin(TSLP).From November 2019 to November 2020,3 seasonal AR patients,3 perennial AR patients,and 3 healthy controls who attended the Department of Otolaryngology Head and Neck Surgery,China-Japan Union Hospital of Jilin University were analyzed.The patients′primary nasal epithelial cells were cultured in vitro,treated with corresponding allergens for 24 hours.Total RNA was extracted for transcriptome sequencing,and the sequencing results were analyzed by bioinformatics.Results The qPCR results showed that the cytokines IL-6,IL-8,IL-33 and TSLP of HNEpC treated with mugworts extracts and HDM extracts had the same trend of change.After the nasal epithelial cells from patients with seasonal AR and perennial AR were treated with corresponding allergens,there were differences in biological processes and signal pathways between those and control.Gene ontology(GO)enrichment analysis showed that the differentially expressed genes(DEG)in AR patients allergic to mugwort were mainly enriched in the oxidation-reduction process,the negative regulation of apoptosis process,and the cell adhesion;the DEG in AR patients allergic to HDM were mainly enriched in cell adhesion,the negative regulation of cell proliferation and the response to drug.Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway showed that the DEG of AR patients allergic to mugwort were significantly enriched in arachidonic acid metabolism,p53 signaling pathway and transforming growth factorβ(TGF-β)signaling pathway,while the DEG of AR patients allergic to HDM were mainly enriched in cells cycle,Fanconi anemia pathway and DNA replication.Gene Set Enrichment Analysis(GSEA)showed that the inflammatory response,TNF-α/NF-κB signaling pathway and IL-2/STAT5 signaling pathway were significantly up-regulated in AR patients allergic to mugwort,indicating the promotion of inflammatory response;and AR patients allergic to HDM had significant down-regulation of G2M,E2F,and MYC,indicating the inhibition of cell proliferation.The protein-protein interaction network showed that TNF and CDK1 were the most interacting proteins in mugwort and HDM allergic AR patients,respectively.Conclusion Seasonal AR and perennial AR may affect the different biological processes and signal pathways of nasal epithelial cells,leading to differences in the occurrence and development of AR.