基于高通量转录组测序探究儿童支气管哮喘发病的新途径

New method of genetic mechanism of children with bronchial asthma based on high-throughput transcriptome sequencing

目的探究支气管哮喘患儿的转录组变化。方法共招募9名哮喘患儿以及9名健康儿童作为研究对象,并将该18名儿童的外周血样本进行RNA测序分析。进行基因富集分析以鉴定受差异表达基因影响的途径。在另外单独招募的20例哮喘儿童及20例健康儿童中,通过定量RT-PCR(qRT-PCR),western印迹验证靶基因的变化。结果共有592个基因在实验组中差异表达。在实验组中差异表达基因主要富集于炎症相关反应,例如CSF3、IL-1b,IL-6。KEGG通路分析证实,上调的基因参与炎症途径,如NF-KB和AGE-RAGE。在氧化应激途径中通过实验验证了两个具有显著意义的基因(FRA1和PTX3)。结论本研究为哮喘患儿外周血样本的转录组测序分析提供了新的研究方向。而鉴定的差异表达基因与氧化应激的关系,能够进一步揭示诱发支气管哮喘的新途径。

Objective To evaluate the change of bronchial asthma tissue transcriptome.Methods The samples were obtained from subjects with bronchial asthma(n=9)and healthy controls(n=9).RNA-sequencing analysis was performed in these samples.Gene enrichment analysis was conducted to identify pathways affected by the differentially expressed genes.Changes of top genes were verified by quantitative RT-PCR(qRT-PCR)and Western blot.Results A total of 592 genes were differentially expressed(DEGs)in bronchial asthma.DEGs were mainly enriched in inflammatory genes,such as Colony Stimulating Factor 3(CSF3),Interleukin-1b(IL-1b),and IL-6.KEGG pathway analysis confirmed that upregulated genes were involved in inflammatory pathways,such as Tumor Necrosis Factor(TNF),Nuclear Factor-KB(NF-KB)and advanced glycation end-products-receptor advanced glycation end products(AGE-RAGE).Two genes of significant significance(FRA1 and PTX3)were experimentally validated in an oxidative stress pathway.Conclusion This study provides new directions for sequencing bronchial asthma transcriptome.The identification of differentially expressed genes and oxidative stress can further reveal the new pathways leading to bronchial asthma.