含Tu GTP结合结构域延伸因子2(Eftud2)增强小鼠巨噬细胞功能的生物信息学分析

Bioinformatic analysis of Eftud2 enhancing mouse macrophage function

目的通过生物信息学方法分析含Tu GTP结合结构域延伸因子2(Eftud2)增强小鼠巨噬细胞免疫功能的作用机制。方法分别收集10例Eftud2髓系细胞特异性敲除(MKO)小鼠及野生型(WT)小鼠骨髓来源巨噬细胞(BMDM),经100 ng/mL脂多糖(LPS)刺激2 h。采用生物信息学方法对样本进行基因表达差异以及mRNA水平可变剪接差异的检测。分别利用RNA序列差异表达基因(DEGseq)软件包、复制转录本剪接多变量分析(rMATS)软件对两组小鼠BMDM样本中基因表达及可变剪接差异进行统计;利用京都基因与基因组百科全书(KEGG)分类与富集法统计两组小鼠BMDM样本中基因表达与可变剪接差异所影响的信号通路。结果与WT小鼠相比,MKO小鼠的BMDM经LPS刺激后白细胞介素6(IL-6)、IL-1β、肿瘤坏死因子α(TNF-α)及与免疫反应相关的基因显著下调;KEGG通路分析表明,BMDM基因表达差异及可变剪接差异主要影响信号转导及免疫系统相关代谢通路,且与磷脂酰肌醇3激酶/蛋白激酶B(PI3K-AKT)信号通路相关性较强。其中可变剪接差异还存在于泛素化及细胞内吞作用中。与WT小鼠相比,MKO小鼠BMDM可变剪接差异共232处,其中跳过外显子的可变剪接共有125处,占比最多。此外,对可变剪接差异的分析也证实了前期实验结果,即Eftud2可通过影响Toll样受体4/核因子κB(TLR4/NF-κB)信号通路中髓样分化因子88(MyD88)等关键分子的可变剪接,调控相关炎性信号通路的激活,增强巨噬细胞的功能。结论Eftud2通过影响基因的表达及可变剪接促进巨噬细胞释放炎性细胞因子,从而增强巨噬细胞免疫功能。

Objective To elucidate the mechanisms by which elongation factor Tu GTP binding domain containing 2(Eftud2)enhances the immune function of murine macrophages by bioinformatics analysis.Methods The bone marrow-derived macrophages(BMDMs)of Eftud2 myeloid cell-specific knockout(MKO)mice(n=10)and wild-type(WT)littermates(n=10)were collected and stimulated by lipopolysaccharide(LPS)(100 ng/mL)for 2 hours.Bioinformatics analysis was conducted to examine the differences in gene expression and mRNA transcription levels.The the differences in gene expression and alternative splicing of mRNA transcription in BMDMs were analyzed by DEGseq and rMATS,respectively.The signaling pathways affected were clarified by Kyoto Encyclopedia of Genes and Genomes(KEGG)classification and enrichment methods.Results Compared with WT counterparts,the expression levels of IL-6,IL-1β,TNF-α,and the genes related to immune response in MKO BMDMs were down-regulated following LPS stimulation.KEGG pathway analysis showed that the differently expressed genes in BMDMs and alternative splicing mainly affected the signal transduction and immune system-related metabolic pathways,and had a strong correlation with PI3K-AKT signaling pathway.The difference in alternative splicing also existed in ubiquitination and endocytosis.Compared with WT counterparts,there were 232 differences in alternative splicing in MKO BMDMs,among which 125 were skipping exons,accounting for the largest proportion.In addition,the analysis of alternative splicing differences also confirmed the previous experimental results,that is,Eftud2 could participate in the activation of inflammatory signaling pathways by enhancing the alternative splicing of key molecules such as MyD88 in TLR4-NF-κB signaling pathway,thereby augmenting the function of macrophages.Conclusion Eftud2 can promote the release of inflammatory cytokines in BMDMs by regulating gene expression and alternative splicing,and consequently enhance the immune function of macrophages.