广西医学

目的　基于生物信息学探讨核受体亚家族4A组成员1（NR4A1）对急性移植物抗宿主疾病（aGVHD）的调控机制。方法　（1）从GEO数据库中筛选与人类aGVHD相关的转录组学数据集，分析差异表达基因（DEGs）。针对DEGs进行基因本体（GO）功能富集分析、京都基因和基因组百科全书（KEGG）通路富集分析，并构建蛋白⁃蛋白相互作用（PPI）网络，分析NR4A1与其他蛋白的关系。（2）选择20只C57BL/6J（H⁃2b）小鼠作为受鼠，将其随机分为正常组、Syn组、Allo⁃bm组、Allo⁃sp组，每组5只。Syn组、Allo⁃bm组、Allo⁃sp组给予清髓和移植处理，正常组不给予相应处理。取移植第14天各组小鼠的结肠组织，利用实时荧光定量PCR检测NR4A1 mRNA表达水平。结果　（1）筛选出上调DEGs 1 089个，下调DEGs 944个，其中NR4A1为表达下调DEGs。GO功能富集分析结果显示DEGs主要涉及淋巴细胞分化、T淋巴细胞活化调节、炎症反应调节等生物过程，含有线粒体蛋白的复合物、溶酶体膜等细胞组分，RNA聚合酶Ⅱ特异性DNA结合转录因子结合、核转录因子κＢ（NF⁃κB）结合等分子功能。KEGG通路富集分析结果显示，DEGs富集在T淋巴细胞受体信号通路、NF⁃κB信号通路等信号通路。PPI网络分析显示，NR4A1与凋亡相关通路中的关键蛋白B淋巴细胞瘤2、蛋白激酶2等联系紧密。（2）在移植第14天，Syn组与正常组小鼠肠组织中NR4A1 mRNA表达水平差异无统计学意义（P>0.05）；与正常组相比，Allo⁃bm组和Allo⁃sp组小鼠肠组织中的NR4A1 mRNA表达水平降低（P<0.05）。结论　NR4A1可能在aGVHD的发生与发展中发挥关键作用，有望成为aGVHD的潜在分子标志物。

Objective To investigate the regulatory mechanism of nuclear receptor subfamily 4 group A member 1 (NR4A1) on acute graft versus host disease (aGVHD) based on bioinformatics. Methods (1) Transcriptome datasets related to human aGVHD were screened from the GEO database, and differentially expressed genes (DEGs) were analyzed. Gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for DEGs. A protein⁃protein interaction (PPI) network was constructed to analyze the relation between NR4A1 and other proteins. (2) Twenty C57BL/6J (H⁃2b) mice were selected as recipients and randomly divided into normal, Syn, Allo⁃bm or Allo⁃sp groups, with 5 mice in each group. The Syn, Allo⁃bm and Allo⁃sp groups were treated with myeloablative and transplantation, while the normal group was not treated. The colon tissues of mice in each group were collected on day 14 of transplantation, and the expression of NR4A1 mRNA was detected by real⁃time fluorescent quantitative PCR. Results (1) There were 1089 up⁃regulated DEGs and 944 down⁃regulated DEGs, therein NR4A1 was the down⁃regulated DEGs. The results of GO functional enrichment analysis revealed that DEGs were mainly involved in biological processes such as lymphocyte differentiation, regulation of T lymphocyte activation, and regulation of inflammatory response, in cellular components such as complexes containing mitochondrial proteins and lysosomal membranes, and in molecular functions such as RNA polymerase Ⅱ⁃specific DNA binding transcription factor binding and nuclear transcription factor κB (NF⁃κB) binding. The results of KEGG pathway enrichment analysis depicted that DEGs were enriched in T lymphocyte receptor signaling pathway, NF⁃κB signaling pathway and other signaling pathways. PPI network analysis indicated that NR4A1 was closely related to B⁃cell lymphoma 2 and protein kinase 2, which were key proteins in apoptosis⁃related pathways. (2) On the 14th day after transplantation, there was no statistically significant difference in the expression of NR4A1 mRNA between the Syn group and the normal group (P>0.05). Compared with the normal group, the Allo⁃bm group and the Allo⁃sp group exhibited decreased expression of NR4A1 mRNA in colon tissues of mice (P<0.05). Conclusion NR4A1 may play a key role in the occurrence and development of aGVHD, and may be a potential molecular marker for aGVHD.

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