复发性流产合并胰岛素抵抗蜕膜组织中免疫细胞关键基因鉴定及机制分析

Identification and mechanism analysis of key genes of immune cells in decidua of recurrent abortion complicated with insulin resistance

目的本研究旨在通过转录组测序分析复发性流产合并胰岛素抵抗患者的蜕膜组织与正常对照的差异基因的表达,揭示潜在的关键通路与主要基因及免疫细胞的关联。方法2023年12月至2024年5月于中山大学孙逸仙纪念医院计划生育门诊收集4例复发性流产合并胰岛素抵抗孕妇(实验组)和4例人工流产孕妇(对照组)的蜕膜组织样本。应用高通量测序技术构建测序文库,筛选差异表达基因,进行基因本体(GO)、基因组百科全书分析(KEGG)、基因集富集分析(GSEA)及蛋白质互作网络(PPI),经Cytoscape筛选出主要基因后qRT-PCR进行实验验证,最后联合人类蛋白质图谱数据库(HPA)探究主要基因与免疫细胞的关系。结果差异基因表达分析共筛选1204个差异表达基因,其中上调基因486个,下调基因718个。GO分析显示差异表达基因生物过程富集于T细胞分化与活化及自然杀伤细胞(NK细胞)趋化性,细胞组分位于含细胞外基质和质膜内侧,分子功能作用于受体与配体活性。KEGG分析显示,差异基因主要富集于PI3K-Akt信号通路和MAPK信号通路。GSEA富集于IL-2炎症通路及IL-17信号通路。PPI网络联合Cytoscape筛选出4个主要基因为EGFR、FN1、IGF1和IGFBP3。经qRT-PCR验证,EGFR、FN1和IGFBP3在实验组患者蜕膜组织中高表达(P<0.05),IGF1则呈现低表达(P<0.05),经HPA数据库检索发现EGFR、FN1和IGFBP3与NK细胞表达增强相关,IGF1与Th17细胞表达增强相关。结论在实验组患者的蜕膜组织中,其转录组基因表达与对照组患者的蜕膜组织表现出显著的差异性,EGFR、FN1、IGF1和IGFBP3可能是影响复发性流产合并胰岛素抵抗的关键基因,其可能通过PI3K-Akt信号通路和MAPK信号通路调节免疫细胞功能进而导致复发性流产。

Objective To study the differential gene expression of transcriptome sequencing of ecchymosis tissues from patients with recurrent abortion complicated with insulin resistance and normal ecchymosis tissues,and to investigate the relationship between the key pathways and the major genes and immune cells.Methods From December 2023 to May 2024,meconium tissue samples were taken from four pregnant women(the experimental group)who had recurrent abortion complicated with insulin resistance,and four pregnant women(the control group)who had induced abortions,at Sun Yat-Sen Memorial Hospital,Sun Yat-Sen University.After screening the main genes using Cytoscape,high-throughput sequencing technology was used to build sequencing libraries,screen differentially expressed genes,perform Gene Ontology(GO),Genome Encyclopedia Analysis(KEGG),and Protein Interaction Network(PPI),and finally,qRT-PCR for experimental validation.Lastly,the Human Protein Atlas(HPA)database was used to study the connection between the main genes and the immune cells.Results A total of 1204 differentially expressed genes,486 upregulated and 718 downregulated were investigated by differential gene expression analysis.The biological processes of differentially expressed genes were found to be enriched in T cell differentiation and activation as well as NK-cell chemotaxis,according to GO analysis.The extracellular matrix and the inside of the plasma membrane were found to be the locations of the cellular components,and the activities of receptors and ligands were found to be the molecular functions.The KEGG analysis revealed that the MAPK and PI3K-Akt signaling pathways contained a high concentration of the differentially expressed genes.The IL-17 signaling pathway and the IL-2 inflammatory pathway were shown to be enhanced in GSEA.The PPI network combined with Cytoscape screened out four major genes,namely EGFR,FN1,IGF1 and IGFBP3.The results of qRT-PCR showed that EGFR,FN1 and IGFBP3 were highly expressed in the decidua tissue of the experimental group(P<0.05),while IGF1 was low expressed(P<0.05).The HPA database search revealed that EGFR,FN1 and IGFBP3 were related to the increased expression of NK cells and IGF1 was related to the increased expression of Th17 cells.Conclusion In the decidua tissue of the experimental group,the expression of transcriptome genes is significantly different from that of the control group.EGFR,FN1,IGF1 and IGFBP3 may be the key genes affecting recurrent abortions associated with insulin resistance.They can regulate immune cell function through the PI3K-Akt signaling pathway and the MAPK signaling pathway,thereby leading to recurrent abortions.