结合基因芯片与单细胞转录组鉴定反复种植失败患者子宫内膜的细胞间通讯

doi:10.12280/gjszjk.20240037

摘要/Abstract

摘要：

目的：利用生物信息学分析法挖掘胚胎反复种植失败（recurrent implantation failure，RIF）患者的相关关键基因，鉴定人子宫内膜中细胞亚群及细胞间通讯，探讨其对子宫内膜容受性的影响。方法：从高通量基因表达（Gene Expression Omnibus，GEO）数据库中下载GSE103465数据集、GSE183837数据集和GSE223672数据集为研究样本，以RIF患者及对照组女性的子宫内膜组织数据为研究对象，使用R语言及Perl软件将原始数据经过质量分析后利用limma包筛选差异基因，使用筛选的差异基因进行基因本体论（gene ontology，GO）及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genome，KEGG）分析，构建蛋白互作网络（Protein-Protein Interaction Networks，PPI网络），筛选关键基因，利用R语言相关包进行单细胞注释与分类，使用筛选的差异基因构建细胞间通讯图谱。结果：筛选出关键基因RPF2、DDX27、PWP2、CDC5L、NOP2、DCAF13、CEBPZ、FTSJ1、GNL3L和NSUN2，鉴定RIF患者子宫内膜的主要细胞类型分别为上皮细胞、自然杀伤细胞、粒单核祖细胞、内皮细胞和平滑肌细胞，差异基因富集的GRN、SPP1、PTN、MIF、MK信号通路通过受体-配体对参与细胞间通讯。结论：RIF患者子宫内膜容受性受损与RNA的修饰与加工异常，出现表观遗传修饰错误相关，还与炎症反应及代谢下降密切关联，细胞间通讯显示RIF患者子宫内膜上皮细胞骨桥蛋白-整合素介导的黏附与侵袭能力下降，可能是导致胚胎种植失败的原因。

关键词: 胚胎植入, 胚胎移植, 计算生物学, 基因, 细胞间通讯, 子宫内膜容受性

Abstract:

Objective: To explore the key genes related to patients with recurrent implantation failure (RIF) using bioinformatics analysis, to identify cell subpopulations and cell-cell communications in human endometrium, and to discuss the impact on endometrial receptivity. Methods: The GSE103465 dataset, GSE183837 dataset and GSE223672 dataset were downloaded from the Gene Expression Omnibus (GEO) database as research samples. Endometrial tissue data from RIF patients and control females were utilized. R language and Perl scripts were employed to conduct the quality analysis on the raw data, and the limma package was utilized for differential gene selection. The selected differentially expressed genes were subjected to gene ontology (GO) and Kyoto encyclopedia of genes and genome (KEGG) analysis, Protein-Protein Interaction Networks (PPI) construction and core gene selection. Using R language-related packages, single-cell annotation and classification were performed, followed by the construction of an intercellular communication map. Results: The identified key genes included RPF2, DDX27, PWP2, CDC5L, NOP2, DCAF13, CEBPZ, FTSJ1, GNL3L, and NSUN2. The main cell types in the endometrium of patients with RIF were identified as epithelial cells, NK cells, granulocyte-monocyte progenitors, endothelial cells, and smooth muscle cells. The enriched pathways involving GRN, SPP1, PTN, MIF, MK signaling pathways participate in the intercellular communication through receptor-ligand pairs. Conclusions: The impaired endometrial receptivity in RIF patients is associated with aberrant RNA modification and processing and epigenetic modification errors, as well as inflammatory response and decreased metabolism. Intercellular communication reveals that endometrial epithelial cells in RIF patients have a decreased capacity for osteoblastin-integrin-mediated adhesion and invasion, which may be the cause of embryo implantation failure.

Key words: Embryo implantation, Embryo transfer, Computational biology, Genes, Cell communication, Endometrial receptivity

吴春蕾, 赵晓丽, 邱韵桓, 王宝娟, 董融, 李凯茜, 夏天. 结合基因芯片与单细胞转录组鉴定反复种植失败患者子宫内膜的细胞间通讯[J]. 国际生殖健康/计划生育杂志, 2024, 43(4): 265-273.

WU Chun-lei, ZHAO Xiao-li, QIU Yun-huan, WANG Bao-juan, DONG Rong, LI Kai-xi, XIA Tian. Integration of Gene Expression Microarrays and Single-Cell Transcriptomics to Identify Intercellular Communication in the Endometrium of Recurrent Implantation Failure Patients[J]. Journal of International Reproductive Health/Family Planning, 2024, 43(4): 265-273.

图/表 10

表1 基因测序数据基本信息

数据集ID	平台	组织来源	RIF组/ 对照组（例）	取材时间	数据类型
GSE103465	GPL16043	子宫内膜	3/3	排卵后5 d	基因芯片
GSE183837	GPL24676	子宫内膜	6/3	排卵后5 d	单细胞转录组
GSE223672	GPL24676	子宫内膜	3/0	排卵后5 d	单细胞转录组

表2 RIF子宫内膜上调和下调的前10个差异表达基因

上调基因		下调基因
Gene Symbol	logFC	Gene Symbol	logFC
SNORD89	4.20	PAEP	-4.95
HOXA6	2.71	C3	-3.55
KIAA1731	2.55	ESYT1	-2.49
LOC728741	2.54	NNMT	-2.49
SNORA54	2.36	DEPDC6	-2.49
POMZP3	2.35	NFKBIZ	-2.47
HOXD4	2.33	FHDC1	-2.40
BEND5	2.12	RP2	-2.20
POSTN	2.07	ATP13A3	-2.18
CIR1	1.80	PYGL	-2.17

图1 GO功能富集分析图

图2 KEGG分析图

图3 PPI网络图

图4 PPI关键基因网络图注：CDC5L不和其他9个互作，但与其他未上榜基因互作。

图5 细胞注释图注：t-SNE用于高维数据的可视化，通过将高维数据点转换为二维或三维空间中的点，使得原始数据点之间的相似性在新空间中以距离的形式得以保留。在细胞注释图中，“tSNE_1”和“tSNE_2”指的是转换后的二维空间坐标轴。

表3 细胞亚群的特征基因

上皮细胞		自然杀伤细胞		GMP		内皮细胞		平滑肌细胞
表达情况	基因	表达情况	基因	表达情况	基因	表达情况	基因	表达情况	基因
下调	MT-CO2	上调	NKG7	上调	CFD	上调	IGFBP7	下调	S100A10
下调	MALAT1	上调	CCL4	上调	IGFBP2	上调	A2M	下调	ATP5F1E

图6 细胞通讯的受体-配体图注：横坐标为细胞间通讯关系，纵坐标表示参与通讯的受体-配体对。圆圈大小表示P值大小，圆圈越大，差异越显著。颜色表示通讯强度，颜色越红表示通讯强度越强。

图7 通路水平参与受体-配体对的细胞通讯图注：a GRN通路，b SPP1通路，c PTN通路，d MIF通路，e MK通路。通讯网络图中节点代表细胞类型，节点越大，就说明这个细胞类型里面细胞的数目越多，如果两个细胞之间有连线，说明这两个细胞之间存在通讯，连线的颜色代表配体细胞的颜色，连线的另一端作为受体细胞负责接收信号。网格图中横坐标代表细胞类型，纵坐标代表作用的类型。从上到下依次为发送器（sender，向其他的细胞发送信号），接收器（receiver，接收信号），桥梁（mediator），信息流（influencer），颜色越深表明功能越强。小提琴图中横坐标代表细胞类型，纵坐标代表基因名称，小提琴宽度说明基因的高表达与低表达。

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