基于RNA-seq不明原因复发性流产绒毛组织mRNA基因差异性表达研究

doi:10.12280/gjfckx.20230584

国际妇产科学杂志 ›› 2024, Vol. 51 ›› Issue (3): 322-328.doi: 10.12280/gjfckx.20230584

• 产科生理及产科疾病：论著 • 上一篇下一篇

基于RNA-seq不明原因复发性流产绒毛组织mRNA基因差异性表达研究

杨丽, 杨静, 叶尔登切切克, 韩锐, 腊晓琳()

830054 乌鲁木齐，新疆医科大学第一附属医院生殖医学中心（杨丽，叶尔登切切克，韩锐，腊晓琳），生殖疾病与出生缺陷综合防治研究中心（杨丽，腊晓琳），新疆生殖免疫临床医学研究中心（杨丽，腊晓琳），省部共建中亚高发病成因与防治国家重点实验室（腊晓琳）;新疆医科大学第五附属医院妇科（杨静）

收稿日期:2023-07-29 出版日期:2024-06-15 发布日期:2024-06-25
通讯作者: 腊晓琳 E-mail:909232905@qq.com
基金资助:
新疆维吾尔自治区自然科学基金(2022D01C467);省部共建中亚高发病成因与防治国家重点实验室开放课题(SKL-HIDCA-2020-JZ11)

RNA Sequencing of Villus Tissue Reveals Differentially Expressed Genes in Unexplained Recurrent Spontaneous Abortion

YANG Li, YANG Jing, Yeerdengqieqieke , HAN Rui, LA Xiao-lin()

Center for Reproductive Medicine (YANG Li, Yeerdengqieqieke, HAN Rui, LA Xiao-lin), Research Center for Integrated Prevention and Treatment of Reproductive Diseases and Birth Defects (YANG Li, LA Xiao-lin), Xinjiang Clinical Research Center for Reproductive Immunology (YANG Li, LA Xiao-lin), State Key Laboratory of Pathogenesis, Prevention, and Treatment of High Incidence Diseases in Central Asia (LA Xiao-lin), The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China; Department of Gynecology, The Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China (YANG Jing)

Received:2023-07-29 Published:2024-06-15 Online:2024-06-25
Contact: LA Xiao-lin E-mail:909232905@qq.com

摘要/Abstract

摘要：

目的：构建不明原因复发性流产（unexplained recurrent spontaneous abortion，URSA）孕妇绒毛组织的mRNA差异性表达谱并进行功能分析，为阐明URSA的发生发展机制提供依据。方法：收集2022年3月于新疆医科大学第一附属医院妇科门诊收治的4例URSA（试验组）和4例选择性终止妊娠孕妇（对照组）的绒毛组织样本。应用转录组测序技术（RNA-seq）筛选2组差异表达基因（differentially expressed genes，DEGs），并对其进行GO功能注释、KEGG通路分析及蛋白质-蛋白质相互作用（PPI）网络分析，进一步研究差异表达mRNA的功能，并筛选出URSA的关键基因。结果：RNA-seq分析共筛选出3 350 个DEGs，其中上调基因2 259个，下调基因1 091个。通过PPI网络分析，筛选出与URSA有关的前10个关键基因：TYROBP、CD74、GH1、GH2、STAT5A、HLA-DRA、STAT5B、HLA-DRB1、CISH和HLA-A。通过GO和KEGG生物学功能分析，发现差异性表达的mRNA参与到多种白细胞分化相关免疫反应通路和同种异体免疫应答等生物学过程。结论：利用RNA-seq获得的URSA的mRNA差异性表达谱，筛选出可能与URSA有关的关键基因及生物学通路，为进一步研究URSA的发病机制和诊疗靶点提供了理论依据。

关键词: 流产，习惯性, 绒毛组织, RNA，信使, 转录组测序, 免疫调节

Abstract:

Objective: To discover the differentially expressed genes (DEGs) in villus tissue of patients with unexplained recurrent spontaneous abortion (URSA) and to analyze their functions, aiming to elucidate the mechanisms behind the onset and progression of URSA. Methods: Villus tissue samples were collected from four URSA patients and four elective termination of pregnancy(ETP) at Department of Gynecology Outpatient of the First Affiliated Hospital of Xinjiang Medical University in March 2022. RNA-seq was employed to screen for DEGs between the two groups, followed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to identify the biological functions and pathways of the identified DEGs. A protein-protein interaction (PPI) network was constructed using the STRING database, and screen out the key genes of URSA. Results: RNA-seq analysis identified a total of 3 350 DEGs, of which 2 259 were upregulated genes and 1 091 were downregulated genes in the URSA group. Through PPI network analysis, the top 10 key genes related to URSA were identified, including TYROBP, CD74, GH1, GH2, STAT5A, HLA-DRA, STAT5B, HLA-DRB1, CISH, and HLA-A. Through GO and KEGG biological functional analysis, it was found that differentially expressed mRNA is involved in various immune response pathways related to white blood cell differentiation and allogeneic immune responses. Conclusions: This study utilized transcriptome sequencing technology to obtain the mRNA differential expression profile of URSA, and screened for key genes and biological pathways that may be related to URSA. Our data provide a theoretical basis for elucidating the pathogenesis and diagnostic and therapeutic targests of URSA.

Key words: Abortion, habitual, Villus tissue, RNA, messenger, RNA-Seq, Immunomodulation

杨丽, 杨静, 叶尔登切切克, 韩锐, 腊晓琳. 基于RNA-seq不明原因复发性流产绒毛组织mRNA基因差异性表达研究[J]. 国际妇产科学杂志, 2024, 51(3): 322-328.

YANG Li, YANG Jing, Yeerdengqieqieke , HAN Rui, LA Xiao-lin. RNA Sequencing of Villus Tissue Reveals Differentially Expressed Genes in Unexplained Recurrent Spontaneous Abortion[J]. Journal of International Obstetrics and Gynecology, 2024, 51(3): 322-328.

图/表 7

表1 2组患者的临床特征比较（$\bar{x}±s$）

组别	n	年龄（岁）	孕前BMI （kg/m²）	月经周期（d）	孕龄（d）
试验组	4	30.5±3.4	21.0±1.3	30.3±2.1	54.8±1.7
对照组	4	30.0±3.7	21.0±1.0	29.3±1.5	52.5±1.7
t		0.20	-0.03	0.46	1.85
P		0.85	0.98	0.78	0.11

图1 2组样本关联性注：A 2组样本线性相关程度；B 样本聚类分析图，PT为试验组样本，RL为对照组样本。

图2 URSA和ETP绒毛组织中mRNA的表达谱注：A DEGs火山图，图中横坐标表示表达量FC（以2为底取log值），纵坐标表示表达量变化差异的统计学显著性（以10为底取log值）；B 不同样本差异表达基因分级聚类热图。

图3 DEGs的GO功能富集分析图注：A 上调DEGs中前10个显著富集的生物学过程的GO分析；B.下调DEGs中前10个显著富集的生物学过程的GO分析。

图4 DEGs的KEGG信号通路富集分析图注：A 上调DEGs中前10个显著富集的KEGG通路富集分析：B 下调DEGs中前10个显著富集的KEGG通路富集分析。

图5 URSA和ETP绒毛组织中差异表达mRNA的PPI网络分析注：A PPI网络同心圆；B 具有高连接度的前10个关键基因的最短路径网络图。

表2 前10位关键基因的表达量

基因名称	log₂(FC)	URSA_2	URSA_3	URSA_4	URSA_5	ETP_1	ETP_3	ETP_4	ETP_5
TYROBP	2.920 470	17.19	39.88	28.09	20.91	0.75	0.20	1.27	13.30
CD74	5.277 949	12.56	19.26	20.93	4.10	0.21	0.09	0.59	0.70
GH1	3.259 979	35.81	43.56	27.80	92.55	8.50	3.62	4.54	7.11
GH2	2.337 459	702.77	562.54	921.77	1 211.00	333.29	89.32	139.94	186.22
STAT5A	2.498 606	5.22	4.99	6.44	7.24	1.05	0.81	1.23	1.73
HLA-DRA	6.050 772	51.18	65.28	82.84	16.28	0.21	0.12	2.25	1.02
STAT5B	1.442 519	19.52	20.66	21.93	29.65	11.81	6.84	8.98	10.40
HLA-DRB1	3.704 165	11.65	11.18	17.43	4.68	1.26	0.06	0.90	1.47
CISH	1.900 941	5.17	6.94	6.67	9.41	2.83	1.46	1.50	2.64
HLA-A	2.658 422	42.37	62.90	53.30	16.44	3.34	10.97	4.26	12.22

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基于RNA-seq不明原因复发性流产绒毛组织mRNA基因差异性表达研究

RNA Sequencing of Villus Tissue Reveals Differentially Expressed Genes in Unexplained Recurrent Spontaneous Abortion

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