溃疡性结肠炎发病机制的单细胞转录组生物信息学研究

doi:10.3969/j.issn.1006-5725.2024.15.006

摘要/Abstract

摘要：

目的利用单细胞转录组生物信息学方法挖掘溃疡性结肠炎（UC）发病的潜在机制。方法从高通量基因表达数据库中下载UC样本的单细胞转录组数据集GSE125527作为研究对象，利用R语言的Seurat数据包对数据进行过滤和标准化，将具有相似特征基因的细胞进行聚类和注释，筛选与UC发病相关的细胞亚群，提取差异表达基因（DEGs），对DEGs进行富集分析，挖掘UC发病的潜在信号通路，对差异最大的细胞亚群进行高维加权基因共表达网络分析（hdWCNA），筛选与UC相关的基因模块；下载GSE36807、GSE42911、GSE65114和GSE6731数据集，筛选DEGs，结合单细胞DEGs和与UC相关的基因模块，筛选UC发病的核心基因，绘制受试者工作特征曲线（ROC）评价核心基因预测UC发病的价值。复制UC模型大鼠验证UC核心基因的表达。结果过滤获得UC样本细胞18 642个，正常结肠组织样本细胞4 298个，共聚类成10个亚群，UC样本中B细胞占比高。筛选获得单核细胞相关的差异表达上调基因81个，差异表达下调基因21个。DEGs显著富集于肿瘤坏死因子（TNF）?α、核转录因子（NF）?κB、白细胞介素（IL）?2和信号传导与转录活化因子（STAT）5，以及干扰素（IFN）?α信号通路上。hdWGCNA筛选出2个UC相关基因模块，经筛选并验证，LAPTM5为UC的核心基因。与对照组大鼠相比，UC模型大鼠结肠组织中LAPTM5表达水平显著升高。结论 UC与正常结肠组织样本细胞分布存在较大差异，B细胞与UC发病密切相关，是预防和治疗UC的潜在靶细胞。LAPTM5是UC发病的潜在核心基因。

关键词: 溃疡性结肠炎, 单细胞转录组, 生物信息学

Abstract:

Objective To explore the underlying mechanisms of ulcerative colitis （UC） pathogenesis using single-cell transcriptomic bioinformatics approaches. Methods Single-cell transcriptome dataset GSE125527， consisting of UC samples， was downloaded from a high-throughput Gene Expression Omnibus for this study. Data filtering and normalization were conducted using the Seurat package in R. Cells with similar gene expression profiles were clustered and annotated. Cell subgroups associated with UC pathogenesis were identified， and differentially expressed genes （DEGs） were extracted. Enrichment analysis of these DEGs was performed to uncover potential signaling pathways involved in UC. High-dimensional weighted gene co-expression network analysis （hdWGCNA） was applied to the cell subgroup with the greatest differences to select UC-related gene modules. Additional datasets， GSE36807， GSE42911， GSE65114， and GSE6731， were downloaded for DEG screening. By integrating single-cell DEGs with UC-related gene modules， core genes involved in UC pathogenesis were identified. Receiver operating characteristic （ROC） curves were plotted to evaluate the predictive value of these core genes for UC pathogenesis. The expression of core genes was validated in a UC rat model. Results After filtering， 18 642 cells from UC samples and 4 298 cells from normal colonic tissue were obtained and clustered into ten subgroups， with a high proportion of B cells in UC samples. Eighty-one upregulated and twenty-one downregulated DEGs related to mononuclear cells were identified. Significant enrichment of DEGs was observed in pathways related to tumor necrosis factor-alpha， nuclear factor Kappa B， interleukin-2， signal transducer and activator of transcription 5， and interferon-alpha. Two UC-related gene modules were identified through hdWGCNA. After screening and validation， LAPTM5 was confirmed as a core gene in UC pathogenesis. Compared to control rats， LAPTM5 expression was significantly higher in the colonic tissues of UC model rats. Conclusions There are significant differences in cell distribution between UC and normal colonic tissues， with B cells closely related to UC pathogenesis， serving as potential targets for prevention and treatment. LAPTM5 is identified as a potential core gene in UC pathogenesis.

Key words: ulcerative colitis, single-cell transcriptome, bioinformatics

中图分类号:

R656.9

庞雪,郭建壮. 溃疡性结肠炎发病机制的单细胞转录组生物信息学研究[J]. 实用医学杂志, 2024, 40(15): 2075-2083.

Xue PANG,Jianzhuang. GUO. Single-cell transcriptomic bioinformatics study of the pathogenesis of ulcerative colitis[J]. The Journal of Practical Medicine, 2024, 40(15): 2075-2083.

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参考文献 27

1	SEGAL J P， LEBLANC J F， HART A L. Ulcerative colitis： an update［J］. Clin Med （Lond）， 2021，21（2）：135-139. doi:10.7861/clinmed.2021-0080 doi: 10.7861/clinmed.2021-0080
2	PASVOL T J， HORSFALL L， BLOOM S， et al. Incidence and prevalence of inflammatory bowel disease in UK primary care： a population-based cohort study［J］. BMJ Open， 2020，10（7）：e036584. doi:10.1136/bmjopen-2019-036584 doi: 10.1136/bmjopen-2019-036584
3	QIAO Y， RAN Z. Potential influential factors on incidence and prevalence of inflammatory bowel disease in mainland China［J］. JGH Open， 2019，4（1）：11-15. doi:10.1002/jgh3.12238 doi: 10.1002/jgh3.12238
4	KOBAYASHI T， SIEGMUND B， LE BERRE C， et al. Ulcerative colitis［J］. Nat Rev Dis Primers， 2020，6（1）：74. doi:10.1038/s41572-020-0205-x doi: 10.1038/s41572-020-0205-x
5	YU Y R， RODRIGUEZ J R. Clinical presentation of Crohn's， ulcerative colitis， and indeterminate colitis： Symptoms， extraintestinal manifestations， and disease phenotypes［J］. Semin Pediatr Surg， 2017，26（6）：349-355. doi:10.1053/j.sempedsurg.2017.10.003 doi: 10.1053/j.sempedsurg.2017.10.003
6	UNGARO R， COLOMBEL J F， LISSOOS T， et al. A Treat-to-Target Update in Ulcerative Colitis： A Systematic Review［J］. Am J Gastroenterol， 2019，114（6）：874-883. doi:10.14309/ajg.0000000000000183 doi: 10.14309/ajg.0000000000000183
7	BOLAND B S， HE Z， TSAI M S， et al. Heterogeneity and clonal relationships of adaptive immune cells in ulcerative colitis revealed by single-cell analyses［J］. Sci Immunol， 2020，5（50）：eabb4432. doi:10.1126/sciimmunol.abb4432 doi: 10.1126/sciimmunol.abb4432
8	SATIJA R， FARRELL J A， GENNERT D， et al. Spatial reconstruction of single-cell gene expression data［J］. Nat Biotechnol， 2015，33（5）：495-502. doi:10.1038/nbt.3192 doi: 10.1038/nbt.3192
9	LIBERZON A， BIRGER C， THORVALDSDÓTTIR H， et al. The Molecular Signatures Database （MSigDB） hallmark gene set collection［J］. Cell Syst， 2015，1（6）：417-425. doi:10.1016/j.cels.2015.12.004 doi: 10.1016/j.cels.2015.12.004
10	张士伟，程慎令，邢启峰. 茯苓酸通过抑制cGAS-STING信号通路减轻溃疡性结肠炎大鼠结肠上皮细胞损伤［J］. 免疫学杂志， 2023， 39（8）： 672-680.
11	ALI A， TAN H， KAIKO G E. Role of the Intestinal Epithelium and Its Interaction With the Microbiota in Food Allergy［J］. Front Immunol， 2020，11：604054. doi:10.3389/fimmu.2020.604054 doi: 10.3389/fimmu.2020.604054
12	TSANG D K L， WANG R J， DE SA O， et al. A single cell survey of the microbial impacts on the mouse small intestinal epithelium［J］. Gut Microbes， 2022，14（1）：2108281. doi:10.1080/19490976.2022.2108281 doi: 10.1080/19490976.2022.2108281
13	SAYOC-BECERRA A， KRISHNAN M， FAN S， et al. The JAK-Inhibitor Tofacitinib Rescues Human Intestinal Epithelial Cells and Colonoids from Cytokine-Induced Barrier Dysfunction［J］. Inflamm Bowel Dis， 2020，26（3）：407-422. doi:10.1093/ibd/izz266 doi: 10.1093/ibd/izz266
14	EASTAFF-LEUNG N， MABARRACK N， BARBOUR A， et al. Foxp3+ regulatory T cells， Th17 effector cells， and cytokine environment in inflammatory bowel disease［J］. J Clin Immunol， 2010，30（1）：80-89. doi:10.1007/s10875-009-9345-1 doi: 10.1007/s10875-009-9345-1
15	UZZAN M， MARTIN J C， MESIN L， et al. Ulcerative colitis is characterized by a plasmablast-skewed humoral response associated with disease activity［J］. Nat Med， 2022，28（4）：766-779. doi:10.1038/s41591-022-01680-y doi: 10.1038/s41591-022-01680-y
16	王小天，王月，彪雅宁，等. 燮理汤对溃疡性结肠炎小鼠TLR4/NF-κB/HIF-1α通路的影响［J］. 中国实验方剂学杂志，2023，29（8）：142-149.
17	高飞，徐琪，林怡，等. 柴胡皂苷A对溃疡性结肠炎大鼠IKK/IKB/NF-κB信号通路及肠上皮细胞凋亡的影响［J］. 中国老年学杂志，2022，42（17）：4289-4294. doi:10.3969/j.issn.1005-9202.2022.17.041 doi: 10.3969/j.issn.1005-9202.2022.17.041
18	DONG G， YANG Y， ZHANG H， et al. Protein Kinase CK2 Maintains Reciprocal Balance Between Th17 and Treg Cells in the Pathogenesis of UC［J］. Inflamm Bowel Dis， 2022，28（6）：830-842. doi:10.1093/ibd/izab312 doi: 10.1093/ibd/izab312
19	WANG M， HUANG X， KANG Z， et al. Mechanism of Sishen-Pill-Regulated Special Memory T and mTfh Cell via Involving JAK/STAT5 Pathway in Colitis Mice［J］. Evid Based Complement Alternat Med， 2022，2022：6446674. doi:10.1155/2022/6446674 doi: 10.1155/2022/6446674
20	刘滨，刘雅清，宋红新，等. 黄芩汤对溃疡性结肠炎小鼠Th17/Treg、Th1/Th2细胞平衡的影响［J］. 中国实验方剂学杂志，2022，28（22）：7-15.
21	LI X， LIU X， ZHANG Y， et al. Protective effect of Gloeostereum incarnatum on ulcerative colitis via modulation of Nrf2/NF-κB signaling in C57BL/6 mice［J］. Mol Med Rep， 2020，22（4）：3418-3428.
22	WANG H， ZHOU Q， XIE D F， et al. LAPTM4B-mediated hepatocellular carcinoma stem cell proliferation and MDSC migration： implications for HCC progression and sensitivity to PD-L1 monoclonal antibody therapy［J］. Cell Death Dis，2024，15（2）：165. doi:10.1038/s41419-024-06542-8 doi: 10.1038/s41419-024-06542-8
23	DING Y， JIANG Y， ZENG H， et al. Identification of a robust biomarker LAPTM4A for glioma based on comprehensive computational biology and experimental verification［J］. Aging （Albany NY）， 2024，16. doi： 10.18632/aging.205736 . doi: 10.18632/aging.205736
24	PAK Y， GLOWACKA W K， BRUCE M C， et al. Transport of LAPTM5 to lysosomes requires association with the ubiquitin ligase Nedd4， but not LAPTM5 ubiquitination［J］. J Cell Biol， 2006，175（4）：631-645. doi:10.1083/jcb.200603001 doi: 10.1083/jcb.200603001
25	KAWANO Y， OUCHIDA R， WANG J Y， et al. A novel mechanism for the autonomous termination of pre-B cell receptor expression via induction of lysosome-associated protein transmembrane 5［J］. Mol Cell Biol， 2012，32（21）：4462-4471. doi:10.1128/mcb.00531-12 doi: 10.1128/mcb.00531-12
26	ZOUALI M. Transcriptional and metabolic pre-B cell receptor-mediated checkpoints： implications for autoimmune diseases［J］. Mol Immunol， 2014，62（2）：315-320. doi:10.1016/j.molimm.2014.01.009 doi: 10.1016/j.molimm.2014.01.009
27	SONG Z， WANG X， HE L， et al. Suppression of lysosomal-associated protein transmembrane 5 ameliorates cardiac function and inflammatory response by inhibiting the nuclear factor-kappa B （NF-κB） pathway after myocardial infarction in mice［J］. Exp Anim， 2022，71（4）：415-425. doi:10.1538/expanim.22-0008 doi: 10.1538/expanim.22-0008

数据包	函数
Seurat	Normalizedata
	LogNormalize
	ScaleData
	RunPCA
	FindAllMarkers
SingleR	HumanPrimaryCellAtlasData（）
SingleR	scores within cells
limma
clusterProfiler
org.Hs.eg.db
hdWGCNA

变量	Control组（n = 8）	UC组（n = 7）
年龄（x ± s，岁）	48.1 ± 17.8	43.3 ± 13.6
男性（例）	6	3
体质量指数（x ± s，kg/m²）	26.9 ± 3.4	26.5 ± 4.3
病程（x ± s，年）		8.3 ± 6.8

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