胃印戒细胞癌的H3K27me3沉默子重塑特征及转录调控功能  

Remodeling characteristics of H3K27me3-marked silencers in gastric signet-ring cell carcinoma and its transcriptional regulatory function

作　　者：杜艾贝 任原锋 储召乐 刘碧颖 李先锋 向俊宇 陈东风[2] 王涛[2] 王斌[2] 郭海英 张璇 李玉红 DU Aibei;REN Yuanfeng;CHU Zhaole;LIU Biying;LI Xianfeng;XIANG Junyu;CHEN Dongfeng;WANG Tao;WANG Bin;GUO Haiying;ZHANG Xuan;LI Yuhong(Department of Cell Biology,College of Basic Medical Sciences,Army Medical University(Third Military Medical University),Chongqing;Department of Gastroenterology,Chongqing Key Laboratory of Precise Prevention and Treatment of Digestive Malignancies,Army Medical Center of PLA/Daping Hospital of Third Military Medical University,Chongqing;Department of Oncology,Chongqing Hospital of Traditional Chinese Medicine,Chongqing,China)

机构地区：[1]陆军军医大学基础医学院细胞生物学教研室,重庆 [2]陆军特色医学中心(第三军医大学大坪医院)消化内科,消化系统肿瘤精准防治重庆市重点实验室,重庆 [3]重庆市中医院肿瘤科,重庆

出　　处：《陆军军医大学学报》2025年第5期417-425,共9页Journal of Army Medical University

基　　金：国家自然科学基金优秀青年科学项目(81822032);重庆市自然科学基金面上项目(CSTB2022NSCQ-MSX0162)。

摘　　要：目的通过表观遗传组学技术,解析胃印戒细胞癌(signet-ring cell carcinoma of the stomach,SRCC)的H3K27me3沉默子的全基因组特征及其对基因转录的调控作用,以阐明SRCC恶性进展的表观调控机制。方法采集2021年1月至2023年12月陆军特色医学中心消化内科35例胃镜样本(正常胃窦/胃体15例,SRCC 20例)。采用以下多组学分析:染色质可及性测序(assay for transposase-accessible chromatin with high-throughput sequencing,ATAC-seq)检测染色质开放区域;染色质靶向捕获测序(cleavage under targets and tagmentation,CUT&Tag)捕获H3K27me3沉默子区域;转录组测序(transcriptome sequencing,RNA-seq)分析转录组。测序于Illumina NovaSeq 6000平台完成(符合深度标准)。通过DESeq2筛选H3K27me3相关差异基因(|Log_(2)FC|>1,FDR<0.05),并通过基因本体论(Gene Oncology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析进行功能富集,Homer鉴定转录因子基序,Cytoscape构建调控网络,并使用免疫组化进行验证,探讨其调控机制。结果SRCC沉默子主要分布于基因组远端基因间区(占37.06%)。与正常组织相比,SRCC中H3K27me3沉默子信号显著降低(95%CI:1.34~2.30,P=0.007),共鉴定出6257个丢失位点(FDR<0.01)。CUT&Tag和RNA-seq整合分析显示,380个因沉默子丢失而上调的基因显著富集于免疫相关通路,如T细胞受体信号通路(OR=4.2,95%CI:2.8~6.3,P=0.002)。免疫组化验证表明,转录因子EHF表达显著升高(P<0.05)。结论SRCC中存在H3K27me3沉默子重塑现象,转录因子EHF可能在SRCC恶性进展中发挥关键作用。Objective To draw the genome-wide distribution and remodeling characteristics of H3K27me3 silencers in signet-ring cell carcinoma of the stomach(SRCC)through epigenetic sequencing technology,and to investigate their roles in transcriptional regulation in order to elucidate the regulatory mechanism of SRCC malignant progression.Methods The study was conducted on 35 gastric samples obtained by gastroendoscopic biopsy(15 normal and 20 SRCC tissues)from Department of Gastroenterology of Army Medical Center of PLA between January 2021 and December 2023.Multi-omics analyses,including assay for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq),cleavage under targets and tagmentation(CUT&Tag)and transcriptome sequencing(RNA-seq),were performed to identify chromatin accessibility,H3K27me3 silencer regions,and transcriptional changes,with aid of Illumina NovaSeq 6000.H3K27me3 related differentially expressed genes(|Log_(2)FC|>1,FDR<0.05)were screened using DESeq2.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were employed to analyze the enrichment function,and Homer was employed to identify transcription factor motifs.A regulatory network was constructed using Cytoscape,and then validated using immunohistochemistry to explore its regulatory mechanism.Results H3K27me3 silencers were primarily located in distal intergenic regions(37.06%)in SRCC.Compared with the normal tissues,SRCC showed a significant reduction in H3K27me3 silencer signals(95%CI:1.34~2.30,P=0.007)with 6257 lost sites(FDR<0.01).Integrating CUT&Tag and RNA-seq revealed 380 up-regulated immune-related genes,particularly in T cell receptor signaling(OR=4.2,95%CI:2.8~6.3,P=0.002).Immunohistochemistry confirmed elevated expression of transcription factor EHF(P<0.05).Conclusion There is the remodeling of H3K27me3 silencers in SRCC,and EHF may potentially play a crucial role in the SRCC malignant progression.

关 键 词：胃印戒细胞癌 表观遗传 组蛋白修饰 沉默子 H3K27me3 

分 类 号：R394.3[医药卫生—医学遗传学] R730.23[医药卫生—基础医学] R735.2