基于SMART-seq2测序分析结直肠癌外周血DNT细胞转录组的表达差异及功能初探  

A preliminary analysis of transcriptome expression differences and functions of colorectal cancer peripheral blood DNT cells based on SMART-seq2 sequencing

作　　者：代玉玲 魏云波 武静 朱惠茹 刘晓斐 DAI Yuling;WEI Yunbo;WU Jing;ZHU Huiru;LIU Xiaofei(Department of Medical Laboratory,Weifang Medical University,Weifang 261053,China;Department of Laboratory Medicine,the 960th Hospital of PLA Joint Logistics Support Force,Ji’nan 250031,China;Laboratory of Immunology for Environment and Health,Shandong Analysis and Test Center,Qilu University of Technology(Shandong Academy of Sciences),Ji’nan 250031,China)

机构地区：[1]潍坊医学院医学检验学院,261053 [2]齐鲁工业大学(山东省科学院)山东省分析测试中心环境与健康免疫研究室,济南250031 [3]联勤保障部队第九六〇医院检验科,济南250031

出　　处：《免疫学杂志》2023年第10期900-909,共10页Immunological Journal

摘　　要：目的探讨结直肠癌患者外周血中双阴性T细胞差异基因及功能。方法选取2例结直肠癌患者和2例健康体检者,首先采用流式细胞术分选外周血双阴性T细胞,然后利用单细胞全长转录组(SMART-seq2)测序技术获得测序数据,筛选差异表达基因,并对筛选出的差异表达基因进行基因本体富集分析(GO)和京都基因与基因组百科全书(KEGG)途径富集分析。利用STRING数据库构建蛋白质互作网络,并通过Cytoscape软件鉴定关键基因。采用RT-qPCR验证差异表达基因。结果与健康体检者相比,结直肠癌患者外周血双阴性T细胞差异基因共有1276个,其中141个上调基因,1135个下调基因。GO分析显示差异基因主要参与甲基化、代谢过程以及转移酶活性等生物学功能。KEGG通路分析显示差异基因主要涉及自噬、P53信号通路和磷酸肌醇代谢等信号通路。蛋白质互作网络包含1154个节点和1022条边,并鉴定出10个Hub基因:PIK3C3、WIPI1、ATG101、PIK3R4、DDX10、RBM28、SDAD1、ATG16L1、UVRAG、ATG7。RT-qPCR验证10个差异表达基因,其中7个差异基因表达变化趋势与测序结果一致,3个基因表达与测序结果不符。结论DNT细胞可能通过甲基化、P53信号通路和自噬等作用参与了结直肠癌的发生发展,同时,DNT细胞可能通过对基因的调控抑制结直肠癌的发生发展。本研究为进一步探讨DNT细胞在恶性肿瘤中的功能提供理论依据。This study was performed to explore the differential genes and functions of double-negative T cells in peripheral blood of patients with colorectal cancer.Two colorectal cancer patients and two healthy physical examiners were selected,and peripheral blood double-negative T cells were firstly sorted by flow cytometry,and then sequencing data were obtained using single cell full-length transcriptome(SMART-seq2)sequencing technology to screen differentially expressed genes.The screened differentially expressed genes were subjected to Gene Ontology Enrichment(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.The STRING database was used to construct the protein interaction network and identify key genes by Cytoscape software;RT-qPCR was used to verify the differential expression genes in DNT cells.Compared with healthy subjects,there were 1276 peripheral blood double-negative T-cell differential genes in colorectal cancer patients,including 141 up-regulated genes and 1135 down-regulated genes.GO analysis showed that the differential genes were mainly involved in biological functions such as methylation,metabolic processes and transferase activity;KEGG pathway analysis showed that the differential genes were mainly involved in signaling pathways such as autophagy,P53 signaling pathway and phosphatidylinositol metabolism.The protein interaction network contains 1154 nodes and 1022 edges,in addition,10 hub genes were identified:PIK3C3,WIPI1,ATG101,PIK3R4,DDX10,RBM28,SDAD1,ATG16L1,UVRAG,ATG7.RT-qPCR validated 10 differentially expressed genes,of which 7 differentially expressed genes showed trends consistent with sequencing results,and 3 genes showed expression inconsistent with sequencing results.DNT cells may be involved in the development of colorectal cancer through methylation,P53 signaling pathway and autophagy,and at the same time,DNT cells may inhibit the development of colorectal cancer through the regulation of genes.This study provides a theoretical basis for further investigation

关 键 词：结直肠癌 双阴性T细胞 差异基因 SMART-seq2 

分 类 号：R735.3[医药卫生—肿瘤] R392.12[医药卫生—临床医学]