| 摘要: |
| 目的 基于早期和晚期胰腺导管腺癌（PDAC）患者肿瘤组织巨噬细胞相关的差异基因，探索PDAC肿瘤免疫微环境的特征，为PDAC提供早诊早治新靶点。方法 收集3例早期与4例晚期PDAC患者肿瘤组织样本，利用单细胞测序技术分析组织转录组数据，结合癌症基因组图谱（TCGA）数据库，获取与预后有关的早期与晚期PDAC差异表达的巨噬细胞相关基因，采用最小绝对收缩和选择算子（LASSO）-Cox回归方法构建预后风险评分模型。利用基因表达谱交互分析2（GEPIA2）在线生存分析工具筛选出与PDAC预后相关的风险基因，使用通过估计RNA转录本相对子集进行细胞类型鉴定（CIBERSORT）方法对其免疫浸润情况进行分析，通过t-分布随机近邻嵌入（tSNE）聚类降维和Monocle包对其在不同时期不同亚群巨噬细胞中的表达情况进行解析，并进一步借助STRING数据库及人类蛋白质图谱（HPA）数据库对其蛋白质-蛋白质相互作用和免疫组织化学染色图片进行分析。结果 共发现48个早期与晚期PDAC差异表达的巨噬细胞相关基因与预后有关。筛选出11个差异表达的巨噬细胞基因作为PDAC预后基因，并利用这11个基因构建PDAC预后风险评分模型。筛选出2个不良预后基因，其中2'-5'-寡腺苷酸合成酶2（OAS2）基因主要在分泌型磷蛋白1（SPP1）阳性巨噬细胞中表达，人类异常纺锤体样小头畸形相关蛋白（ASPM）基因主要在增殖标志物Ki-67（MKI67）阳性巨噬细胞中表达，并且OAS2在不同时期PDAC巨噬细胞中的表达具有明显差异。免疫浸润研究表明OAS2和ASPM在调节性T细胞、M2型巨噬细胞中高表达（均P＜0.05）。蛋白质-蛋白质相互作用网络显示了OAS2和ASPM间的表达关系以及更多其他共表达的蛋白。免疫组织化学分析表明OAS2在肿瘤组织中的表达水平高于正常组织。结论 OAS2和ASPM均为不良预后基因，分别在SPP1阳性巨噬细胞和MKI67阳性巨噬细胞中特异性表达并发挥负性免疫调节作用，进而促进PDAC的进展并导致不良预后，这有望为PDAC的防治提供新的靶点。 |
| 关键词: 胰腺导管腺癌 巨噬细胞 预后 肿瘤微环境 单细胞测序 拟时序分析 2'-5'-寡腺苷酸合成酶2 |
| DOI：10.16781/j.CN31-2187/R.20240258 |
| 投稿时间：2024-04-19修订日期：2024-09-25 |
| 基金项目:国家自然科学基金面上项目（82273317），上海市科技创新行动计划生物医药科技支撑项目（22S11901600），上海市细胞工程重点实验室项目（14DZ2272300）. |
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| Role of 2'-5'-oligoadenylate synthetase 2 in tumor-associated macrophages of pancreatic ductal adenocarcinoma revealed by single-cell sequencing-based technology |
| WANG Zixin1△,YANG Yujie2△,WANG Yue3,ZHAO Yunpeng3* |
(1. The Sixth Student Team, College of Basic Medical Sciences, Naval Medical University (Second Military Medical University), Shanghai 200433, China;
2. Department of Anatomy, College of Basic Medical Sciences, Naval Medical University (Second Military Medical University), Shanghai 200433, China;
3. Laboratory of Stem Cell and Regenerative Medicine, Center of Translational Medicine, Naval Medical University (Second Military Medical University), Shanghai 200433, China
△Co-first authors.
* Corresponding author) |
| Abstract: |
| Objective To explore the characteristics of tumor immune microenvironment of pancreatic ductal adenocarcinoma (PDAC) based on the differentially expressed genes associated with macrophages in early and late tumor tissues of PDAC patients, so as to provide new targets for early diagnosis and treatment of PDAC. Methods Three early-stage and 4 late-stage tumor samples were collected from PDAC patients. Tissue transcriptome data were analyzed by single-cell sequencing technology. With The Cancer Genome Atlas (TCGA) database, macrophage related genes differentially expressed in early and late PDAC related to prognosis were obtained. The prognostic risk scoring model was constructed using the least absolute shrinkage and selection operator (LASSO)-Cox method. The risk genes associated with PDAC prognosis were screened using the Gene Expression Profiling Interaction Analysis 2 (GEPIA2) online survival analysis tool. Their immune infiltration was analyzed using the cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method, and their expression in different subpopulations of macrophages at different periods was resolved by t-distributed stochastic nearest-neighbour embedding (tSNE) clustering downscaling and the Monocle package. The protein-protein interactions and immunohistochemistry were further analyzed with the help of STRING database and The Human Protein Atlas (HPA) database. Results A total of 48 differentially expressed macrophage-related genes in early and late PDAC were found to be associated with prognosis. Eleven differentially expressed macrophage genes were screened as PDAC prognostic genes, and they were used to construct a prognostic risk scoring model for PDAC. Two prognostic genes with significant risk indicators were screened: 2'-5'-oligoadenylate synthetase 2 (OAS2) was mainly expressed in secreted phosphoprotein 1 (SPP1)-positive macrophages, and assembly factor for spindle microtubules (ASPM) was mainly expressed in proliferation marker protein Ki-67 (MKI67)-positive macrophages. There were significant differences in the expression of OAS2 in PDAC macrophages at different stages. Immune infiltration studies showed that OAS2 and ASPM were highly expressed in regulatory T cells and M2-type macrophages (both P＜0.05). Protein-protein interaction network showed the expression relationship between OAS2 and ASPM and other co-expressed proteins. Immunohistochemistry results showed that the expression of OAS2 was higher in tumor tissues than in normal tissues. Conclusion OAS2 and ASPM are both poor prognostic genes that are specifically expressed and exert negative immune effects in SPP1+ macrophages and MKI67+ macrophages, respectively, promoting the progression of PDAC and ultimately leading to a poor prognosis, and it is expected to provide new targets for the prevention and treatment of PDAC. |
| Key words: pancreatic ductal adenocarcinoma macrophages prognosis tumor microenvironment single cell sequencing pseudotime analysis 2'-5'-oligoadenylate synthetase 2 |
