黏蛋白1与肿瘤相关蛋白的调控网络研究

doi:10.3969/j.issn.1674-8115.2022.08.007

摘要/Abstract

摘要：

目的·研究黏蛋白1（mucin1，MUC1）在不同肿瘤组织中的表达水平及其对患者生存期的影响；通过分析与MUC1存在相互作用的肿瘤相关蛋白的调控网络，预测MUC1参与肿瘤发生发展的可能机制。方法·使用GEPIA 2在线平台对MUC1在33种肿瘤中的mRNA水平，以及其与患者生存期的关系进行分析。在HEK293T细胞中转染MUC1-HA质粒，用HA抗体进行免疫共沉淀（co-immunoprecipitation，Co-IP）实验，对MUC1结合蛋白进行液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry，LCMS/MS）分析。使用String 11.5在线平台对MUC1结合蛋白的亚细胞定位、分子功能、涉及的疾病、参与的生物学过程以及这些蛋白之间的相互作用调控网络进行分析。结果·MUC1在乳腺癌、宫颈癌、弥漫性大B细胞瘤、多发性胶质细胞瘤、低级别脑胶质瘤、卵巢癌、胰腺癌、胸腺癌和子宫内膜癌等9种肿瘤中高表达；对这9种肿瘤中MUC1表达与生存期的关系分析发现，MUC1表达与生存期呈负相关，其中在乳腺癌、宫颈癌、多发性胶质细胞瘤、低级别脑胶质瘤、胰腺癌和胸腺癌等6种肿瘤中具有统计学意义。质谱分析共检测到526个MUC1结合蛋白，这些蛋白定位于细胞器、细胞质和膜结构最多；主要分子功能包括蛋白结合、离子结合和酶活性等；涉及的疾病有解剖实体性疾病、细胞增殖性疾病、代谢性疾病和癌症等；参与的生物学过程主要包括细胞应激、代谢、发育和生物合成等。MUC1结合蛋白主要参与代谢、癌症、cGMP-依赖cGMP的蛋白激酶（cGMP-dependent protein kinase，PKG）、肿瘤坏死因子（tumor necrosis factor，TNF）和细胞周期等信号通路；深入分析后发现，MUC1结合蛋白一方面大量参与到Wnt/β-catenin、Notch和cAMP等癌症相关信号通路，另一方面通过调控代谢、凋亡和细胞周期促进肿瘤的发生发展。结论·MUC1在多种肿瘤组织中高表达并与患者预后不良相关；MUC1通过蛋白相互作用参与调控细胞代谢和肿瘤相关信号通路。该研究创新性地发现多个新的MUC1结合蛋白，可为进一步研究MUC1生物学新功能奠定基础。

关键词: 黏蛋白1, 肿瘤, 生存分析, 蛋白质组, 信号通路

Abstract:

Objective ·To investigate the level of the expression of mucin 1 (MUC1) in different cancers and its impact on patient survival, and predict the possible mechanisms of MUC1 involvement in oncogenesis and tumor progression by analyzing the regulatory network of tumor-associated proteins that interact with MUC1. Methods ·The mRNA levels of MUC1 in 33 tumors and the relationship between the expression of MUC1 and patient survival were analyzed by using the GEPIA 2 online platform. MUC1-HA plasmids were transfected in HEK293T cells, and then MUC1-binding proteins were obtained by co-immunoprecipitation (Co-IP) experiments with HA antibody, which was later analyzed by liquid chromatography-tandem mass spectrometry (LCMS/MS). The subcellular localization, molecular function, diseases involved, biological process of MUC1-binding proteins and the regulatory network of interactions between these proteins were analyzed by using String 11.5 online platform. Results ·MUC1 was highly expressed in 9 kinds of tumors, including breast cancer, cervical cancer, diffuse large B-cell tumor, glioblastoma multiforme, brain lower grade glioma, ovarian carcinoma, pancreatic carcinoma, thymoma, and uterine corpus carcinoma. The relationship between MUC1 expression and overall survival in these 9 kinds of tumors was analyzed, and MUC1 expression was found to be negatively correlated with the overall survival. Higher MUC1 expression was related to significantly shorter survival time in 6 kinds of tumors, including breast cancer, cervical cancer, glioblastoma multiforme, brain lower grade glioma, pancreatic carcinoma, and thymoma. A total of 526 MUC1-binding proteins were detected by mass spectrometry analysis, and these proteins were mostly localized in organelles, cytoplasm, and membrane structures. The main molecular functions of these proteins included protein binding, ion binding, and enzymatic activity. They were primarily involved in anatomical entity diseases, cell proliferation diseases, metabolic diseases, and cancers. The related biological processes largely consisted of cellular stress, metabolism, development, and biosynthesis. MUC1-binding proteins played various roles in signaling pathways of metabolism, cancer, cGMP-cGMP-dependent protein kinase (PKG), tumor necrosis factor (TNF), and cell cycle. Further analysis of MUC1-binding-protein-associated signaling pathways revealed that they were heavily implicated in cancer-related signaling pathways such as Wnt/β-catenin, Notch, and cAMP on the one hand, and promoted tumor development by regulating metabolism, apoptosis, and cell cycle on the other hand. Conclusion ·MUC1 is highly expressed in a variety of tumor issues and is associated with poor prognosis of patients. MUC1 regulates cellular metabolism and plays a part in tumor-related signaling pathways possibly through protein interactions. This study innovatively identifies several new MUC1-binding proteins, which lay the foundation for further investigation of new biological functions of MUC1.

Key words: mucin 1 (MUC1), tumor, survival analysis, proteome, signaling pathway

中图分类号:

褚云开, 廖春华, 邓华云, 黄雷. 黏蛋白1与肿瘤相关蛋白的调控网络研究[J]. 上海交通大学学报（医学版）, 2022, 42(8): 1024-1033.

CHU Yunkai, LIAO Chunhua, DENG Huayun, HUANG Lei. Study of the regulatory network of MUC1 and tumor-associated proteins[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(8): 1024-1033.

图/表 5

图1 MUC1 在不同肿瘤组织中的转录水平Note： Red indicates high expression of MUC1. Green indicates low expression of MUC1. Black indicates no significant difference. T—tumor tissue. N—normal tissue.CHOL—cholangiocarcinoma; COAD—colon adenocarcinoma; ESCA—esophageal carcinoma; HNSC—head and neck squamous cell carcinoma; KICH—kidney chromophobe; KIRC—kidney renal clear cell carcinoma; KIRP—kidney renal papillary cell carcinoma; LAML—acute myeloid leukemia; LIHC—liver hepatocellular carcinoma; LUAD—lung adenocarcinoma; LUSC—lung squamous cell carcinoma; MESO—mesothelioma; PCPG—pheochromocytoma and paraganglioma; PRAD—prostate adenocarcinoma; READ—rectum adenocarcinoma; SARC—sarcoma; SKCM—skin cutaneous melanoma; STAD—stomach adenocarcinoma; TGCT—testicular germ cell tumors; THCA—thyroid carcinoma; UCS—uterine carcinosarcoma; UVM—uveal melanoma.

Fig 1 Transcription levels of MUC1 in different tumor tissues

图2 9种癌症中 MUC1 表达与患者OS的关系

Fig 2 Relationship between MUC1 expression and OS in 9 kinds of cancers

图3 MUC1结合蛋白的亚细胞定位和分子功能分析Note：A. Subcellular localization of MUC1-binding proteins. B. Molecular function of MUC1-binding proteins.

Fig 3 Subcellular localization and molecular function of MUC1-binding proteins

图4 与MUC1结合蛋白有关的疾病、生物学进程和信号通路Note：A. Diseases associated with MUC1-binding proteins. B. Biological processes associated with MUC1-binding proteins. C. Signaling pathways associated with MUC1-binding proteins.

Fig 4 Diseases, biological processes and signaling pathways associated with MUC1-binding proteins

图5 MUC1结合蛋白参与的信号通路Note：A. Pathways in cancer. B. Metabolic pathway. C. cGMP-PKG signaling pathway. D. TNF signaling pathway. E. Cell cycle.

Fig 5 Signaling pathways of MUC1-binding proteins

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