通过生物信息学分析THBS2基因在结肠癌中的表达及临床意义

Bioinformatics Analysis of THBS2 Gene Expression and Its Clinical Significance in Colon Cancer

摘要: 目的：通过生物信息学方法筛选出与结肠癌表达及预后相关的基因并研究其临床意义。方法：从基因综合表达数据库(Gene Expression Omnibus, GEO)中分别下载人类结直肠癌数据集GSE21510、GSE21815和GSE37364，利用R语言相关软件包对数据集进行筛选和分析获取差异表达基因(differentially expressed genes, DEGs)。将DEGs输入到STRING数据库构建蛋白质–蛋白质相互作用(protein-protein interaction, PPI)网络，并通过Cytoscape软件筛选出核心基因。通过GEPIA数据库进行进一步表达验证和生存分析，最终筛选出与预后显著相关的目标基因。通过TIMER数据库、HPA数据库和UALCAN数据库分析目标基因的表达、临床意义、启动子甲基化水平以及与结肠癌组织中免疫细胞浸润的相关性。通过LinkedOmics数据库获取与目标基因的共表达基因并进行GO和KEGG富集分析，以探究其潜在生物学功能和信号通路。结果：通过差异分析从三个数据集中共鉴定出199个共同DEGs，其中63个表达上调，136个表达下调。通过PPI网络最终筛选出MMP2，MMP3，MMP11，MMP7，THBS2，PLAU，COL11A1，INHBA，CCL20，CXCL11共10个核心基因。通过GEPIA数据库进一步分析最终筛选出目标基因：血小板反应蛋白2 (thrombospondin-2, THBS2)，GEPIA数据库显示THBS2基因在结肠癌患者中表达升高有统计学意义且与预后相关(P < 0.05)。通过TIMER2.0数据库分析发现THBS2在包括结直肠癌在内的多种癌症中表达水平均高于正常组织，HPA数据库的免疫组化结果提示THBS2蛋白在正常组织中染色呈弱阳性，而在肿瘤组织中染色呈高度阳性并显示蛋白产物位于细胞质和膜质中。UALCAN数据库分析得出THBS2的表达与结直肠癌的病理分期、组织学亚型、患者年龄相关(P < 0.05)，与淋巴结转移程度无明显相关(P > 0.05)，且结肠癌组织中的启动子甲基化水平显著低于正常结肠组织(P < 0.01)。TIMER数据库检索发现THBS2的表达与细胞纯度呈负相关，与CD4⁺ T细胞、CD8⁺ T细胞、中性粒细胞、巨噬细胞和树突状细胞的浸润水平呈正相关(P < 0.01)，而与B细胞的浸润水平无相关性(P > 0.05)。LinkedOmics数据库中的共表达基因GO富集分析显示THBS2及其共表达基因主要参与细胞外基质组装、细胞黏附、骨骼形成等生物学过程，位于细胞外基质和细胞–基质连接结构处，影响细胞外基质成分结合和黏附分子结合等分子功能。这些结果与THBS2作为细胞外基质糖蛋白的特性相一致。KEGG通路分析发现，THBS2及其共表达基因主要富集在肌动蛋白细胞骨架、PI3K-AKT信号通路、整合素信号通路和黏着斑等信号通路。

Abstract: Objective: To screen genes associated with the expression and prognosis of colon cancer using bioinformatics methods and analyze their clinical significance. Methods: Human colorectal cancer datasets GSE21510, GSE21815, and GSE37364 were downloaded from the Gene Expression Omnibus (GEO) database. R language-related software packages were used to screen and analyze the datasets to obtain differentially expressed genes (DEGs). The DEGs were input into the STRING database to construct a protein-protein interaction (PPI) network, and core genes were screened using Cytoscape software. Further expression validation and survival analysis were performed through the GEPIA database to finally screen out target genes significantly associated with prognosis. The TIMER database, Human Protein Atlas (HPA) database, and UALCAN database were used to analyze the expression, clinical significance, promoter methylation levels, and correlation with immune cell infiltration in colon cancer tissues of the target gene. Co-expressed genes of the target gene were obtained through the LinkedOmics database, and GO and KEGG enrichment analyses were performed to explore their potential biological functions and signaling pathways. Results: A total of 199 common DEGs were identified from the three datasets through differential analysis, including 63 up-regulated and 136 down-regulated genes. Through the PPI network, 10 core genes were finally screened: MMP2, MMP3, MMP11, MMP7, THBS2, PLAU, COL11A1, INHBA, CCL20, and CXCL11. Through further analysis using the GEPIA database, the target gene thrombospondin-2 (THBS2) was finally selected. The GEPIA database showed that elevated expression of the THBS2 gene in colon cancer patients was statistically significant and associated with prognosis (P < 0.05). Analysis through the TIMER2.0 database revealed that THBS2 expression levels were higher in tumor tissues than in normal tissues in various cancers, including colorectal cancer. Immunohistochemical results from the HPA database indicated that THBS2 protein staining was weakly positive in normal tissues but highly positive in tumor tissues, with protein products located in the cytoplasm and membrane. UALCAN database analysis showed that THBS2 expression was associated with pathological stage, histological subtype, and patient age in colorectal cancer (P < 0.05), but not significantly associated with lymph node metastasis (P > 0.05). Promoter methylation levels in colon cancer tissues were significantly lower than those in normal colon tissues (P < 0.01). TIMER database retrieval found that THBS2 expression was negatively correlated with cell purity and positively correlated with the infiltration levels of CD4⁺ T cells, CD8⁺ T cells, neutrophils, macrophages, and dendritic cells (P < 0.01), but not with B cell infiltration levels (P > 0.05). GO enrichment analysis of co-expressed genes from the LinkedOmics database showed that THBS2 and its co-expressed genes were mainly involved in biological processes such as extracellular matrix organization, cell adhesion, and bone formation, located in the extracellular matrix and cell-matrix junction structures, affecting molecular functions such as extracellular matrix component binding and adhesion molecule binding. These results are consistent with the characteristics of THBS2 as an extracellular matrix glycoprotein. KEGG pathway analysis revealed that THBS2 and its co-expressed genes were mainly enriched in signaling pathways including actin cytoskeleton, PI3K-AKT signaling pathway, integrin signaling pathway, and focal adhesion.