生物信息学方法筛选胰腺癌进展相关的核心基因

doi:10.3969/j.issn.1674-8115.2021.05.003

摘要/Abstract

摘要：

目的·筛选胰腺癌进展相关的核心基因和关键通路。方法·通过GEO（Gene Expression Omnibus）数据库检索筛选得到包含45例胰腺癌组织和45例癌旁正常组织的基因芯片数据集GSE28735。采用GEO2R在线分析工具提取癌组织及癌旁正常组织的基因，联合RStudio软件筛选差异表达基因（differentially expressed genes，DEGs）并对其进行可视化处理。通过基因功能注释在线工具DAVID对差异表达显著的基因进行基因本体（gene ontology，GO）功能富集和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集分析。利用交互基因检索工具STRING及Cytoscape软件构建蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络，结合节点度值进一步筛选核心基因。通过基因表达谱分析数据库GEPIA对179例胰腺癌中核心基因的表达水平与患者总生存期（overall survival，OS）、无病生存期（disease free survival，DFS）及肿瘤分期的关系进行分析。结果·从GSE28735芯片中筛选得到131个DEGs，其中表达上调的基因115个，表达下调的基因16个。GO功能富集分析显示DEGs主要在细胞黏附、质膜、蛋白质结合方面富集。KEGG通路富集提示磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase，PI3K）/蛋白激酶B（protein kinase B，AKT）是DEGs富集的主要信号通路。通过PPI网络筛选得到5个核心基因，分别为纤维连接蛋白1（fibronectin 1，FN1）、间质表皮转化因子（mesenchymal to epithelial transition factor，MET）、层粘连蛋白β3（polyclonal antibody to laminin β3，LAMB3）、层粘连蛋白α3（laminin subunit α3，LAMA3）、整合素亚单位α3（integrin subunit α3，ITGA3）。MET、LAMA3、LAMB3和ITGA3的表达水平均与胰腺癌患者OS有关，仅MET、LAMB3和ITGA3的表达水平与胰腺癌患者DFS有关，且基因低表达组的预后明显优于基因高表达组。FN1、MET、LAMA3和LAMB3的表达水平在不同分期胰腺癌组织中的差异存在统计学意义。结论·胰腺癌中异常表达的FN1、MET、LAMB3、LAMA3、ITGA3与细胞黏附、质膜组分、蛋白质结合功能的改变和PI3K/AKT通路相关，MET和LAMB3的表达升高可能预示胰腺癌患者的不良预后。

关键词: 胰腺癌, 生物信息学, 差异表达基因, 肿瘤发生

Abstract:

Objective·To select pancreatic cancer progression-related core genes and key pathways.

Methods·The dataset GSE28735 for pancreatic cancer was obtained by searching and screening in the Gene Expression Omnibus (GEO) database. Genes in 45 cases of cancer tissues and 45 cases of normal tissues adjacent to cancer were extracted by GEO2R and combined with RStudio to screen and visualize differentially expressed genes (DEGs). Genes with significant differential expression were analyzed by gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis through the gene utilization function annotation online tool DAVID. The protein-protein interaction (PPI) network was constructed by using the interactive gene retrieval tool, STRING and Cytoscape, to further screen core genes based on the node degree value. The relationship between the expression levels of core genes and overall survival (OS), disease-free survival (DFS) and tumor stage of 179 cases of patients was analyzed through the gene expression profile analysis database GEPIA.

Results·A total of 131 DEGs were screened from the dataset GSE28735, including 115 up-regulated genes and 16 down-regulated genes. GO function enrichment analysis showed that DEGs were mainly enriched in cell adhesion, plasma membrane, and protein binding. KEGG pathway enrichment suggested that phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) was the main signaling pathway for DEG enrichment. Five core genes, fibronectin1 (FN1), mesenchymal to epithelial transition factor (MET), polyclonal antibody to laminin β3 (LAMB3), laminin subunit α3 (LAMA3), and integrin subunit α3 (ITGA3),were obtained through PPI network screening. The expression levels of MET, LAMA3, LAMB3 and ITGA3 were associated with OS of patients, and the expression levels of MET, LAMB3 and ITGA3 were associated with DFS. The prognosis of low gene expression group was significantly better than that of high gene expression group. There were significant differences in the expression levels of FN1, MET, LAMA3 and LAMB3 in the different stages of pancreatic cancer.

Conclusion·The abnormal expression of FN1, MET, LAMB3, LAMA3 and ITGA3 is related to the changes of cell adhesion, plasma membrane component, protein binding function and PI3K/Akt pathway. The increased expression of MET and LAMB3 may predict poor prognosis of patients with pancreatic cancer.

Key words: pancreatic cancer, bioinformatics, differentially expressed gene, tumorigenesis

中图分类号:

R735.9

杨鹿笛, 王高明, 胡仁豪, 蒋小华, 崔然. 生物信息学方法筛选胰腺癌进展相关的核心基因[J]. 上海交通大学学报(医学版), 2021, 41(5): 571-578.

Lu-di YANG, Gao-ming WANG, Ren-hao HU, Xiao-hua JIANG, Ran CUI. Identification of core genes in pancreatic cancer progression by bioinformatics analysis[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(5): 571-578.

图/表 7

图1 胰腺癌组织及癌旁组织中DEGs的可视化分析Note: A. Volcanic map of top 250 genes. Green dots represent significantly down-regulated genes in pancreatic cancer; red dots represent significantly up-regulated genes in pancreatic cancer; black dots represent no significant difference in expression levels of genes between pancreatic cancer and adjacent tissues. B. Heatmap of 131 DEGs. Green box represents significantly down-regulated genes in pancreatic cancer; red box represents significantly up-regulated genes in pancreatic cancer.

Fig 1 Visual analysis of the DEGs in pancreatic cancer and adjacent tissues

图2 DEGs的GO功能富集和KEGG通路富集分析Note: A. Biological processes of DEG enrichment. B. Cell component of DEG enrichment. C. Molecular function of DEG enrichment. D. KEGG pathway of DEG enrichment.

Fig 2 Enrichment analysis of GO function and KEGG pathway of DEGs

图3 DEGs对应蛋白的PPI网络图

Fig 3 PPI network diagram of DEGs corresponding proteins

表1 PPI网络中5个DEGs的生物学特点

Tab 1 Biological characteristics of the five DEGs in PPI network

Gene	Degree	Biological characteristic
FN1	24	Involved in cell adhesion, cell movement, wound healing and maintenance of cell morphology
MET	15	Regulating cell proliferation, dispersion, proliferation and differentiation
LAMB3	11	Involved in cell adhesion, migration and interaction with other extracellular matrix components; playing an important role in regulating cell migration and mechanical signal transduction
LAMA3	11	Involved in cell adhesion, migration and interaction with other extracellular matrix components; playing an important role in regulating cell migration and mechanical signal transduction
ITGA3	11	Involved in cell adhesion, invasion foot formation and matrix degradation

图4 5个DEGs在179例胰腺癌组织和171例正常组织中的表达Note: FN1 (A), MET (B), LAMA3 (C), LAMB3 (D), ITGA3 (E) mRNA expression levels determined by the GEPIA database. The cutoff of |log2FC| is 1; the default cutoff of P value is 0.01.

Fig 4 Expression of the five DEGs in 179 cases of pancreatic cancer tissues and 171 cases of adjacent normal tissues

图5 Kaplan-Meier曲线分析评估5个DEGs对胰腺癌患者预后的意义Note: A?E. OS of pancreatic cancer patients with high FN1 (A), MET (B), LAMA3 (C), LAMB3 (D), ITGA3 (E) expression and low FN1 (A), MET (B), LAMA3 (C), LAMB3 (D), ITGA3 (E) expression. F?J. DFS of pancreatic cancer patients with high FN1 (F), MET (G), LAMA3 (H), LAMB3 (I), ITGA3 (J) expression and low FN1 (F), MET (G), LAMA3 (H), LAMB3 (I), ITGA3 (J) expression. Red curve represents high gene expression group; blue curve represents low gene expression group. KPM—transcripts per kilobase of exon model per million mapped reads.

Fig 5 Prognostic significance of the five DEGs for pancreatic cancer patients assessed via Kaplan-Meier analysis

图6 5个DEGs在不同病理分期胰腺癌中的表达Note: Analysis of FN1 (A), MET (B), LAMA3 (C), LAMB3 (D), and ITGA3 (E) gene expression in stage Ⅰ, Ⅱ, Ⅲ, and Ⅳ pancreatic cancer.

Fig 6 Expression of the five DEGs in pancreatic carcinoma with different pathological stages

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