循环肿瘤细胞FCGBP和BIGH3作为结直肠癌潜在生物标志物的可行性研究

doi:10.19401/j.cnki.1007-3639.2024.08.004

摘要/Abstract

摘要：

背景与目的：结直肠癌（colorectal cancer，CRC）是全球常见的恶性肿瘤之一，且晚期患者的预后较差，寻找新的CRC潜在生物标志物的需求日益增加。循环肿瘤细胞（circulating tumor cell，CTC）从原发肿瘤脱落并进入循环系统，可在血液中被检测到，被认为是CRC的重要生物标志物。本研究旨在探讨CRC中的FCGBP和BIGH3是否可作为结直肠癌的潜在生物标志物。方法：本研究从基因表达综合数据库（Gene Expression Omnibus，GEO）获取了3个有CTC的CRC数据集，分别为GSE74369、GSE117606和GSE164191。通过生物信息学分析在CTC和正常样本之间筛选出差异表达基因。其中一个包含临床信息的数据集用于WGCNA分析，并鉴定了两个关键基因模块，共包含1 148个基因。然后对这些模块中的基因进行了功能富集分析。使用Venn图、PPI调控网络构建分析和筛选候选基因。最后，使用癌症基因组图谱（The Cancer Genome Atlas，TCGA）和基因型-组织表达数据库（Genotype-Tissue Expression，GTEx）数据进行生存分析，并鉴定出与CRC相关的关键基因。在结直肠癌组织中通过实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RTFQ-PCR）验证BIGH3基因表达，并通过克隆形成、划痕实验以及transwell实验等功能实验在HCT116和SW620细胞系中验证BIGH3基因与结直肠癌的关系。结果：通过GEO中CRC数据集分析，共筛选出了2 214个差异基因，通过WGCNA分析和PPI网络构建鉴定出4个与CRC相关的CTC基因。通过GEPIA数据库进行生存分析发现， FCGBP及BIGH3与总生存期及无疾病生存期具有相关性。进一步实验表明，BIGH3基因在30对配对的结直肠癌样本中呈高表达，在HCT116和SW620细胞系中敲低BIGH3的表达能够减慢细胞增殖和迁移的速度，并降低侵袭性，而上调BIGH3的表达则会增加其侵袭性或提高迁移率。结论：本研究结果表明，FCGBP及BIGH3与TNM分期呈正相关，这暗示它们在CRC发展中的重要作用，具有较好的预后价值，其可以作为潜在的CRC生物标志物，并可能作为潜在的治疗靶点。同时，我们的实验数据也揭示了BIGH3在结直肠癌中的重要角色，它可能影响结直肠癌的生物学行为。

关键词: 结直肠癌, 循环肿瘤细胞, 生物标志物

Abstract:

Background and purpose: Colorectal cancer (CRC) is globally recognized as one of the most prevalent malignant tumors. Advanced CRC is marked by a relatively poor prognosis for patients, signifying an urgent need to identify novel potential biomarkers for CRC. A particular focus has been given to circulating tumor cells (CTC), which are cells that have detached from the primary tumor mass and subsequently entered the circulatory system. These cells can be detected within the blood and are currently considered significant potential biomarkers for CRC. This study aimed to investigate if FCGBP and BIGH3 in CRC could be potential markers for colorectal cancer. Methods: This study obtained 3 CRC datasets (GSE74369, GSE117606, and GSE164191) with CTC from the GEO database. Bioinformatics analysis was conducted to screen differentially expressed genes between CTC and normal samples. One dataset that includes clinical information was used for WGCNA analysis, and two key gene modules were identified, containing 1 148 genes in total. Then, functional enrichment analysis was carried out for these genes in the modules. Venn diagrams, PPI network construction analysis, and candidate gene screening were employed. Finally, survival analysis was performed using TCGA and GTEx data in the GEPIAS database, and key genes associated with CRC were identified. The expression of BIGH3 gene was validated in colorectal cancer tissues by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR), and its association with colorectal cancer was verified through clonogenic, scratch, and transwell assays in HCT116 and SW620 cell lines. Results: Through the analysis of CRC datasets from GEO, we screened a total of 2 214 differential genes. With the help of WGCNA analysis and PPI network construction, 4 CTC-related genes associated with CRC were identified. Survival analysis from the GEPIA database revealed that FCGBP and BIGH3 are associated with overall survival and disease-free survival. Further experiments indicated that BIGH3 gene is overexpressed in 30 matched colorectal cancer samples. The downregulation of BIGH3 expression could slow down cell proliferation and migration rates, as well as decrease invasiveness in HCT116 and SW620 cell lines. In contrast, upregulation of BIGH3 expression could increase its invasiveness or accelerate migration rate. Conclusion: FCGBP and BIGH3 are positively correlated with TNM staging, indicating their pivotal roles in CRC progression. They bear good prognostic value and may serve as potential biological markers for CRC clinically, and could provide potential therapeutic targets. Moreover, our experimental data revealed the crucial role of BIGH3 in colorectal cancer, suggesting it may influence the biological behavior of this disease.

Key words: Colorectal cancer, Circulating tumor cells, Biomarkers

中图分类号:

R735.3+4

葛祖荫, 宋坤, 林云霄, 钟烨凌, 郝敬铎. 循环肿瘤细胞FCGBP和BIGH3作为结直肠癌潜在生物标志物的可行性研究[J]. 中国癌症杂志, 2024, 34(8): 745-752.

GE Zuyin, SONG Kun, LIN Yunxiao, ZHONG Yeling, HAO Jingduo. The feasibility study of FCGBP and BIGH3 in circulating tumor cells as potential markers for colorectal cancer[J]. China Oncology, 2024, 34(8): 745-752.

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Gene	Forward primer (5' to 3')	Reverse primer (5' to 3')
BIGH3	GGGAAGCTCCTTACTGTGGG	CACCCGGGGATGGAACTAAT
GAPDH	GTCTCCTCTGACTTCAACAGCG	ACCACCCTGTTGCTGTAGCCAA

RNA oligos	Sequences
si-NC	Sense: 5'- UUCUCCGAACGUGUCACGUTT -3' Anti-sense: 5'- ACGUGACACGUUCGGAGAATT -3'
si-BIGH3#1	Sense: 5'-GGGACAUGCUCACUAUCAATT -3' Anti-sense: 5'-UUGAUAGUGAGCAUGUCCCTT -3'
si-BIGH3#2	Sense: 5'-GUGCUGUCCUGGAUAUGAATT -3' Anti-sense: 5'- UUCAUAUCCAGGACAGCACTT -3'
si-BIGH3#3	Sense: 5'- AUACGGUUCAAAGUCUCACTT -3' Anti-sense: 5'- GUGAGACUUUGAACCGUAUTT -3'