Pan Yang, Wang Chenci, Wang Shengnan, Wu Xingwei, Sheng Lili, Qi Zhilin
Department of Oncology, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, China.
Anhui Province Key Laboratory of Biological Macro-molecules Research, Wannan Medical College, Wuhu, China.
J Gastrointest Oncol. 2023 Jun 30;14(3):1331-1345. doi: 10.21037/jgo-23-304. Epub 2023 Jun 27.
The purpose of this study is to understand the CLEC5A mechanism in colon cancer's proliferation and migration.
The CLEC5A expression levels in colon cancer tissues were analyzed using bioinformatics method based on Oncomine and The Cancer Genome Atlas (TCGA) databases, which were further tested by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). The CLEC5A expression levels in 4 types of colon cancer cell lines (HCT116, SW620, HT29, and SW480) were also examined by qRT-PCR. We constructed CLEC5A knockdown cell lines and used colony formation, Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assays for investigating the CLEC5A function in colon cancer's proliferation and migration. A CLEC5A silencing nude mice model was established to measure the scale, weight, and growth rate of tumor xenograft. In CLEC5A knockdown cell lines and xenograft tissues, the levels of cell cycle and epithelial-mesenchymal transition (EMT)-related proteins were detected using Western blot (WB), and the phosphorylation levels of AKT/mTOR pathway key proteins were also detected by WB. On the basis of gene expression data retrieved from TCGA database, a relevance between CLEC5A and AKT/mTOR pathway in colon cancer was examined by gene set enrichment analysis (GSEA), and correlation analysis of CLEC5A and COL1A1 was employed to confirm their interaction.
Bioinformatics analysis, IHC staining, and qRT-PCR assay results all showed the significant high levels of CLEC5A expression in colon cancer tissues and cells, and positive links between CLEC5A levels and lymph node metastasis, vascular metastasis, and tumor-node-metastasis (TNM) stages of colon cancer patients. The suppressive effects of CLEC5A knockdown on colon cancer's proliferation and migration were verified in cell function and nude mice tumorigenesis assays. WB analysis further indicated that CLEC5A knockdown could inhibit cell cycle, and EMT processes, as well as AKT/mTOR pathway phosphorylation in colon cancer. On the basis of TCGA data, CLEC5A's activation effect on AKT/mTOR pathway had been confirmed by GSEA analysis, and the interaction between CLEC5A and COL1A1 was also revealed through correlation analysis in colon cancer.
CLEC5A may promote the development and migration of colon cancer by triggering the AKT/mTOR signaling pathway. Furthermore, COL1A1 could serve as the target gene of CLEC5A.
本研究旨在了解CLEC5A在结肠癌增殖和迁移中的机制。
基于Oncomine和癌症基因组图谱（TCGA）数据库，采用生物信息学方法分析结肠癌组织中CLEC5A的表达水平，并通过免疫组织化学（IHC）和定量实时聚合酶链反应（qRT-PCR）进一步检测。还通过qRT-PCR检测了4种结肠癌细胞系（HCT116、SW620、HT29和SW480）中CLEC5A的表达水平。我们构建了CLEC5A敲低细胞系，并使用集落形成、细胞计数试剂盒-8（CCK-8）、5-乙炔基-2'-脱氧尿苷（EdU）、伤口愈合和transwell实验来研究CLEC5A在结肠癌增殖和迁移中的功能。建立了CLEC5A沉默裸鼠模型，以测量肿瘤异种移植的大小、重量和生长速率。在CLEC5A敲低细胞系和异种移植组织中，使用蛋白质免疫印迹法（WB）检测细胞周期和上皮-间质转化（EMT）相关蛋白的水平，并通过WB检测AKT/mTOR通路关键蛋白的磷酸化水平。基于从TCGA数据库检索到的基因表达数据，通过基因集富集分析（GSEA）研究结肠癌中CLEC5A与AKT/mTOR通路之间的相关性，并采用CLEC5A与COL1A1的相关性分析来确认它们之间的相互作用。
生物信息学分析、IHC染色和qRT-PCR检测结果均显示，结肠癌组织和细胞中CLEC5A表达水平显著升高，且CLEC5A水平与结肠癌患者的淋巴结转移、血管转移和肿瘤-淋巴结-转移（TNM）分期呈正相关。在细胞功能和裸鼠肿瘤发生实验中证实了CLEC5A敲低对结肠癌增殖和迁移的抑制作用。WB分析进一步表明，CLEC5A敲低可抑制结肠癌的细胞周期、EMT过程以及AKT/mTOR通路的磷酸化。基于TCGA数据，通过GSEA分析证实了CLEC5A对AKT/mTOR通路的激活作用，并且通过结肠癌中的相关性分析也揭示了CLEC5A与COL1A1之间的相互作用。
CLEC5A可能通过激活AKT/mTOR信号通路促进结肠癌的发展和迁移。此外，COL1A1可能是CLEC5A的靶基因。
