PO.TB03.02 · 肿瘤生物学
C6ORF15作为结直肠癌上皮-间充质转化及淋巴结转移标志物的特征研究
Characterisation of C6ORF15 as a marker for epithelial-mesenchymal transition and lymph node metastasis in colorectal cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言 结直肠癌（CRC）的淋巴结转移与不良预后相关，但其分子驱动因素仍不明确。本研究旨在鉴定与淋巴结转移相关的新型靶点。
方法 利用TCGA数据将结肠癌分为"扩张型"和"转移型"表型。通过RNA-seq和GSEA分析转录组图谱。通过CRISPR-Cas9和慢病毒转导实现HCT116和SW480细胞中C6ORF15的敲除（KO）和过表达（OE），并经Sanger测序和Western blot验证。通过增殖、迁移和侵袭实验以及针对EMT标志物的Western blot评估功能效应。在C6ORF15-OE HCT116细胞中进行免疫沉淀-质谱（IP-MS），以鉴定潜在的相互作用蛋白。
结果 TCGA分析确定CRC肿瘤中位大小为5.375 cm，据此可分为"扩张型"（>5 cm，无淋巴结受累）和"转移型"（≤5 cm，早期淋巴结受累）表型。Kaplan-Meier分析显示"转移型"组的生存显著较差（p = 0.0145）。GSEA确定上皮-间充质转化（EMT）为"转移型"肿瘤中富集程度最高的Hallmark（NES = 1.81，FDR q = 0），其中C6ORF15高度上调（LogFC = 2.37，p = 2.06E-07），并在患者来源组织标本中经免疫组化证实。
在同基因细胞系中，C6ORF15 KO下调了EMT基因集（NES = -1.44，FDR q = 0.1），减少了SNAI1/2表达而保留了CDH1/2，使细胞向非转移性上皮表型转变，伴随增殖、迁移和侵袭能力降低。相反，OE细胞表现出加重的部分EMT特征，标志为CDH1下调以及部分间充质标志物（CDH2、SLUG和SNAI1）在细胞系依赖方式下的上调，使细胞向更具侵袭性的转移表型转变，增殖、迁移和侵袭能力增强。IP-MS鉴定FAT1为两个潜在C6ORF15相互作用蛋白之一。
已知FAT1 OE可上调EMT并与CRC相关。在同基因细胞系中，C6ORF15 OE在FAT1 KO细胞中挽救了EMT活性，而C6ORF15 KO则在FAT1 OE细胞中下调了EMT，提示C6ORF15对EMT的作用可能独立于FAT1表达而持续存在。
结论 C6ORF15可能促进EMT及涉及淋巴结的早期CRC转移，显示出作为未来治疗靶点的潜力。
查看英文原文 English abstract
Introduction Lymph node metastasis in colorectal cancer (CRC) correlates with poor prognosis, yet its molecular drivers remain unknown. This study aimed to identify novel targets associated with lymph node metastasis.
Methods Colon cancers were classified into "expanding" and "metastatic" phenotypes using TCGA data. Transcriptomic profiles were analyzed by RNA-seq and GSEA. C6ORF15 knockout (KO) and overexpression (OE) in HCT116 and SW480 cells were achieved via CRISPR-Cas9 and lentiviral transduction, validated by Sanger sequencing and Western blot. Functional effects were assessed through proliferation, migration, and invasion assays, and Western blot for EMT markers. Immunoprecipitation-mass spectrometry (IP-MS) was performed in C6ORF15 -OE HCT116 cells to identify potential interactors.
Results TCGA analysis identified a median CRC tumor size of 5.375 cm, enabling classification into ‘expanding' (>5 cm, no lymph node involvement) and ‘metastatic' (≤5 cm, early lymph node involvement) phenotypes. Kaplan-Meier analysis showed significantly poorer survival in the ‘metastatic' group (p = 0.0145). GSEA identified epithelial-mesenchymal transition (EMT) as the top enriched Hallmark in ‘metastatic' tumors (NES = 1.81, FDR q = 0), with C6ORF15 highly upregulated (LogFC = 2.37, p = 2.06E-07), confirmed by immunohistochemistry in patient-derived tissue specimens.
In isogenic cell lines, C6ORF15 KO downregulated the EMT gene set (NES = -1.44, FDR q = 0.1), reducing SNAI1/2 while preserving CDH1/2, shifting cells toward a non-metastatic epithelial phenotype with reduced proliferation, migration, and invasion. Conversely, OE cells exhibited aggravated partial EMT characteristics, marked by downregulation of CDH1 and upregulation of selected mesenchymal markers (CDH2, SLUG, and SNAI1) in a cell line-dependent manner, shifting cells toward a more aggressive metastatic phenotype with enhanced proliferation, migration, and invasion. IP-MS identified FAT1 as one of two potential C6ORF15 interactors.
FAT1 OE is known to upregulate EMT and is associated with CRC. In isogenic cell lines, C6ORF15 OE rescued EMT activity in FAT1 KO cells, while C6ORF15 KO downregulated EMT in FAT1 OE cells, suggesting that C6ORF15 effects on EMT may persist independent of FAT1 expression.
Conclusion C6ORF15 may promote EMT and early CRC metastasis involving lymph nodes, showing potential as a target for future therapies.
利益披露 Disclosure
D. Chan, None.