DataSheet_2_Identification of necroptosis-related genes for predicting prognosis and exploring immune infiltration landscape in colon adenocarcinoma.xlsx

BackgroundNecroptosis is a recently discovered form of cell death that plays an important role in the occurrence and development of colon adenocarcinoma (COAD). Our study aimed to construct a risk score model to predict the prognosis of patients with COAD based on necroptosis-related genes.
MethodsThe gene expression data of COAD and normal colon samples were obtained from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). The least absolute shrinkage and selection operator (LASSO) Cox regression analysis was used to calculate the risk score based on prognostic necroptosis-related differentially expressed genes (DEGs). Based on the risk score, patients were classified into high- and low-risk groups. Then, nomogram models were built based on the risk score and clinicopathological features. Otherwise, the model was verified in the Gene Expression Omnibus (GEO) database. Additionally, the tumor microenvironment (TME) and the level of immune infiltration were evaluated by “ESTIMATE” and single-sample gene set enrichment analysis (ssGSEA). Functional enrichment analysis was carried out to explore the potential mechanism of necroptosis in COAD. Finally, the effect of necroptosis on colon cancer cells was explored through CCK8 and transwell assays. The expression of necroptosis-related genes in colon tissues and cells treated with necroptotic inducers (TNFα) and inhibitors (NEC-1) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR).
ResultsThe risk score was an independent prognostic risk factor in COAD. The predictive value of the nomogram based on the risk score and clinicopathological features was superior to TNM staging. The effectiveness of the model was well validated in GSE152430. Immune and stromal scores were significantly elevated in the high-risk group. Moreover, necroptosis may influence the prognosis of COAD via influencing the cancer immune response. In in-vitro experiments, the inhibition of necroptosis can promote proliferation and invasion ability. Finally, the differential expression of necroptosis-related genes in 16 paired colon tissues and colon cancer cells was found.
ConclusionA novel necroptosis-related gene signature for forecasting the prognosis of COAD has been constructed, which possesses favorable predictive ability and offers ideas for the necroptosis-associated development of COAD.

背景 坏死性凋亡（Necroptosis）是近年来发现的一种细胞死亡形式，在结肠腺癌（colon adenocarcinoma, COAD）的发生与发展中发挥关键作用。本研究旨在基于坏死性凋亡相关基因构建风险评分模型，以预测结肠腺癌患者的预后。
方法 从癌症基因组图谱（The Cancer Genome Atlas, TCGA）与基因型组织表达（Genotype-Tissue Expression, GTEx）数据库获取结肠腺癌及正常结肠样本的基因表达数据。基于预后相关坏死性凋亡差异表达基因（differentially expressed genes, DEGs），采用最小绝对收缩和选择算子（least absolute shrinkage and selection operator, LASSO）Cox回归分析计算风险评分。根据风险评分将患者划分为高风险组与低风险组，随后基于风险评分及临床病理特征构建列线图模型。本研究同时在基因表达综合数据库（Gene Expression Omnibus, GEO）中对所建模型进行验证。此外，通过"ESTIMATE"算法与单样本基因集富集分析（single-sample gene set enrichment analysis, ssGSEA）评估肿瘤微环境（tumor microenvironment, TME）及免疫浸润水平。开展功能富集分析以探究坏死性凋亡在结肠腺癌中发挥作用的潜在机制。最后，通过CCK8实验与Transwell实验探究坏死性凋亡对结肠癌细胞的影响。采用实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测经坏死性凋亡诱导剂（肿瘤坏死因子α, TNF-α）及抑制剂（NEC-1）处理后的结肠组织与细胞中，坏死性凋亡相关基因的表达水平。
结果 风险评分可作为结肠腺癌患者的独立预后风险因素。基于风险评分与临床病理特征构建的列线图，其预测价值优于TNM分期。该模型在GSE152430数据集中得到了良好验证。高风险组的免疫评分与基质评分显著升高。此外，坏死性凋亡可能通过调控癌症免疫应答影响结肠腺癌患者的预后。体外实验结果显示，抑制坏死性凋亡可增强结肠癌细胞的增殖与侵袭能力。最后，本研究发现16对配对结肠组织及结肠癌细胞中，坏死性凋亡相关基因存在差异表达。
结论 本研究构建了一种新型坏死性凋亡相关基因特征模型，可用于预测结肠腺癌患者的预后，该模型具备良好的预测能力，可为结肠腺癌坏死性凋亡相关的发病机制研究提供新思路。