Table4_Generation and Analysis of Pyroptosis-Based and Immune-Based Signatures for Kidney Renal Clear Cell Carcinoma Patients, and Cell Experiment.DOCX

Background: Pyroptosis is a programmed cell death caused by inflammasomes, which is closely related to immune responses and tumor progression. The present study aimed to construct dual prognostic indices based on pyroptosis-associated and immune-associated genes and to investigate the impact of the biological signatures of these genes on Kidney Renal Clear Cell Carcinoma (KIRC).
Materials and Methods: All the KIRC samples from the Cancer Genome Atlas (TCGA) were randomly and equally divided into the training and testing datasets. Cox and Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis were used to screen crucial pyroptosis-associated genes (PAGs), and a pyroptosis-associated genes prognostic index (PAGsPI) was constructed. Immune-associated genes (IAGs) related to PAGs were identified, and then screened through Cox and LASSO regression analyses, and an immune-associated genes prognostic index (IAGsPI) was developed. These two prognostic indices were verified by using the testing and the Gene Expression Omnibus (GEO) datasets and an independent cohort. The patients’ response to immunotherapy was analyzed. A nomogram was constructed and calibrated. qRT-PCR was used to detect the expression of PAGs and IAGs in the tumor tissues and normal tissues. Functional experiment was carried out.
Results: 86 PAGs and 1,774 differentially expressed genes (DEGs) were obtained. After intersecting PAGs with DEGs, 22 differentially expressed PAGs (DEPAGs) were included in Cox and LASSO regression analyses, identifying 5 crucial PAGs. The PAGsPI was generated. Patients in the high-PAGsPI group had a poor prognosis. 82 differentially expressed IAGs (DEIAGs) were highly correlated with DEPAGs. 7 key IAGs were screened out, and an IAGsPI was generated. Patients in the high-IAGsPI group had a poor prognosis. PAGsPI and IAGsPI were verified to be robust and reliable. The results revealed patients in low-PAGsPI group and high-IAGsPI group may be more sensitive to immunotherapy. The calibrated nomogram was proved to be reliable. An independent cohort study also proved that PAGsPI and IAGsPI performed well in prognosis prediction. We found that the expression of AIM2 may affect proliferation of KIRC cells.
Conclusion: PAGsPI and IAGsPI could be regarded as potential biomarkers for predicting the prognosis of patients with KIRC.

背景：细胞焦亡（Pyroptosis）是一种由炎症小体介导的程序性细胞死亡，与免疫应答及肿瘤进展密切相关。本研究旨在基于细胞焦亡相关基因与免疫相关基因构建双重预后指标，并探究这些基因的生物学特征在肾透明细胞癌（Kidney Renal Clear Cell Carcinoma, KIRC）中的影响。
材料与方法：将癌症基因组图谱（The Cancer Genome Atlas, TCGA）中所有肾透明细胞癌样本随机均分为训练集与测试集。采用Cox回归与最小绝对收缩和选择算子（Least Absolute Shrinkage and Selection Operator, LASSO）回归分析筛选关键细胞焦亡相关基因（pyroptosis-associated genes, PAGs），并构建细胞焦亡相关基因预后指数（pyroptosis-associated genes prognostic index, PAGsPI）。筛选与PAGs相关的免疫相关基因（immune-associated genes, IAGs），再通过Cox回归与LASSO回归分析进一步筛选，进而构建免疫相关基因预后指数（immune-associated genes prognostic index, IAGsPI）。利用测试集、基因表达综合数据库（Gene Expression Omnibus, GEO）数据集及独立队列对这两种预后指数进行验证，并分析患者对免疫治疗的响应情况。构建并校准列线图（nomogram）。采用定量实时聚合酶链反应（quantitative Real-Time Polymerase Chain Reaction, qRT-PCR）检测肿瘤组织与正常组织中PAGs与IAGs的表达水平，并开展功能实验。
结果：共获得86个PAGs与1774个差异表达基因（differentially expressed genes, DEGs）。将PAGs与DEGs取交集后，纳入22个差异表达PAGs（differentially expressed PAGs, DEPAGs）进行Cox与LASSO回归分析，最终筛选得到5个关键PAGs，并生成PAGsPI。高PAGsPI组患者预后较差。共获得82个与DEPAGs高度相关的差异表达IAGs（differentially expressed IAGs, DEIAGs），经筛选得到7个关键IAGs并生成IAGsPI，高IAGsPI组患者同样预后较差。经验证，PAGsPI与IAGsPI具备良好的稳定性与可靠性。结果显示，低PAGsPI组与高IAGsPI组患者可能对免疫治疗更为敏感。校准后的列线图表现可靠，独立队列研究同样证实PAGsPI与IAGsPI在预后预测中表现优异。本研究发现AIM2的表达可能影响肾透明细胞癌细胞的增殖能力。
结论：PAGsPI与IAGsPI可作为预测肾透明细胞癌患者预后的潜在生物标志物。