焦亡相关基因与胃癌预后及免疫浸润的相关性分析

Correlation analysis of pyroptosis-related genes with prognosis and immune infiltration in gastric cancer

目的通过生物信息学筛选胃癌预后焦亡相关基因(PRGs),构建和评估风险模型并分析PRGs与免疫浸润的相关性,验证胃癌治疗的新靶点。方法从癌症基因组图谱(TCGA)数据库获取胃癌的转录组和临床数据并筛选差异表达的PRGs。采用单因素Cox回归及LASSO回归分析差异表达的PRGs并构建预后模型。Kaplan-Meier生存曲线、受试者操作特征曲线评估模型的准确性及预后价值。根据风险评分中位数将患者分为高、低风险组,Cox回归风险模型评估风险评分与预后的相关性。胃癌基因芯片数据集GSE84437作为预测模型的外部验证。RT-qPCR和Western blot检测SERPINE1、Caspase-1、NLRP3 mRNA和蛋白表达量。结果筛选35个差异表达PRGs,其中5个PRGs(CRTAC1、GPX3、IRGM、RGS2和SERPINE1)被纳入风险模型构建。年龄、M分期、N分期和风险评分是TCGA队列患者生存率的独立影响因素(P<0.05);年龄、T分期和N分期是基因表达综合(GEO)队列患者生存率的独立影响因素(P<0.05)。TCGA、GEO队列中高风险组免疫细胞浸润水平、免疫相关途径活性总体均高于低风险组(P<0.05)。CRTAC1与CD4+T细胞(r=0.488)、巨噬细胞(r=0.588)和树突状细胞(r=0.332)的浸润水平呈正相关(P<0.01);GPX3与CD4+T细胞(r=0.372)、巨噬细胞(r=0.572)和树突状细胞(r=0.406)的浸润水平呈正相关(P<0.01);RGS2与CD4+T细胞(r=0.343)、巨噬细胞(r=0.526)和树突状细胞(r=0.326)的浸润水平呈正相关(P<0.01);IRGM(r=0.327)、SERPINE1(r=0.310)与巨噬细胞的浸润水平呈正相关(P<0.01)。胃癌组织中SERPINE1表达水平高于正常胃组织(P<0.05);高表达SERPINE1组的生存时间短于低表达SERPINE1组(P<0.01)。干扰组SERPINE1、Caspase-1、NLRP3 mRNA和蛋白表达量均低于阴性对照组(P<0.05)。结论通过生物信息学方法筛选5个PRGs构建胃癌预后模型,SERPINE1高表达与胃癌不良预后相关,可能参与调控NLRP3/Caspase-1细胞焦亡途径。

Objective To screen prognostic pyroptosis-related genes(PRGs)in gastric cancer through bioinformatics,construct and evaluate risk models,analyze the correlation between PRGs and immune infiltration,and validate new targets for gastric cancer treatment.Methods The transcriptome and clinical data of gastric cancer were obtained from the cancer genome atlas(TCGA)database,and differentially expressed PRGs were screened.Univariate Cox regression and LASSO regression were used to analyze differentially expressed PRGs and construct a prognostic model.Kaplan-Meier survival curve and receiver operator characteristic curve were used to evaluate the accuracy and prognostic value of the model.Patients were divided into the high-risk group and the low-risk group based on the median risk score,and the correlation between risk score and prognosis was evaluated by using Cox regression risk model.The gastric cancer gene chip dataset GSE84437 was used as an external validation for prediction models.The mRNA and protein expression levels of SERPINE1,Caspase-1,and NLRP3 were detected by using RT-qPCR and Western blott.Results A total of 35 differentially expressed PRGs were screened,among which five PRGs(CRTAC1,GPX3,IRGM,RGS2,and SERPINE1)were included in the risk model construction.Age,M-stage,N-stage,and risk score were independent influencing factors for survival in patients in TCGA cohort(P<0.05);age,T-stage,and N-stage were independent influencing factors for survival in patients in gene expression omnibus(GEO)cohort(P<0.05).The immune cell infiltration level and immune related pathway generally activity of the high-risk group in TCGA and GEO cohorts were higher than those of the low-risk group(P<0.05).CRTAC1 was positively correlated with the infiltration levels of CD4+T cells(r=0.488),macrophages(r=0.588),and dendritic cells(r=0.332)(P<0.01);GPX3 was positively correlated with the infiltration levels of CD4+T cells(r=0.372),macrophages(r=0.572),and dendritic cells(r=0.406)(P<0.01);RGS2 was positively correlated with the infiltration levels of CD4+T cells(r=0.343),macrophages(r=0.526),and dendritic cells(r=0.326)(P<0.01);IRGM(r=0.327)and SERPINE1(r=0.310)were positively correlated with the infiltration level of macrophages(P<0.01).The expression level of SERPINE1 in gastric cancer tissue was higher than that in normal gastric tissue(P<0.05);the survival time of the high expression SERPINE1 group was shorter than that of the low expression SERPINE1 group(P<0.01).The mRNA and protein expression levels of SERPINE1,Caspase-1,and NLRP3 in the interference group were lower than those in the negative control group(P<0.05).Conclusion Screening five PRGs by using bioinformatics methods to construct a gastric cancer prognosis model.High expression of SERPINE1 is associated with poor prognosis of gastric cancer and may be involved in regulating the NLRP3/Caspase-1 cell pyroptosis pathway.