NTN4对肾透明细胞癌的临床应用价值

Clinical application value of NTN4 in clear cell renal cell carcinoma

目的探讨神经导向因子4(NTN4)在肾透明细胞癌(ccRcc)中的临床应用价值。方法运用TCGA、UALCAN和HPA数据库,比较NTN4的信使RNA(mRNA)在癌旁组织及ccRcc组织中的表达水平,以及NTN4蛋白在正常肾脏组织和ccRcc组织中的表达水平,采用survminer包的surv_cutpoint()函数确定NTN4表达水平的最佳截断值,并以此为截点将患者分为NTN4高表达组与NTN4低表达组,并分析NTN4表达水平与ccRcc患者临床特征的关系及其对ccRcc患者预后的影响。绘制受试者工作特征(ROC)曲线分析NTN4对ccRcc的诊断价值。采用Kaplan-Meier生存曲线和Log-Rank检验分析NTN4高表达组和NTN4低表达组的生存情况。采用单因素和多因素Cox回归分析ccRcc预后的危险因素。比较ccRcc组织和正常肾脏组织的NTN4的启动子甲基化水平(Beta值),利用基因集富集分析(GSEA)探讨NTN4在ccRcc组织中的潜在机制并构建列线图预测模型,且采用Calibration校准曲线进行模型验证。结果从TCGA数据库中筛选出72例癌旁组织和533例ccRcc组织样本数据(其中72例样本为配对组织)。533例ccRcc组织中NTN4的mRNA表达水平低于癌旁组织,差异有统计学意义(P<0.05);72例配对ccRcc组织中NTN4的mRNA表达水平低于癌旁组织,差异有统计学意义(P<0.05)。UALCAN数据库分析结果显示,ccRcc组织中NTN4蛋白表达水平低于正常肾脏组织,差异有统计学意义(P<0.05)。NTN4高表达组有384例患者,NTN4低表达组有149例患者。NTN4低表达组和NTN4高表达组ccRcc患者组织学分级、病理学分期、肿瘤大小及局部浸润范围(T分期)、远处转移情况(M分期)比较,差异均有统计学意义(P<0.05)。ROC曲线分析结果显示,NTN4诊断ccRcc的曲线下面积为0.881(95%CI:0.847~0.916),最佳截断值为6.68,灵敏度为0.65,特异度为0.94。Kaplan-Meier生存曲线分析结果显示,NTN4高表达组ccRcc患者的预后情况优于NTN4低表达组ccRcc患者(P<0.05)。单因素与多因素Cox回归分析结果显示,NTN4低表达、年龄>60岁、M分期为M1是ccRcc患者预后不良的独立危险因素(P<0.05)。与正常肾脏组织相比,ccRcc组织中NTN4的Beta值明显升高(P<0.05)。列线图模型的Calibration校准曲线分析结果显示,列线图模型的C-index为0.728。GSEA结果显示,NTN4相关基因主要富集在RNA剪接、胞吞作用、泛素介导的蛋白质降解、神经营养因子信号通路等相关途径。结论NTN4在ccRcc组织中表达水平下降,且有望作为预测ccRcc患者预后的生物标志物。

Objective To investigate the clinical application value of nerve guidance factor 4(NTN4)in clear cell renal cell carcinoma(ccRcc).Methods TCGA,UALCAN and HPA databases were used to compare the expression level of NTN4 messenger RNA(mRNA)in adjacent tissues and ccRcc tissues,and the expression level of NTN4 protein in normal kidney tissues and ccRcc tissues.The surv_cutpoint()function of survminer package was used to determine the best cut-off value of NTN4 expression level,and the patients were divided into NTN4 high expression group and NTN4 low expression group according to the best cut-off value.The relationship between NTN4 expression level and clinical characteristics of ccRcc patients and its effect on prognosis of ccRcc patients were analyzed.Receiver operating characteristic(ROC)curve was drawn to analyze the diagnostic value of NTN4 in ccRcc patients.Kaplan-Meier survival curve and Log-Rank test were used to analyze the survival of NTN4 high expression group and NTN4 low expression group.Univariate and multivariate Cox regression analysis were used to analyze the risk factors of ccRcc prognosis.NTN4 promoter methylation level(Beta value)was compared between ccRcc and normal kidney tissues.Gene set enrichment analysis(GSEA)was used to explore the potential mechanism of NTN4 in ccRcc tissues and construct a nomogram prediction model,and Calibration curve was used to verify the model.Results A total of 72 adjacent tissues and 533 ccRcc tissues(including 72 paired tissues)were selected from TCGA database.The expression level of NTN4 mRNA in 533 ccRcc tissues was significantly lower than that in adjacent tissues(P<0.05).The expression level of NTN4 mRNA in 72 ccRcc tissues was significantly lower than that in adjacent non-cancerous tissues(P<0.05).The results of UALCAN database analysis showed that the expression level of NTN4 protein in ccRcc tissues was significantly lower than that in normal kidney tissues(P<0.05).There were 384 patients in the NTN4 high expression group and 149 patients in the NTN4 low expression group.There were significant differences in histological grade,pathological stage,tumor size,local invasion(T stage)and distant metastasis(M stage)between NTN4 low expression group and NTN4 high expression group(P<0.05).ROC curve analysis showed that the area under the curve of NTN4 for the diagnosis of ccRcc was 0.881(95%CI:0.847-0.916)The best cut-off value was 6.68,the sensitivity was 0.65,and the specificity was 0.94.Kaplan-Meier survival curve analysis showed that the prognosis of ccRcc patients in the NTN4 high expression group was better than that in the NTN4 low expression group(P<0.05).Univariate and multivariate Cox regression analysis showed that low expression of NTN4,age>60 years and M stage of M1 were independent risk factors for poor prognosis of ccRcc patients(P<0.05).Compared with normal kidney tissues,the Beta value of NTN4 was significantly increased in ccRcc tissues(P<0.05).The Calibration curve analysis results of the nomogram prediction model showed that the C-index of the nomogram model was 0.728.GSEA results showed that NTN4-related genes were mainly enriched in RNA splicing,endocytosis,ubiquitin-mediated protein degradation,neurotrophic factor signaling pathway and other related pathway.Conclusion The expression of NTN4 is decreased in ccRcc,and it may be used as a biomarker for predicting the prognosis of ccRcc patients.