基于加权基因共表达网络分析鉴定肾透明细胞癌免疫相关基因

Identification of immune-related genes in renal clear cell carcinoma based on weighted gene co-expression network analysis

目的应用加权基因共表达网络分析(WGCNA)方法鉴定肾透明细胞癌(ccRCC)的关键基因及免疫通路, 并进行初步验证。方法在癌症基因组图谱(TCGA)数据库下载ccRCC的RNA-seq表达数据和临床数据, 使用Wilcoxon检验和fold change方法筛选差异表达基因。基于Metascape对差异表达基因进行富集分析;使用R包survival功能进行生存分析;使用R包WGCNA功能进行WGCNA分析。取30例患者的肿瘤组织及正常肾组织, 实时荧光定量反转录聚合酶链反应(RT-qPCR)检测筛选出基因的mRNA水平。应用R软件分析网络数据, 采用t检验、Spearman相关分析等方法进行统计分析。结果肿瘤和正常肾组织的差异基因筛选结果显示, 肿瘤组织中有2 943个基因表达增高, 2 669个基因表达降低。对这些基因进行生存分析, 得到882个与患者预后显著相关的基因。进一步对影响生存的基因进行WGCNA分析, 得到5个重要网络模块, 富集分析结果显示turquoise模块与免疫功能相关。其中的免疫相关基因细胞毒性T淋巴细胞相关抗原4(CTLA4)、CD276、细胞间黏附分子-1(ICAM1)均与程序性死亡受体1(PD-1)表达正相关(r=0.80、0.24、0.46, 均P<0.01)。RT-qPCR结果显示, 肿瘤组织中PD-1、CTLA4、CD276、ICAM1的mRNA(2.74±0.90、3.42±1.00、1.57±0.13、0.86±0.20)均高于正常肾组织(0.73±0.19、0.41±0.15、0.34±0.13、0.23±0.05, t=11.56、15.75、35.40、16.17, 均P<0.01);CTLA4、ICAM1均与PD-1表达呈正相关(r=0.58、0.43, P<0.05)。结论免疫检查点基因CTLA4、ICAM1通过与PD-1的共同作用在ccRCC进展过程中发挥重要作用。

Objective To identify the key genes and immune pathways in clear cell renal carcino-ma(ccRCC)by using the weighted gene co-expression network analysis(WCCNA),and the preliminary verification was detected.Methods The RNA-seq expression data and clinical data of ccRCC were down-loaded from The cancer genome map(TCGA)database,and the differentially expressed genes(DEGs)were screened by Wilcoxon test and fold change method.Enrichment analysis of DEGs was done based on Metascape.The R package was used to carry out survival analysis.WGCNA analysis was also treated with R package.The mRNA levels of the screened genes were detected by fluorescence real-time quantitative re-verse transcription polymerase chain(RT-qPCR)in ccRCC tissues and normal kidney tissues of 30 pa-tients.The network data were analyzed by R package,and the clinical samples'verification data were ana-lyzed by SPSS 25.0 statistical software,then statistical analysis was carried out by t test and Spearman cor-relation analysis.Results The results of differential gene screening between tumor and normal kidney tis-sue showed that 2943 genes in ccRCC tissue were up-regulated and 669 genesdown-regulated.The survival analysis of these genes showed that 882 genes were significantly related to the prognosis of patients.There were 5 important network modules obtained with WGCNA analysis of survival related genes,and the enrich-ment analysis showed that the turquoise module was related to immune function,and among them,cytotoxic T lymphocyte-associated antigen 4(CTLA4),CD276 and intercellular adhesion molecule 1(ICAM1)were all positively correlated with programmed death receptor 1(PD-1)(r=0.80,0.24,0.46,all P<0.01).The results of RT-qPCR showed that the expression of PD-1,CTLA4,CD276 and ICAM1 RNA in ccRCC tissues(2.74±0.90,3.42±1.00,1.57±0.13,0.86±0.20)was higher than that in normal kidney tissues(0.73±0.19,0.41±0.15,0.34±0.13,0.23±0.05,t=11.56,15.75,35.40,t=16.17,all P<0.01).Both CTLA4 and ICAM1 were positively correlated with PD-1 expression(r=0.58,0.43,P<0.05).Conclusion Immunocheckpoint genes CTLA4 and ICAM1 may play an important role in the progress of ccRCC through their interaction with PD-1 gene.