肿瘤坏死因子受体超家族成员21在肝细胞癌中的表达及临床意义

Expression and clinical significance of tumor necrosis factor receptor superfamily 21 gene in hepatocellular carcinoma

目的基于生物信息学分析探讨肿瘤坏死因子受体超家族成员21(TNFRSF21)在肝细胞癌(HCC)中的表达和预后价值、潜在的生物学功能及对免疫微环境的影响。方法从高通量基因表达(GEO)数据库获取HCC细胞株数据集筛选共交集基因。从癌症基因组图谱(TCGA)数据库获取HCC样本数据信息,分析TNFRSF21表达与总生存时间(OS)、临床病理特征的关系,以及在癌组织和癌旁组织中的表达情况。收集2009年7月至2010年10月手术切除的67例HCC组织及配对癌旁组织,采用实时荧光定量PCR(qPCR)检测TNFRSF21水平,分析HCC组织中TNFRSF21表达水平及与HCC患者OS的关系。通过STRING构建蛋白互作网络,采用基因本体(GO)和京都基因和基因组百科全书(KEGG)富集分析,探讨TNFRSF21的生物学功能和肿瘤相关通路。使用TIMER和TISIDB数据库分析HCC中TNFRSF21表达与肿瘤免疫浸润细胞的相关性。结果基于GEO数据集,筛选出CSN5基因敲低的HepG2、Huh7细胞株与COP1基因敲低的HepG2、Huh7细胞株共同的差异表达基因TNFRSF21、CROT、APPBP2和CPD。基于TCGA数据库,TNFRSF21在50例癌旁组织中的中位表达量为3.125,而在374例HCC组织中为3.953(P<0.001)。HCC组织中TNFRSF21 mRNA的相对表达量为1.77±1.60,高于配对癌旁组织的1.07±0.83(P=0.002)。生存分析显示,仅TNFRSF21基因表达与OS有关(P=0.030)。67例HCC患者中,TNFRSF21低表达组的中位OS未达,显著优于高表达组的35.0个月(P=0.039)。根据TCGA数据库TNFRSF21表达中位值2.678将374例HCC患者分为TNFRSF21高表达组和低表达组,发现TNFRSF21表达与血清AFP水平(P<0.001)和血管侵犯(P=0.029)有关。STRING及富集分析发现,TNFRSF21可能通过APP、TARDD等配体参与NF-κB及JNK信号通路调控肿瘤的发生发展。TIMER分析显示,TNFRSF21表达与B细胞(r=0.299,P<0.001)、CD8+T细胞(r=0.242,P<0.001)、CD4+T细胞(r=0.417,P<0.001)、巨噬细胞(r=0.527,P<0.001)、中性粒细胞(r=0.389,P<0.001)及树突细胞(r=0.363,P<0.001)免疫浸润呈正相关;TISIDB分析显示,TNFRSF21表达与活化的树突状细胞(r=0.271,P<0.001)、滤泡辅助性T细胞(r=0.333,P<0.001)、肥大细胞(r=0.270,P<0.001)和CD4+中央记忆性T细胞(r=0.393,P<0.001)丰度呈正相关。结论TNFRSF21在HCC组织中表达升高,高表达者预后不良。TNFRSF21可能通过内皮细胞坏死和肿瘤免疫浸润细胞改变肿瘤微环境,促进HCC的发生发展,有望成为HCC预后不良的分子标记物。

Objective To explore the expression of tumor necrosis factor receptor superfamily 21(TNFRSF21)in hepatocellular carcinoma(HCC),related biological function and its relationships with prognosis and immune system.Methods The data sets of HCC cell lines were obtained from Gene Expression Omnibus(GEO)database to screen the common differentially expressed genes.The data of HCC patients were obtained from The Cancer Genome Atlas(TCGA)database,the expression of TNFRSF21 of HCC and paracancerous tissues was compared,and the relationship between TNFRSF21 and overall survival(OS),as well as clinicopathological characteristics was analyed.Moreover,expression level of TNFRSF21 in 67 patients with HCC from July 2009 to October 2010 were detected by real-time quantitative PCR(qPCR).The relationship of expression level of TNFRSF21 with OS of HCC patients was analyzed.The TNFRSF21 protein-protein interaction network was constructed by STRING,and the biological functions and tumor-related pathways of TNFRSF21 were enriched by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).TIMER and TISIDB were used to analyze the correlation between TNFRSF21 and immune infiltrating cells in HCC.Results Common differentially expressed genes TNFRSF21,CROT,APPBP2 and CPD in HepG2 and Huh7 cell lines with CSN5 knockdown,as well as HepG2 and Huh7 cell lines with COP1 knockdown were screened based on GEO database.Based on TCGA database,the median mRNA of TNFRSF21 was 3.125 in paracancerous tissues and 3.953 in 374 HCC tissues(P<0.001).TNFRSF21 mRNA in HCC tissues was 1.77±1.60,which was higher than 1.07±0.83 in paracancerous tissues(P=0.002).Survival analysis showed that only TNFRSF21 gene expression level was correlated with OS(P=0.030).In 67 HCC patients,the median OS in the high-expression group was unreached,superior to 35.0 months in the low-expression group(P=0.039).According to TCGA database,374 HCC patients were divided into high and low expression groups based on the median TNFRSF21 expression of 2.678.It was found that TNFRSF21 expression level was significantly correlated with serum AFP level(P<0.001)and vascular invasion(P=0.029)of HCC patients.According to STRING and GO&KEGG enrichment analysis,TNFRSF21 may be involved in NF-κB and JNK signaling pathways through APP and TARDD,thus regulating the occurrence and development of tumors.TIMER analysis showed that the high expression of TNFRSF21 was associated with B cells(r=0.299,P<0.001),CD8+T cells(r=0.242,P<0.001),CD4+T cells(r=0.417,P<0.001),macrophages(r=0.527,P<0.001),neutrophils(r=0.389,P<0.001)and dendritic cells(r=0.363,P<0.001).TISIDB analysis showed that the expression of TNFRSF21 was positively correlated with the abundance of activated dendritic cells(r=0.271,P<0.001),follicular helper T cells(r=0.333,P<0.001),mast cells(r=0.270,P<0.001)and CD4+central memory T cells(r=0.393,P<0.001).Conclusion TNFRSF21 gene is up-regulated in HCC,and related to poor prognosis.TNFRSF21 may promote the occurrence and development of HCC by inducing endothelial cell necrosis and tumor microenvironment through tumor immune infiltrating cells.TNFRSF21 can be used as a molecular marker for prognosis in HCC.