P2RY8在肝细胞癌组织中表达的生物信息学分析及其临床意义

A bioinformatics analysis of P2RY8 expression in hepatocellular carcinoma and its clinical significance

目的本研究旨在探讨人B淋巴细胞限制受体P2RY8在肝细胞癌(HCC)诊断和预后中的应用价值,并分析了其与肿瘤免疫的关系。方法利用肿瘤基因组图谱(TCGA)数据库比较P2RY8的表达情况。采用R软件包分析P2RY8与肿瘤分期的相关性,建立诊断及生存的受试者工作特征(ROC)曲线和Nomogram模型。利用肿瘤免疫评估资源(TIMER)分析免疫细胞浸润、免疫细胞生物标志物和免疫检查点。使用STRING数据库分析蛋白质-蛋白质相互作用网络信息。通过基因本体论(GO)分析和京都基因与基因组百科全书(KEGG)分析P2RY8及其互作基因的功能。收集2007年6月—2008年11月行肝癌根治性手术的64例患者HCC组织及其中35例对应的癌旁组织(组织芯片购自上海芯超生物科技有限公司)进行验证,统计其临床资料及随访资料,采用免疫组化法检测HCC及癌旁组织标本中P2RY8的表达情况。符合正态分布的计量资料两组间比较采用t检验;非正态分布的计量资料两组间比较采用Mann-Whitney U检验,多组间比较采用Kruskal-Wallis H检验。计数资料两组间比较采用χ^(2)检验。两变量间的相关性采用Spearman相关分析。应用Kaplan-Meier绘制生存曲线,log-rank检验计算生存率。结果P2RY8在HCC中过表达,可通过P2RY8的表达水平准确鉴别肿瘤与正常组织(ROC曲线下面积为0.794)。P2RY8高表达的患者预后较好(P=0.005)。64例临床样本数据同样证实P2RY8高表达的肝癌患者术后3年生存率(78.9%vs 46.2%,P=0.007)、5年生存率(76.3%vs 38.5%,P=0.002)和OS[92.5(48.8~102.0)个月vs 33.0(25.0~95.5)个月,P=0.022]显著高于低表达者,Kaplan-Meier生存曲线进一步表明P2RY8表达水平与HCC患者预后相关。此外,P2RY8在HCC中与免疫细胞浸润和免疫检查点呈正相关。GO/KEGG通路富集分析显示P2RY8在体液免疫、细胞免疫等信号转导通路中均有富集。结论P2RY8不仅参与了HCC的发生发展,还参与其免疫调节。因此,P2RY8可以作为一个潜在的HCC诊断和预后的生物标志物和治疗靶点。

Objective To investigate the application value of the human B cell-confinement receptor P2RY8 in the diagnosis and prognosis of hepatocellular carcinoma(HCC)and its association with tumor immunity.Methods The Cancer Genome Atlas database was used to compare the expression of P2RY8.R software package was used to analyze the correlation between P2RY8 and tumor staging,and the receiver operating characteristic(ROC)curve and a nomogram model were established for diagnosis and survival.Tumor Immune Evaluation Resource was used to analyze immune cell infiltration,immune cell biomarkers,and immune checkpoints.STRING database was used to analyze protein-protein interaction network information.Gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were used to analyze the function of P2RY8 and its interacting genes.For the purpose of validation,HCC tissue samples were collected from 64 patients who underwent radical surgery for HCC from June 2007 to November 2008,and the corresponding adjacent tissue samples were collected from 35 patients out of these patients(tissue chips were purchased from Shanghai Outdo Biotech Co.,Ltd.);related clinical data and follow-up data were analyzed,and immunohistochemistry was used to measure the expression of P2RY8 in HCC and adjacent tissue samples.The t-test was used for comparison of normally distributed continuous data between two groups;the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups,and the Kruskal-Wallis H test was used for comparison between multiple groups.The chi-square test was used for comparison of categorical data between two groups.A Spearman correlation analysis was used to investigate the correlation between two variables.The Kaplan-Meier method was used to plot survival curves,and the log-rank test was used to calculate survival rates.Results P2RY8 was overexpressed in HCC,and the expression level of P2RY8 could accurately differentiate tumor from normal tissue,with an area under the ROC curve of 0.794.The patients with a higher expression level of P2RY8 tended to have a better prognosis(P=0.005).The data from 64 clinical samples also confirmed that compared with the patients with a low expression level of P2RY8,the patients with a high expression level of P2RY8 had significantly higher 3-year survival rate(78.9%vs 46.2%,P=0.007),5-year survival rate(76.3%vs 38.5%,P=0.002),and overall survival time[92.5(48.8-102.0)months vs 33.0(25.0-95.5)months,P=0.022],and the Kaplan-Meier survival curve further indicated that the expression level of P2RY8 was associated with the prognosis of HCC patients.In addition,P2RY8 was positively correlated with immune cell infiltration and immune checkpoint in HCC.The GO/KEGG pathway enrichment analyses showed that P2RY8 was enriched in the signal transduction pathways such as humoral immunity and cellular immunity.Conclusion P2RY8 participates in the development,progression,and immune regulation of HCC,and therefore,P2RY8 can serves as a potential biomarker and a therapeutic target for the diagnosis and prognosis of HCC.