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

梅儒齐; 鞠倩; 李曰平; 刘诚聪

doi:10.3969/j.issn.1001-5256.2022.08.020

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

DOI: 10.3969/j.issn.1001-5256.2022.08.020

梅儒齐¹,
鞠倩²,
李曰平²,
刘诚聪^2, , ,

1.
北京大学肿瘤医院暨北京市肿瘤防治研究所重症医学科，恶性肿瘤发病机制及转化研究教育部重点实验室，北京 100142
2.
青岛市中心医院胃肠外科，山东青岛 266000

基金项目:

青岛市民生科技计划项目 (15-9-2-80-nsh);

青岛市2020年度中医药科研计划项目 (2020-zyy026)

伦理学声明：本研究方案于2017年1月6日经由上海芯超生物科技有限公司伦理委员会审批，批号：SHYJS-CP-1701002，所纳入患者均签署知情同意书。

利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突，特此声明。

作者贡献声明：梅儒齐负责课题设计，资料分析，撰写论文；梅儒齐、鞠倩、李曰平参与收集数据，修改论文；刘诚聪负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
刘诚聪，84285161@163.com

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出版历程
- 收稿日期: 2022-04-27
- 录用日期: 2022-05-28
- 出版日期: 2022-08-20

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

1.
Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing 100142, China
2.
Department of Gastrointestinal Surgery, Qingdao Central Hospital, Qingdao, Shandong 266000, China

Research funding:

People's Livelihood Science and Technology Project of Qingdao （15-9-2-80-nsh）

Traditional Chinese Medicine Scientific Research Project of Qingdao in 2020 （2020-zyy026）

More Information

Corresponding author: LIU Chengcong, 84285161@163.com(ORCID: 0000-0001-7399-3312)

摘要

摘要: 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χ²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。
- 癌，肝细胞 /
- 人B淋巴细胞限制受体P2RY8 /
- 肿瘤基因组图谱 /
- 生物标记
Abstract: 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.
- Carcinoma, Hepatocellular /
- Human B cell restriction receptor P2RY8 /
- TCGA /
- Biomarkers

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图 1 P2RY8表达与HCC分期的关系

Figure 1. Relationship between P2RY8 expression levels and staging of hepatocellular carcinoma

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图 2 P2RY8表达与HCC预后的关系

注：P2RY8在HCC中高和低表达患者的OS(a)、疾病特异性生存期(b)和无进展间期(c)生存曲线；d，P2RY8表达的多因素Cox分析。

Figure 2. Relationship between P2RY8 expression levels and prognosis of hepatocellular carcinoma

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图 3 P2RY8的ROC曲线分析及Nomogram模型

注：a，诊断区分肿瘤与正常组织的ROC曲线；b，时间依赖生存ROC曲线分析预测1、3、5年生存率；c，Nomogram模型，结合临床分期和P2RY8水平预测1、3、5年生存率。

Figure 3. ROC analysis and Nomogram model of P2RY8

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图 4 HCC中P2RY8与免疫细胞浸润相关性的棒棒糖图

Figure 4. Correlations between P2RY8 expression levels and immune cell infiltration in hepatocellular carcinoma

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图 5 预测P2RY8高表达组与低表达组间免疫反应得分的分布情况

Figure 5. Predicting the distribution of immune response score between P2RY8 high expression group and low expression group

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图 6 HCC组织内P2RY8的表达情况(DAB显色+苏木紫染色，×200)

注：a，阴性表达；b，弱阳性表达；c，中阳性表达；d，强阳性表达。

Figure 6. Expression of P2RY8 in hepatocellular carcinoma (DAB+Sappanwood violet staining, ×200)

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图 7 64例HCC患者P2RY8表达水平与术后总生存期的Kaplan-Meier生存曲线分析

Figure 7. Kaplan-Meier survival curve analysis of P2RY8 expression level and postoperative overall survival in 64 HCC patients

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表 1 TCGA数据库中HCC的单因素和多因素生存分析

Table 1. Univariate and multivariate survival analysis of hepatocellular carcinoma

参数	例数	单因素分析		多因素分析
参数	例数	HR(95%CI)	P值	HR(95%CI)	P值
T分期	370
T1	183
T2	94	1.428 (0.901~2.264)	0.129	1.558 (0.865~2.808)	0.140
T3+T4	93	2.949 (1.982~4.386)	＜0.001	3.031 (1.832~5.016)	＜0.001
N分期	258
N0	254
N1	4	2.029 (0.497~8.281)	0.324
M分期	272
M0	268
M1	4	4.077 (1.281~12.973)	0.017	2.769 (0.826~9.283)	0.099
P2RY8	373	0.766 (0.627~0.935)	0.009	0.754 (0.589~0.966)	0.025

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表 2 HCC中P2RY8表达与免疫细胞标志物的相关性分析

Table 2. Correlation analysis of P2RY8 expression and immune cell markers in hepatocellular carcinoma

免疫细胞	生物标志物	r值	P值
B淋巴细胞	CD19	0.271	＜0.001
	CD20(KRT20)	-0.040	0.443
	CD38	0.315	＜0.001
CD8⁺T淋巴细胞	CD8A	0.379	＜0.001
	CD8B	0.321	＜0.001
Tfh	BCL6	0.086	0.095
	ICOS	0.364	＜0.001
	CXCR5	0.300	＜0.001
Th1	T-bet(TBX21)	0.451	＜0.001
	STAT1	0.250	＜0.001
	STAT4	0.237	＜0.001
	IL12RB2	0.267	＜0.001
	WSX1(IL27RA)	0.290	＜0.001
	IFNγ(IFNG)	0.230	＜0.001
	TNFα(TNF)	0.259	＜0.001
Th2	CCR3	0.064	0.215
	GATA3	0.369	＜0.001
	STAT5A	0.375	＜0.001
	STAT6	0.149	0.004
Th9	IRF4	0.390	＜0.001
	PU.1(SPI1)	0.346	＜0.001
	TGFBR2	0.444	＜0.001
Th17	IL-17A	0.127	0.014
	IL-21R	0.369	＜0.001
	IL-23R	0.279	＜0.001
	STAT3	0.269	＜0.001
Th22	AHR	0.109	0.035
	CCR10	0.110	0.034
Treg	CCR8	0.332	＜0.001
	CD25(IL2RA)	0.336	＜0.001
	FOXP3	0.194	＜0.001
M1巨噬细胞	COX2(PTGS2)	0.408	＜0.001
	INOS(NOS2)	0.247	＜0.001
	IRF5	0.147	0.004
M2巨噬细胞	ARG1	0.013	0.795
	CD206(MRC1)	0.376	＜0.001
	CD115(CSF1R)	0.420	＜0.001
肿瘤相关巨噬细胞	PDCD1LG2	0.466	＜0.001
	CD80	0.315	＜0.001
	CD40	0.132	0.011
	TLR7	0.400	＜0.001
自然杀伤细胞	CD7	0.230	＜0.001
	KIR3DL1	0.271	＜0.001
	XCL1	0.146	0.005
中性粒细胞	CD11b(ITGAM)	0.174	＜0.001
	CD15(FUT4)	0.151	0.004
	CD66b(CEACAM8)	-0.039	0.456
树突状细胞	CD1C	0.439	＜0.001
	CD11c(ITGAX)	0.336	＜0.001
	CD141(THBD)	0.537	＜0.001

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表 3 64例HCC患者中P2RY8表达水平与临床特征及预后之间的关系

Table 3. The relationship between P2RY8 expression level and clinical characteristics and outcomes in 64 HCC patients

参数	P2RY8表达水平		统计值	P值
参数	高表达组(n=38)	低表达组(n=26)	统计值	P值
年龄(岁)	52.3±9.0	53.0±10.4	t=0.318	0.751
男[例(%)]	34(89.5)	22(84.6)	χ²=0.333	0.564
既往HBV或HCV感染[例(%)]	31(81.6)	21(80.8)	χ²=0.804	0.804
肝硬化[例(%)]	33(86.8)	23(88.5)	χ²=0.406	0.524
AFP(μg/L)	122.5(7.8~929.5)	242.0(6.5~3133.0)	Z=-0.548	0.584
TBil(μmol/L)	12.3(9.9~20.9)	13.0(10.4~16.5)	Z=-0.358	0.720
ALT(U/L)	38.0(24.0~71.8)	41.0(21.0~70.0)	Z=-0.499	0.618
GGT(U/L)	46.0(31.8~108.8)	69.0(40.5~105.0)	Z=-1.131	0.258
Alb(g/dL)	4.4±0.6	4.2±0.4	t=1.286	0.203
肿瘤数目＞1[例(%)]	3(7.9)	3(11.5)	χ²=0.241	0.623
肿瘤大小(cm)	3.0(2.0~5.3)	6.0(3.8~8.3)	Z=-3.030	0.002
TNM分期Ⅱ~Ⅲ期[例(%)]	9(23.7)	10(38.5)	χ²=1.615	0.204
1年生存[例(%)]	37(97.4)	23(88.5)	χ²=2.090	0.148
3年生存[例(%)]	30(78.9)	12(46.2)	χ²=7.359	0.007
5年生存[例(%)]	29(76.3)	10(38.5)	χ²=9.293	0.002
PFS(月)	47.0(14.0~54.0)	21.0(8.0~53.0)	Z=-1.068	0.285
OS(月)	92.5(48.8~102.0)	33.0(25.0~95.5)	Z=-2.295	0.022

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