基于生物信息学分析Midkine在胆管癌中的表达及其对预后的预测价值

谷月月; 于书敏; 常秀娟; 高旭东; 黄加干; 贾晓东; 曾珍

doi:10.12449/JCH240722

基于生物信息学分析Midkine在胆管癌中的表达及其对预后的预测价值

DOI: 10.12449/JCH240722

谷月月^{1, 2},
于书敏³,
常秀娟²,
高旭东²,
黄加干²,
贾晓东⁴,
曾珍^{1, 2, 3, , ,}

1.
安徽医科大学解放军307临床学院，安徽医科大学第五临床医学院，合肥 230032
2.
中国人民解放军总医院第五医学中心肝病医学部，北京 100039
3.
北京大学三〇二临床医学院，北京 100039
4.
中国人民解放军总医院第五医学中心肿瘤医学部，北京 100071

基金项目:

国家“十三五”科技重大专项课题 (2018ZX10725-506-002);

国家卫生健康委医药卫生科技发展研究中心 (WKZX2023CX020011);

北京市自然科学基金 (7222172)

伦理学声明：本研究方案于2022年12月14日经由中国人民解放军总医院第五医学中心伦理委员会审批，批号：KY-2022-12-76-1，所纳入患者均签署知情同意书。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：谷月月负责课题设计，资料分析，撰写论文；于书敏、常秀娟、高旭东、黄加干、贾晓东参与收集数据，修改论文；曾珍负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
曾珍， zengzhen1970@sina.com （ORCID： 0000-0002-1574-7196）

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出版历程
- 收稿日期: 2024-02-01
- 录用日期: 2024-04-25
- 出版日期: 2024-07-25

Expression of Midkine in cholangiocarcinoma and its value in predicting prognosis based on bioinformatics analysis

Yueyue GU^{1, 2},
Shumin YU³,
Xiujuan CHANG²,
Xudong GAO²,
Jiagan HUANG²,
Xiaodong JIA⁴,
Zhen ZENG^{1, 2, 3
, ,
,}

1.
The PLA 307 Clinical College of Anhui Medical University，The Fifth Clinical Medical College of Anhui Medical University，Hefei 230032，China
2.
Senior Department of Hepatology，The Fifth Medical Center of Chinese PLA General Hospital，Beijing 100039，China
3.
Peking University 302 Clinical Medical School，Beijing 100039，China
4.
Senior Department of Oncology，The Fifth Medical Center of Chinese PLA General Hospital，Beijing 100071，China

Research funding:

National Science and Technology Major Project of China (2018ZX10725-506-002);

National Health Commission Medical and Health Science and Technology Development Research Center (WKZX2023CX020011);

Beijing Municipal Natural Science Foundation (7222172)

More Information

Corresponding author: ZENG Zhen， zengzhen1970@sina.com （ORCID： 0000-0002-1574-7196）

摘要

摘要: 目的研究中期因子（MDK）在胆管癌（CCA）中的表达及其对患者预后的预测价值，探索MDK可能影响CCA进展的机制。方法从癌症基因组图谱（TCGA）数据库获取CCA样本数据信息，分析MDK在癌与癌旁组织中的表达差异及其与临床特征的相关性，使用基因表达综合数据库（GEO）和解放军总医院第五医学中心2018年6月—2021年9月经手术切除的11例CCA患者的临床资料进行相关验证。通过STRING和Cytoscape构建蛋白互作网络，使用基因本体（GO）和京都基因与基因组数据库（KEGG）进行富集分析，探讨MDK相关基因所参与的生物学过程和肿瘤相关通路。此外，应用TIMER和TISIDB数据库分析CCA肿瘤组织中MDK的表达与免疫细胞浸润的相关性。计量资料两组间比较采用成组t检验或Mann-Whitney U检验。计数资料组间比较采用Fisher精确概率法。应用Kaplan-Meier法绘制生存曲线，并采用Log-rank检验进行组间比较。两变量间的相关性采用Spearman相关性分析。结果比较TCGA数据库中CCA患者肿瘤组织及癌旁组织中MDK的表达水平，非配对分析结果和配对分析结果均显示，MDK在CCA肿瘤组织中的表达水平高于癌旁组织（P值均<0.001）；对纳入的11例CCA患者肿瘤组织及其配对癌旁组织标本进行转录组测序，结果显示，MDK在CCA肿瘤组织中的表达水平显著高于其配对癌旁组织（P<0.01）。MDK高表达与淋巴结转移（P=0.045）和血管侵犯（P=0.044）有关。生存分析显示，MDK高表达的CCA患者总生存期（χ²=5.30，P=0.028）及疾病特异性生存期（χ²=6.25，P=0.019）均明显短于MDK低表达的患者。GO和KEGG富集分析显示，MDK表达相关的30个基因与泛素介导的蛋白质水解过程密切相关，并影响CCA患者预后；TIMER分析结果显示，MDK表达与B淋巴细胞（r=0.356，P=0.035 6）和树突状细胞（r=0.409，P=0.014 7）在肿瘤组织微环境中的浸润呈正相关；TISIDB分析结果显示，MDK表达水平与CXCL16（r=0.465，P=0.004 67）呈正相关，与CXCL12（r=-0.389，P=0.019 7）及CXCR5（r=-0.393，P=0.018 5）呈负相关；与免疫检查点调节剂VTCN1（r=-0.393，P=0.018 3）、LTA（r=-0.380，P=0.022 7）和PVR（r=-0.350，P=0.037 3）呈负相关。结论 MDK高表达提示CCA患者预后不佳，MDK有潜力成为预测CCA患者预后的分子标志物，其可能通过调控泛素介导的蛋白质水解过程及调控B淋巴细胞和树突状细胞的浸润促进CCA的发生发展。
- 胆管上皮癌 /
- 中期因子 /
- 预后
Abstract: Objective To investigate the expression of Midkine （MDK） in cholangiocarcinoma （CCA） and its value in predicting the prognosis of CCA， as well as the potential mechanism of the effect of MDK on the progression of CCA. Methods The data of CCA samples were obtained from TCGA database to analyze the difference in the expression of MDK between cancer tissue and paracancerous tissue and its association with clinical features， and the data collected from GEO database and 11 CCA patients who underwent surgical resection in The Fifth Medical Center of Chinese PLA General Hospital from June 2018 to September 2021 were used for validation. STRING and Cytoscape were used to construct a protein-protein interaction network， and gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analyses were used to investigate the biological functions and tumor-related pathways involving MDK-related genes. In addition， TIMER and TISIDB databases were used to analyze the correlation between MDK expression and immune cell infiltration in CCA tissue. The independent-samples t test or the Mann-Whitney U test was used for comparison of continuous data between two groups， and the Fisher’s exact test was used for comparison of categorical data between two groups. The Kaplan-Meier method was used to plot survival curves， and the Log-rank test was used for comparison between groups. The Spearman correlation analysis was used to investigate the correlation between two variables. Results The expression level of MDK in cancer tissue and paracancerous tissue of CCA patients was compared based on TCGA database， and the results of the non-paired and paired analyses showed that the expression level of MDK in CCA tumor tissue was significantly higher than that in paracancerous tissue （P<0.001）. Transcriptome sequencing was performed for the tumor tissue and its corresponding paracancerous tissue from 11 CCA patients， and the results showed that the expression level of MDK in CCA tumor tissue was significantly higher than that in corresponding paracancerous tissue （P<0.01）. High expression of MDK was associated with lymph node metastasis （P=0.045） and vascular invasion （P=0.044）. Survival analysis showed that compared with the CCA patients with low MDK expression， the CCA patients with high MDK expression had significantly shorter overall survival time （χ²=5.30， P=0.028） and disease-specific survival time （χ²=6.25， P=0.019）. The GO and KEGG enrichment analyses showed that the 30 MDK-related genes were closely associated with ubiquitin-mediated proteolysis and affected the prognosis of CCA patients. The TIMER analysis showed that the expression level of MDK was positively correlated with the infiltration of B cells （r=0.356， P=0.035 6） and dendritic cells （r=0.409， P=0.014 7） in tumor microenvironment of CCA； the TISIDB analysis showed that the expression level of MDK was positively correlated with CXCL16 （r=0.465， P=0.004 67） and was negatively correlated with CXCL12 （r=-0.389， P=0.019 7） and CXCR5 （r=-0.393， P=0.018 5）， and it was also negatively correlated with the immune checkpoint regulators VTCN1 （r=-0.393， P=0.018 3）， LTA （r=-0.380， P=0.022 7）， and PVR （r=-0.350， P=0.037 3）. Conclusion High expression of MDK is associated with poor prognosis in CCA patients， and MDK has the potential of being used as a molecular marker for predicting the prognosis of CCA. MDK may promote the development and progression of CCA by regulating ubiquitin-mediated proteolysis and the infiltration of B cells and dendritic cells.
- Cholangiocarcinoma /
- Midkine /
- Prognosis

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图 1 MDK在CCA患者癌组织和癌旁组织中的表达情况

注： a，MDK在CCA中表达情况的非配对分析（TCGA数据库）；b，MDK在CCA中表达情况的配对分析（TCGA数据库）；c，MDK在11例CCA患者中表达情况的配对分析（临床病例）；d，MDK在CCA中表达情况的配对分析（GSE132305数据集）。

Figure 1. The expression level of MDK in cancerous and paracancerous tissues of CCA patients

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图 2 CCA患者MDK表达与预后分析

注： a，不同MDK表达水平患者的总生存期比较；b，不同MDK表达水平患者的疾病特异性生存期比较。

Figure 2. The survival analysis of the prognostic significance of MDK expression in cholangiocarcinoma patients

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图 3 MDK在CCA中的诊断和预后价值

注： a，MDK诊断CCA的ROC曲线；b，时间依赖性ROC曲线；c，预后列线图。

Figure 3. The diagnostic and prognostic value of MDK expression in cholangiocarcinoma

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图 4 MDK相关基因的功能聚类及互作网络分析

注： a，与MDK表达正相关的前25个基因热图；b，与MDK表达负相关的前25个基因热图；c，MDK相关基因功能富集分析；d、e，MDK相关基因和CCA生存相关基因韦恩图及功能富集分析；f，MDK相关基因相互作用网络；g，MDK相关基因之间的相关性热图。

Figure 4. MDK functional clustering and interaction network analysis of MDK-related genes

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图 5 CCA肿瘤组织中MDK表达与肿瘤浸润免疫细胞的相关性

Figure 5. The correlation between MDK expression and tumor-infiltrating immune cells in cholangiocarcinoma

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图 6 CCA肿瘤组织中MDK表达与趋化因子和趋化因子受体的相关性分析

注： a，肿瘤组织中MDK表达与趋化因子相关性热图；b，肿瘤组织中MDK表达与趋化因子受体相关性热图；c～e，CCA中MDK表达与CXCL16、CXCL12及CXCR5相关。

Figure 6. Correlation analysis between MDK expression and chemokines and chemokine receptors in CCA tumor tissue

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图 7 CCA肿瘤组织中MDK表达与免疫抑制剂和免疫增强剂的相关性分析

注： a，肿瘤组织中MDK表达与免疫抑制剂的相关性热图；b，肿瘤组织中MDK表达与免疫增强剂的相关性热图；c～e，CCA中MDK表达与VTCN1、LTA及PVR相关。

Figure 7. Correlation analysis between MDK expression and immunoinhibitors and immunostimulators in CCA tumor tissue

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表 1 TCGA数据库中MDK表达与CCA患者临床特征的关系

Table 1. The relationship of MDK expression and clinical characteristics in patients with cholangiocarcinoma from TCGA

临床指标	低表达（n=17）	高表达（n=18）	P值
T分期［例（%）］			>0.05
T1期	9（52.9）	9（50.0）
T2期	6（35.3）	6（33.3）
T3/T4期	2（11.8）	3（16.7）
淋巴结转移［例（%）］^1）			0.045
否	14（100.0）	11（68.8）
是	0（0.0）	5（31.3）
M分期［例（%）］^2）			>0.05
M0期	13（86.7）	14（82.4）
M1期	2（13.3）	3（17.6）
性别［例（%）］			>0.05
女	9（52.9）	10（55.6）
男	8（47.1）	8（44.4）
年龄［例（%）］			0.318
≤65岁	10（58.8）	7（38.9）
>65岁	7（41.2）	11（61.1）
BMI［例（%）］^3）			>0.05
≤25 kg/m²	5（31.3）	5（27.8）
>25 kg/m²	11（68.8）	13（72.2）
血管侵犯［例（%）］^4）			0.044
否	16（100.0）	12（70.6）
是	0（0.0）	5（29.4）
注：1） MDK低表达组3例患者数据缺失，高表达组2例患者数据缺失；2） MDK低表达组2例患者数据缺失，高表达组1例患者数据缺失；3） MDK低表达组1例患者数据缺失；4） MDK低表达组和高表达组各有1例患者数据缺失。

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表 2 临床收集的11例CCA患者肿瘤组织中MDK的表达与临床特征参数的关系

Table 2. The relationship of MDK expression and clinical characteristics in 11 patients with cholangiocarcinoma

临床指标	例数	MDK表达水平	t值	P值
年龄			0.567	0.586
≤60岁	7	3.71±1.95
>60岁	4	4.18±0.77
性别			1.487	0.171
女	5	4.62±1.05
男	6	3.27±1.78
BMI			2.543	0.075
≤25 kg/m²	7	3.15±1.47
>25 kg/m²	4	5.17±0.74
肿瘤直径			0.538	0.601
≤5 cm	3	3.39±0.88
>5 cm	8	4.07±1.80
肿瘤数目			1.146	0.282
单发	3	3.00±1.44
多发	8	4.22±1.59
淋巴结转移			2.416	0.039
否	3	2.34±1.72
是	8	4.47±1.15
血管侵犯			0.578	0.577
否	9	4.02±1.41
是	2	3.28±2.88

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表 3 TCGA数据库中CCA患者的单因素Cox回归分析

Table 3. Univariate Cox regression analyses of cholangiocarcinoma from TCGA

参数	例数	HR（95%CI）	P值
T分期
T1/T2期	30	1.000
T3/T4期	5	0.615（0.140～2.700）	0.520
淋巴结转移^1）
否	25	1.000
是	5	2.147（0.565～8.154）	0.262
M分期^2）
M0期	27	1.000
M1期	5	1.531（0.428～5.475）	0.513
年龄
≤65岁	17	1.000
>65岁	18	1.461（0.570～3.747）	0.430
性别
女	19	1.000
男	16	1.279（0.503～3.255）	0.605
BMI^3）
≤25 kg/m²	10	1.000
>25 kg/m²	24	0.552（0.200～1.527）	0.253
血管侵犯^4）
否	28	1.000
是	5	1.665（0.461～6.012）	0.436
神经侵犯^5）
否	25	1.000
是	7	4.056（1.128～14.582）	0.032
MDK
低表达	17	1.000
高表达	18	3.374（1.141～9.976）	0.028
注：1） 5例患者淋巴结转移数据缺失；2） 3例患者M分期数据缺失；3） 1例患者BMI数据缺失；4） 2例患者血管侵犯数据缺失；5） 3例患者神经侵犯数据缺失。

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