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

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

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MannWhitney 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(χ^(2)=5.30,P=0.028)及疾病特异性生存期(χ^(2)=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=0.0356)和树突状细胞(r=0.409,P=0.0147)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=0.00467)呈正相关,与CXCL12(r=-0.389,P=0.0197)及CXCR5(r=-0.393,P=0.0185)呈负相关;与免疫检查点调节剂VTCN1(r=-0.393,P=0.0183)、LTA(r=-0.380,P=0.0227)和PVR(r=-0.350,P=0.0373)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。

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(χ^(2)=5.30,P=0.028)and disease-specific survival time(χ^(2)=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=0.0356)and dendritic cells(r=0.409,P=0.0147)in tumor microenvironment of CCA;the TISIDB analysis showed that the expression level of MDK was positively correlated with CXCL16(r=0.465,P=0.00467)and was negatively correlated with CXCL12(r=-0.389,P=0.0197)and CXCR5(r=-0.393,P=0.0185),and it was also negatively correlated with the immune checkpoint regulators VTCN1(r=-0.393,P=0.0183),LTA(r=-0.380,P=0.0227),and PVR(r=-0.350,P=0.0373).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.