基于数据库挖掘分析微RNA-129-1在结肠癌中的表达和意义

Expression and significance of microRNA-129-1 in colon cancer based on database mining

目的探讨微RNA(miRNA)-129-1在结肠癌中的表达、调控、潜在作用机制和临床意义。方法利用癌症基因组图谱(TCGA)数据库中结肠癌的甲基化、mRNA表达和miRNA表达数据,分析miRNA-129-1表达和甲基化的变化。利用miRwalk 2.0和TargetScan数据库联合预测miRNA-129-1靶基因,采用DAVID 6.7在线软件对靶基因进行基因本体和京都基因与基因组百科全书富集分析,利用STRING数据库进行蛋白质-蛋白质相互作用网络分析,并再次利用TCGA数据库对miRNA-129-1的关键靶基因进行表达差异分析和预后分析。统计学方法采用配对t检验、独立样本t检验,通过受试者操作特征曲线(ROC)评估miRNA-129-1甲基化对结肠癌的诊断价值,采用Kaplan-Meier法和log-rank检验分析miRNA-129-1表达对患者生存的影响。结果miRNA-129-1在各物种之间序列保守。对TCGA数据库所有结肠癌肿瘤样本和对照样本进行分析,结果显示肿瘤样本中miRNA-129-1的表达与对照样本比较降低(0.98±0.81比5.74±0.59),cg04524088、cg04840800、cg11364290、cg20734982、cg24044186位点的甲基化水平与对照样本比较均降低(0.321±0.130比0.563±0.051、0.432±0.123比0.624±0.064、0.475±0.153比0.768±0.033、0.659±0.180比0.816±0.037、0.862±0.096比0.916±0.019),差异均有统计学意义(t=14.95、11.36、9.39、11.74、5.32、3.47,P均<0.01)。ROC分析结果显示,miRNA-129-1上述5个位点的甲基化水平对结肠癌有较高的诊断效能[曲线下面积分别为0.946、0.915、0.950、0.758、0.667,P均<0.01]。生存分析结果显示,miRNA-129-1低表达与不良预后有关(风险比为0.55,P=0.018)。生物信息学分析发现miRNA-129-1的靶基因显著富集于丝氨酸/苏氨酸激酶受体、丝裂原活化蛋白激酶等与肿瘤密切相关的功能基因簇,其靶基因编码蛋白质之间存在复杂的相互作用网络。miRNA-129-1的潜在关键靶基因肝配蛋白B型受体2(EPHB2)基因高表达与总生存期和无病生存期短有关(风险比分别为1.9和1.6,P均<0.01)。结论miRNA-129-1的表达和甲基化在结肠癌的发生、发展中起着重要调控作用,miRNA-129-1甲基化在结肠癌诊断中具有潜在价值,miRNA-129-1是结肠癌患者预后的影响因素。EPHB2可能是miRNA-129-1的潜在关键靶基因。

Objective To investigate the expression,regulation,potential mechanism and clinical significance of microRNA(miRNA)-129-1 in colon cancer.Methods The changes of expression and methylation of miRNA-129-1 were analyzed from the methylation,mRNA expression and miRNA expression data of colon cancer in the cancer genome atlas(TCGA)database.The target genes of miRNA-129-1 were predicted from miRwalk 2.0 and TargetScan database.DAVID 6.7 online software was used for gene oncology and Kyoto encyclopedia of genes and genomes enrichment analysis.STRING database was used for protein-protein interaction analysis.TCGA data were applied again to analyze the differential expression and prognosis of key target genes of miRNA-129-1.Paired t test and independent sample t test were used for statistical analysis.The receiver operating characteristic curve(ROC)was used to evaluate the diagnostic value of miRNA-129-1 gene methylation in colon cancer.Kaplan-Meier method and log-rank test were used to analyze the effects of miRNA-129-1 expression on survival.Results The sequence of miRNA-129-1 among different species was conserved.After all colon cancer samples,and control samples of TCGA database were analyzed,the results showed that compared with those of control samples,the expression of miRNA-129-1 decreased in cancer samples(0.98±0.81 vs.5.74±0.59),and the methylation levels of cg04524088,cg04840800,cg11364290,cg20734982 and cg24044186 locus of miRNA-129-1 significantly decreased(0.321±0.130 vs.0.563±0.051,0.432±0.123 vs.0.624±0.064,0.475±0.153 vs.0.768±0.033,0.659±0.180 vs.0.816±0.037 and 0.862±0.096 vs.0.916±0.019,respectively)in colon cancer tissues,and the differences were all statistically significant(t=14.95,11.36,9.39,11.74,5.32 and 3.47,all P<0.01).The results of ROC analysis showed that the methylation levels of the above five locus of miRNA-129-1 gene had high diagnostic efficiency in colon cancer(area under curve=0.946,0.915,0.950,0.758 and 0.667,all P<0.01).The results of survival analysis indicated that low expression of miRNA-129-1 was associated with poor prognosis(hazard ratio(HR)=0.55,P=0.018).The results of bioinformatics analysis demonstrated that the target genes of miRNA-129-1 were enriched in serine/threonine kinase receptor,mitogen-activated protein kinase and other functional gene clusters closely related to tumor,and there was a complex interaction network among the target genes proteins.The high expression of ephrin type-B receptor2(EPHB2)gene,a potential key target gene of miRNA-129-1,was associated with the short overall survival and disease-free survival time(HR=1.9 and 1.6,both P<0.01).Conclusions The expression and methylation of miRNA-129-1 play an important regulatory role in the development and development of colon cancer.The methylation of miRNA-129-1 has potential value in the diagnosis of colon cancer,and miRNA-129-1 is an influencing factor for the prognosis of patients with colon cancer.EPHB2 may be a potential key target gene of miRNA-129-1.