【摘要】 目的 探讨miR-204启动子甲基化在结直肠癌(CRC)中的临床意义。方法 收集2018年1月至2020年3月于我院就诊的69例CRC患者血浆样本、肿瘤组织标本和癌旁正常结肠黏膜组织标本；另外招募68例健康志愿者作为正常对照组，采集血浆样本。使用肿瘤基因图谱计划(TCGA)数据库与基因芯片数据(Methyl450K)分析肿瘤和正常组织中识别的CpG位点的甲基化水平。采用定量甲基化特异性PCR法(qMSP)和实时荧光定量PCR法(qPCR)检测样本中miR-204启动子区甲基化状态和基因表达量。结果 TCGA数据表明miR-204表达与宿主基因TRPM3的表达协调下调，并确定了miR-204启动子周围的两个CpG岛。与癌旁组织比较，CRC组织中miR-204启动子甲基化参考百分比(PMR)升高[1.78(0.35,16.61)vs. 1.51(0.45,2.98),P<0.001],且与miR-204表达量呈负相关(r=-0.324,P=0.007),与DNA甲基转移酶1 mRNA呈正相关(r=0.502,P<0.001)。此外，与正常对照组比较，CRC患者血浆cfDNA中miR-204启动子PMR显著升高[6.57(0.76,12.45)vs. 1.60(0.59,3.4),P=0.015],并且与CRC组织miR-204启动子PMR呈正相关(r=0.418,P<0.001)。血浆cfDNA中miR-204启动子PMR用于CRC的诊断的受试者工作特征曲线下面积为0.701(95%CI:0.610～0.791),灵敏度和特异度分别为56.5%和94.1%。此外，进一步依据临床分期、分化程度等进行分层分析，并未观察到miR-204启动子超甲基化与CRC患者主要临床特征的关系(P>0.05)。结论 miR-204启动子甲基化与CRC显著相关，并且血浆cfDNA中miR-204启动子甲基化状态或可作为CRC诊断和监测的潜在生物标记物。更多还原

【Abstract】 Objective To investigate the clinical significance of miR-204 promoter methylation in colorectal cancer(CRC). Methods From January 2018 to March 2020,the blood samples, tumor tissue samples and paracarcinoma normal colon mucosal tissue samples were collected from 69 CRC patients admitted to the department of gastroenterology of our hospital. Another 68 healthy volunteers were recruited as the normal control group. The plasma samples were collected. Methylation levels of CpG sites identified in tumor and normal tissue were analyzed using the Tumor Gene Atlas Project(TCGA) database and gene-on-a-chip data(Methyl450K). The gene expression and promoter methylation status of miR-204 were detected by quantitative methylation-specific PCR(qMSP) and quantitative real-time PCR(qPCR). Results The TCGA data indicated that the expression of miR-204 was coordinately down-regulated with the expression of host gene TRPM3, and two CpG islands around the miR-204 promoter were identified. Compared with the paracancer tissues, the miR-204 promoter methylation reference percentage(PMR) was increased in CRC tissues[1.78(0.35, 16.61) vs. 1.51(0.45, 2.98), P<0.001]. And the methylation level of miR-204 promoter was negatively correlated with the expression level of miR-204(r=-0.324, P=0.007), and positively correlated with DNA methyltransferase 1 mRNA(r=0.502, P<0.001). Moreover, compared with the normal control group, the PMR of miR-204 promoter was significantly higher in plasma cfDNA of CRC patients[6.57(0.76, 12.45) vs. 1.60(0.59, 3.4), P=0.015]. And PMR of miR-204 promoter in plasma cfDNA of CRC patients was positively correlated with PMR of miR-204 promoter in CRC tissues(r=0.418, P<0.001).The area under receiver operating characteristic curve of PMR of miR-204 promoter in plasma cfDNA for CRC diagnosis was 0.701(95%CI: 0.610-0.791), the sensitivity and specificity were 56.5% and 94.1%. In addition, further stratified analysis was conducted according to clinical stage and differentiation degree, there was no significant correlation between the hypermethylation of miR-204 promoter and the main clinical characteristics of CRC patients(P>0.05). Conclusion miR-204 promoter methylation is significantly correlated with CRC, and the status of miR-204 promoter methylation in plasma cfDNA may be used as potential biomarkers for CRC diagnosis and monitoring.更多还原