| 摘要: |
| 目的探讨Wnt6、Wnt3A、LEF1和FZD1基因启动子甲基化在结直肠癌（CRC）筛查及早期诊断中的临床应用。方法选择收治的55例CRC患者为观察组，55例健康体检人群为对照组，分别取观察组CRC组织、术前血标本、术前粪便标本以及对照组正常结直肠组织、外周血样本、粪便标本，使用QIAGEN试剂盒提取结直肠组织基因组DNA以及血液、粪便中脱落细胞DNA，甲基化特异性PCR（MSP）法检测以上4种基因启动子甲基化情况；RT-PCR法检测mRNA水平，Western-blot法检测蛋白水平；进而比较两组受检者以上4种基因启动子甲基化阳性及基因表达情况。结果共提取组织、血液、粪便脱落细胞DNA110例，最终验证存在人基因组DNA并完成MSP110例。观察组粪便脱落细胞DNA中Wnt6、Wnt3A、LEF1和FZD1基因启动子甲基化阳性率分别为69.1%、63.6%、54.5%和56.4%，4种基因总甲基化阳性率为90.9%；对照组粪便脱落细胞DNA中Wnt6、Wnt3A、LEF1和FZD1基因启动子甲基化阳性率分别为0.0%、1.8%、0.0%和1.8%，4种基因总甲基化阳性率为3.6%。粪便脱落细胞DNA中Wnt6、Wnt3A、LEF1和FZD1基因启动子甲基化对21例Ⅰ~Ⅱ期CRC的发现率分别为80.9%,71.4%,57.1%和57.1%，联合检测4种基因启动子甲基化对Ⅰ~Ⅱ期CRC的发现率为90.5%。CRC患者粪便脱落细胞DNA中Wnt6、Wnt3A、LEF1和FZD1基因启动子甲基化与患者性别、年龄、远处转移、肿瘤部位、TNM分期及淋巴结转移均未见相关（均P＞0.05）。观察组与对照组结直肠组织、血标本、粪便标本中Wnt6、Wnt3A、LEF1和FZD1基因启动子甲基化阳性率比较，差异均有统计学意义（均P＜0.01）。两组受检者结直肠组织中Wnt6、Wnt3A、LEF1和FZD1基因启动子甲基化后的mRNA及蛋白水平均有所下调，观察组与对照组的电脉图差异明显。结论MSP法检测结直肠组织、血液及粪便脱落细胞DNA中Wnt6、Wnt3A、LEF1和FZD1基因启动子甲基化是CRC早期筛查的有效方法。 |
| 关键词: 结直肠癌 甲基化特异性 PCR 甲基化 |
| DOI：10.12056/j.issn.1006-2785.2017.40.4.2017-2955 |
| 分类号: |
| 基金项目:宁波市自然科学基金项目（2016A610123） |
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| The clinical application of detecting Wnt6, Wnt3A, LEF1 and FZD1 gene methylation in the screening, early diagnosis of colorectal cancer |
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the Affiliated Hospital,College of Medicine, Ningbo University
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| Abstract: |
| Objective To explore clinical application of Wnt6, Wnt3A, LEF1 and FZD1 gene methylation in colorectal cancer (CRC) screening and early diagnosis. Methods Fifty-five patients with colorectal cancer and 55 healthy people were enrolled in the Affiliated Hospital of Ningbo University Medical College during December 2015 to December 2016. The DNA of colorectal cancer tissue, blood and stool were isolated by QIAGEN kit, and the DNA of the three control groups were also isolated by QIAGEN kit. MS-PCR (methylation-specific PCR) was applied to analyze the promoter hypermethylation of Wnt6, Wnt3A, LEF1 and FZD1 genes. The mRNA levels of Wnt6, Wnt3A, LEF1 and FZD1 were detected by RT-PCR and the protein expression levels of Wnt6, Wnt3A, LEF1 and FZD1 were detected by Western-blot, to explore Whether the promoter hypermethylation of theWnt6, Wnt3A, LEF1 and FZD1 genes causes downregulation gene expression. Results A total of 110 cases of tissue, blood, and fecal abscission cells (DNA) were extracted to verify the existence of human genome DNA and complete MSP in 110 cases. The detection rates of Wnt6, Wnt3A, LEF1 and FZD1 promoter methylation in the DNA of fecal exfoliated cells were 69.1%,63.6%, 54.5% and 56.4% respectively in the observation group. The detection rate of 4 gene promoter methylation of the observation group was 90.9%. The detection rates of Wnt6, Wnt3A, LEF1 and FZD1 promoter methylation in the DNA of fecal exfoliated cells were 0.0% ,1.8% , 0.0% and 1.8% espectively in the control group. The detection rate of 4 gene promoter methylation of the control group was 3.6%. he detection rates of Wnt6, Wnt3A, LEF1 and FZD1 promoter methylation in the DNA of fecal exfoliated cells were 80.9% , 71.4% , 57.1% and 57.1% for 21 cases of the stage I-II CRC respectively. The detection rate of 4 gene promoter methylation for stage I to II CRC was 90.5% . Methylation of Wnt6, Wnt3A, LEF1 and FZD1 genes in DNA of fecal exfoliated cells from CRC patients was not correlated with gender, age, distant metastasis, tumor location, TNM stage and lymph
node metastasis (P ＞0.05). The methylation positive rates of Wnt6, Wnt3A, LEF1 and FZD1 genes in the colorectal tissues, blood
samples and feces samples of the observation group and the control group were statistically significant (all P＜0.01). The mRNA and protein levels of Wnt6, Wnt3A, LEF1 and FZD1 gene promoter in the two groups were all down regulated, and the difference between the observation group and the control group was significant. Conclusion Detection of methylation of Wnt6, Wnt3A, LEF1 and FZD1 genes in DNA and exfoliated cells from blood and feces by MSP is a effective clinical method for early CRC screening. |
| Key words: Colorectal cancer MSP Promoter methylation |
