Association of TFPI-2 methylation at the promoter region with cervical cancer in Uyghur women
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摘要:
目的 研究组织因子抑制物2(TFPI-2)基因在维吾尔族妇女宫颈病变组织中的甲基化水平, 探讨其甲基化水平变化与宫颈病变的关系。 方法 收集维吾尔族妇女慢性宫颈炎、宫颈内瘤变(CIN、cervical intraepithelial neoplasia)Ⅰ/Ⅱ/Ⅲ和宫颈鳞癌(cervical squamous cell carcinoma, CSCC)患者的新鲜组织标本, 提取高质量基因组DNA和RNA, 设计TFPI-2基因启动子区CpG岛片段特异性PCR引物及RT-PCR引物, 应用Sequenom MassARRAY甲基化DNA定量分析平台和半定量RT-PCR技术, 对维吾尔族妇女宫颈组织DNA进行甲基化水平定量分析。 结果 分析Sequenom MassArray质谱数据, TFPI-2基因启动子区CpG岛片段(目的片段)甲基化水平定量差异显著(P < 0.05)。分别对TFPI-2的单点CpG位点甲基化水平差异进行分析, 可见TFPI-2基因的目的片段的CpG-1、CpG-6、CpG-7、CpG-8、CpG-9和CpG-11等六个CpG位点甲基化率在肿瘤与正常对照之间均有统计学差异(P < 0.05)并且两个CpG位点相关性很密切(r < 0.5、P < 0.01);半定量RT-PCR鉴定结果表明, TFPI-2 mRNA表达水平在宫颈炎组增高, 在CINⅠ/Ⅱ/Ⅲ组降低, 而在宫颈鳞癌组最低, 宫颈炎组与宫颈癌组比较, 差异均有统计学意义(P < 0.01), 说明该基因转录表达水平下调伴随着宫颈病变进程。 结论 维吾尔族宫颈癌细胞内的TFPI-2基因启动子区CpG岛高甲基化与该基因表达水平变化密切关联。在维吾尔族妇女宫颈病变进程中TFPI-2基因启动子高甲基化是其基因的转录水平(mRNA)下调的重要原因, 可能是宫颈癌的早期预警指标, 为该基因甲基化相关的表观遗传学研究提供了依据。 Abstract:Objective To investigate the tissue factor pathway inhibitor 2(TFPI-2) gene methylation at the promoter region as well as its association with cervical cancer development. Methods We collected fresh cervical tissues from Uyghur women with cervicitis, cervical intraepithelial neoplasia(CIN), and cervical squamous cell carcinoma(CSCC).High-quality genomic DNA and RNA were then extracted.Polymerase chain reaction(PCR) primers specific to a CpG island in the TFPI-2 promoter region as well as semiquantitative reverse transcription(RT)–PCR primers were designed for the quantitative analysis of methylation in the cervical tissue DNA of Uyghur women by using the Sequenom MassARRAY platform and semiquantitative RT–PCR. Results The methylation level of target CpG islands in the promoter region specific to cervical cancer was significantly different from that of the normal control(P < 0.05).Analysis of TFPI-2 for single-site CpG methylation indicated that the methylation levels of CpG-1, CpG-6, CpG-7, CpG-8, CpG-9, and CpG-11 were higher in cancer tissue DNA compared with normal tissue DNA and associated with the clinical stages(P < 0.05).The two sites are highly correlated(r < 0.5, P < 0.01).Semiquantitative RT–PCR analysis revealed that the mRNAexpression of TFPI-2 was equally high in the cervicitis group, reduced in the CINⅠ/Ⅱ/Ⅲ group, and was very low in CSCC.A significant difference(P < 0.01) between the cervicitis group and the cervical cancer group was indicated, suggesting that the progression of the cervical disease was accompanied by downregulation of gene expression. Conclusion Resultsindicated a close association between methylation of TFPI-2 at the promoter region in Uyghur women and expression of TFPI-2 at the gene level.Hypermethylation of the CpG island in the promoter region contributed to the downregulation at the transcriptional level(mRNA), which may be an early prediction marker for cervical cancer.This study provides evidence for the feasibility of epigenetic research related to hypermethylation of the aforementioned genes. -
Key words:
- tissue factor pathway inhibitor 2 /
- promoter region /
- methylation /
- cervical cancer /
- Uyghur women
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图 1 TFPI-2基因的典型DNA表达
Figure 1. Typical DNA expression map of the TFPI-2 gene
M for 50bp DNA marker, the brightness of a larger stripe 250 bp; TF⁃ PI-2 BSP primer electrophoresis of PCR products, 1 and 2 for cervical tissue DNA template, 3 for negative control
图 2 GAPDH扩增产物琼脂糖凝胶电泳
Figure 2. GAPDH amplification agarose gel electrophoresis
1)1 for cervicitis, 2 for CIN, 3 and 4 for cervical cancer, M for marker; 2)DNA Marker: TAKARA100 bp DNA Ladder
图 3 TFPI-2扩增产物琼脂糖凝胶电泳
Figure 3. TFPI-2 amplification agarose gel electrophoresis
1)1 and 2 for cervical cancer, 3 for CIN, 4 for cervicitis, 5 for the blank control, M for marker; 2)DNA Marker: TAKARA 50 bp DNA Ladder
表 1 cDNA主要合成成份及量
Table 1. Synthesis of the main ingredients and quantities
表 2 反转录反应体系
Table 2. Reverse transcription reaction system
表 3 半定量RT-PCR反应体系
Table 3. Semiquantitative RT–PCR reaction system
表 4 宫颈组织中TFPI-2基因单点甲基化水平分析
Table 4. TFPI-2 gene methylation analysis in cervical tissue
表 5 TFPI-2基因甲基化单点相关性分析
Table 5. Single-point correlation analysis of TFPI-2 gene methylation
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