CAJ | 학술논문

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.
목적:연구조직인자억제물2(TFPI-2)기인재유오이족부녀궁경병변조직중적갑기화수평,탐토기갑기화수평변화여궁경병변적관계.방법:수집유오이족부녀만성궁경염、궁경내류변(CIN、cervical intraepithelial neoplasia)Ⅰ/Ⅱ/Ⅲ화궁경린암(cervical squamous cell carcinoma,CSCC)환자적신선조직표본,제취고질량기인조DNA화RNA,설계TFPI-2기인계동자구CpG도편단특이성PCR인물급RT-PCR인물,응용Sequenom MassARRAY 갑기화DNA정량분석평태화반정량RT-PCR기술,대유오이족부녀궁경조직 DNA 진행갑기화수평정량분석.결과:분석 Sequenom MassArray 질보수거,TFPI-2기인계동자구 CpG 도편단(목적편단)갑기화수평정량차이현저(P<0.05).분별대TFPI-2적단점CpG위점갑기화수평차이진행분석,가견 TFPI-2기인적목적편단적CpG-1、CpG-6、CpG-7、CpG-8、CpG-9화CpG-11등륙개CpG위점갑기화솔재종류여정상대조지간균유통계학차이(P<0.05)병차량개CpG위점상관성흔밀절(r<0.5、P<0.01);반정량RT-PCR감정결과표명,TFPI-2 mRNA표체수평재궁경염조증고,재CINⅠ/Ⅱ/Ⅲ조강저,이재궁경린암조최저,궁경염조여궁경암조비교,차이균유통계학의의(P<0.01),설명해기인전록표체수평하조반수착궁경병변진정.결론:유오이족궁경암세포내적TFPI-2기인계동자구CpG도고갑기화여해기인표체수평변화밀절관련.재유오이족부녀궁경병변진정중TFPI-2기인계동자고갑기화시기기인적전록수평(mRNA)하조적중요원인,가능시궁경암적조기예경지표,위해기인갑기화상관적표관유전학연구제공료의거.
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 mRNA expression 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: Results indicated 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.