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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。
Objective: To investigate the methylation level of tissue factor inhibitor 2 (TFPI-2) gene in cervical lesions of Uigur women and to explore the relationship between the methylation level and cervical lesions. Methods: Fresh tissue samples from Uygur women with chronic cervicitis, cervical intraepithelial neoplasia Ⅰ / Ⅱ / Ⅲ and cervical squamous cell carcinoma (CSCC) were collected, and high quality genomic DNA RNA was used to design the CpG island fragment-specific PCR primers and RT-PCR primers of TFPI-2 promoter region. The DNA of cervical tissues from Uygur women was sequenced by using Sequenom MassARRAY methylation quantitative analysis platform and semi-quantitative RT-PCR The level of the level of quantitative analysis. Results: The methylation level of CpG island fragment (target fragment) of TFPI-2 gene promoter was significantly different (P <0.05) by Sequenom MassArray mass spectrometry. The differences of methylation levels of single point CpG sites in TFPI-2 were analyzed. The results showed that CpG-1, CpG-6, CpG-7, CpG-8, CpG- (P <0.05). There was a significant difference between the two CpG sites (r <0.5, P <0.01). Semiquantitative The results of RT-PCR showed that the expression level of TFPI-2 mRNA increased in cervicitis group and decreased in CINⅠ / Ⅱ / Ⅲ group, while it was the lowest in cervical squamous cell carcinoma group, and there was statistical difference between cervicitis group and cervical cancer group Significance (P <0.01), indicating that the gene transcriptional expression decreased with the progress of cervical lesions. Conclusion: The hypermethylation of CpG island in TFPI-2 promoter region in Uighur cervical cancer cells is closely related to the change of the gene expression level. Hypermethylation of TFPI-2 promoter in cervical lesions of Uygur women is an important reason for the down-regulation of its gene transcription level (mRNA), which may be an early warning index for cervical cancer and an indicator of methylation-related epigenetic Study provides the basis.