摘要
目的探讨维吾尔族妇女宫颈病变及人乳头状瘤病毒(HPV)感染与抗原处理相关转运体(TAP)基因启动子区甲基化水平的关系和意义。方法利用专业软件设计TAP1和TAP2基因启动子区含CpG岛特异性PCR引物,对宫颈癌SiHa细胞DNA进行亚硫酸氢盐修饰、目的片段扩增、质粒载体克隆和测序,确定该区域所含CpG序列的甲基化情况。收集维吾尔族妇女15例正常宫颈上皮、30例宫颈鳞状上皮内肿瘤形成(CIN)和33例宫颈鳞癌患者的新鲜组织标本共78例,提取DNA,采用MassArrayDNA技术平台(质谱分析)定量分析TAP1和TAP2基因启动子甲基化水平,同时以HPV分型芯片鉴定HPV亚型,分析基因甲基化与HPV感染的关系。结果(1)TAP1和TAP2基因相应目的片段各含23个和8个CpG位点,在宫颈癌SiHa细胞基因组DNA中,分别有5个和8个CpG位点发生甲基化。宫颈病变病理过程伴随着TAP1基因CpG片段甲基化水平的改变,其在宫颈鳞癌和CIN组织的甲基化率[(0.048±0.039)和(0.037±0.026)]高于正常宫颈上皮组织(0.035±0.029),差异有统计学意义(P〈0.05)。尽管TAP2基因甲基化水平也发生了相应改变,但其差异无统计学意义(P〉0.05)。HPV分型芯片检出13种HPV基因型,其中HPV16所占比率为66.7%(52/78),HPV16亚型阳性与宫颈病变进程及TAP1基因甲基化率的升高趋势呈正相关(X^2=6.08,P=0.039)。结论TAP1基因启动子区CpG岛甲基化是宫颈癌病变的一种特异性改变,与HPV16感染可能存在密切关系。
Objective To study the relationship between TAP (transporter associated with antigen processing) gene promoter regional methylation level and cervical lesions with HPV infection in Uyghur women. Methods A specialized software was used to design specific primers of CpG island fragments of TAP1 and TAP2 gene promoter for PCR amplification, bisulfitemodified SiHa cancer cell DNA for PCR amplification, cloning and sequencing analysis to obtain the relevant information on the gene base sequence mcthylation of CpG sites. Seventy-eight fresh cervical tissue samples from Uyghur women with cervicitis (number = 15 ), cervical intraepithelial neoplasia (CIN, number = 30 ) and cervical squamous cell carcinoma ( number = 33 ) were collected. The methylation level of TAP1 and TAP2 gene promoter regions was detected using MassArray DNA technology. HPV infection status was determined by HPV gene chips. The relationship between CpG-island methylation of gene promoter regions and HPV infection was then analyzed. Results Each TAP1 and TAP2 gene corresponding target fragment contained 23 and 8 CpG sites. There were 5 and 8 CpG sites methylation occurred in SiHa cervical cancer cells genomic DNA respectively. The TAP1 methylation level increased steadily with the severity of cervical lesions. The methylation levels in cervical squamous cell carcinoma and CIN (0. 048 ± 0. 039 and 0. 037± 0. 026, respectively) were higher than that of normal cervical tissue (0. 035 ± 0. 029, P 〈 0. 05 ). Although TAP2 gene methylation level also demonstrated similar changes, the difference however was not statistically significant ( P 〉 0. 05 ). HPV gene chip detected 13 HPV genotypes, with HPV16 infection rate being 66. 7% (52/78). The methylated proportion of TAPI positively correlated with HPV16 infection ( X^2 = 6. 08, P = 0. 039 ). Conclusion TAP1 methylation is a remarkable phenomenon occurring in a range of cervical lesions and significantly associated with cervical HPV infection.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2012年第7期438-442,共5页
Chinese Journal of Pathology
基金
国家自然科学基金(81060164)
新疆维吾尔自治区自然科学青年基金(2009211B14)