摘要
目的探讨维吾尔族妇女宫颈病变及其人乳头状瘤病毒（humanpa pillomavirus，HPV）感染与低相对分子质量蛋白酶体（low molecular-weight protein，LMP）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LMP2和LMP7基因启动子区含CpG岛特异性PCR引物，对SiHa宫颈癌细胞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序列甲基化情况；收集维吾尔族妇女正常宫颈上皮、宫颈上皮内瘤变（cervical intraepithelial neoplasia，CIN）和宫颈鳞癌（cervical squamous cell carcinoma，CSCC）患者新鲜组织和石蜡组织标本78例，并提取DNA，采用Sequenom MassARRAYDNA技术平台（质谱分析），定量分析LMP2和LMP7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；应用RT—PCR和免疫组织化学方法检测LMP2、LMP7mRNA和蛋白的表达，并分析蛋白表达与基因甲基化的关系。结果LMP2和LMP7基因相应目的片段各含有22个CpG位点，在SiHa宫颈癌细胞基因组DNA中，只有LMP7有2个位点发生甲基化。宫颈病变病理过程伴随着LMP7基因CpG片段甲基化水平改变，其在CSCC和CIN组织的甲基化率（0．1864±0．0893和0．0728±0．0548）高于正常宫颈上皮组织（0．0652±0．0488），差异有统计学意义（P〈0．05）。随着宫颈病变的加重LMP7mRNA和蛋白表达逐渐下调，各组间表达差异具有统计学意义（P〈0．05），LMP7蛋白表达随着该基因甲基化率增高而降低（F=8．69，P=0．035）。而LMP2蛋白表达在3组间差异无统计学意义（P〉0．05）。HPV分型芯片检出12种HPV基因型，其中HPV16感染率构成比为52／78（66．7％），HPV16亚型阳性与宫颈病变进程及LMP7基因甲基化率升高趋势正相关（t=1．996，P=0．049）。结论LMP7基因启动子区CpG岛甲基化是一种宫颈癌病变特异性改变，与HPV16�
Objective To evaluate the relationship and significant between low molecular-weight protein （LMP） methylation and expression, and Uyghur women cervical lesions with human papillomavirus （HPV） infection. Methods Genetic information was obtained from the GenBank database, specialized software was used to scan gene promoter regions, and CpG island fragment specific primers was designed, gene methylation and CpG site sequences related information were gained by the PCR amplification, vector cloning and sequencing of the bisulfate-modified cervical cancer cell DNA. Methylation status of LMP was quantitative evaluated by Sequenom MassARRAY DNA in 78 subjects with different cervical lesions ; mRNA and protein of LMP2 and LMP7 were evaluated by RT-PCR and immunohistoehemistry. HPV infection status determined use HPV gene chips. Results Gene promoter region CpG fragments methylation sequencing it was detected that selected CpG sites of cervical cancer cell genome LMP7 had methylation. However no methylation site was found in gene promoter region of LMP2. The percent of LMP7 methyiation was increased steadily with the severity of cervical lesions, showing 0. 0652±0. 0488, 0. 0728±0. 0548 and 0. 1864± 0. 0893 of which with normal control, CIN and CSCC （ P〈0.01 ）. LMP7 was significantly reduced in CSCC and CIN compared with normal cervical epithelium, and its mRNA expression consistent with the results of proteins,while no significant difference in LMP2 expression. Moreover, the methylated proportion of LMP7 was negatively associated with the protein expression in cervical lesions （ F = 8.69, P = 0. 035 ）. Stratified analysis indicated that the percents of LMP7 methylation in subjects with HPVI6 positive still increased（ t= 1. 996, P= 0. 049）. Conclusion Our findings indicated that LMP7 methylation was significantly associated with cervical lesions and HPV infection.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2012年第3期205-211,共7页
Chinese Journal of Microbiology and Immunology
基金
新疆维吾尔自治区自然科学青年基金（2009211814）
国家自然科学基金（81060164）
