CAJ | 학술논문

目的探讨维吾尔族妇女宫颈病变及其人乳头状瘤病毒( human papillomavirus,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 MassARRAY DNA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LMP7 mRNA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感染可能存在密切关系.
목적 탐토유오이족부녀궁경병변급기인유두상류병독( human papillomavirus,HPV)감염여저상대분자질량단백매체(low molecular-weight protein,LMP)기인계동자구갑기화수평적관계화의의.방법 이용전업연건설계LMP2화LMP7기인계동자구함CpG도특이성PCR인물,대SiHa궁경암세포DNA진행아류산경염수식、목적편단확증、질립재체극륭화측서,학정해구역소함CpG서렬갑기화정황；수집유오이족부녀정상궁경상피、궁경상피내류변(cervical intraepithelial neoplasia,CIN)화궁경린암(cervical squamous cell carcinoma,CSCC)환자신선조직화석사조직표본78례,병제취DNA,채용Sequenom MassARRAY DNA기술평태(질보분석),정량분석LMP2화LMP7기인계동자갑기화수평,동시이HPV분형심편감정HPV아형,분석기인갑기화여HPV감염적관계；응용RT-PCR화면역조직화학방법검측LMP2、LMP7mRNA화단백적표체,병분석단백표체여기인갑기화적관계.결과 LMP2화LMP7기인상응목적편단각함유22개CpG위점,재SiHa궁경암세포기인조DNA중,지유LMP7유2개위점발생갑기화.궁경병변병리과정반수착LMP7기인CpG편단갑기화수평개변,기재CSCC화CIN조직적갑기화솔(0.1864±0.0893화0.0728±0.0548)고우정상궁경상피조직(0.0652±0.0488),차이유통계학의의(P＜0.05).수착궁경병변적가중LMP7 mRNA화단백표체축점하조,각조간표체차이구유통계학의의(P＜0.05),LMP7단백표체수착해기인갑기화솔증고이강저(F=8.69,P=0.035).이LMP2단백표체재3조간차이무통계학의의(P＞0.05).HPV분형심편검출12충HPV기인형,기중HPV16감염솔구성비위52/78(66.7％),HPV16아형양성여궁경병변진정급LMP7기인갑기화솔승고추세정상관(t=1.996,P=0.049).결론 LMP7기인계동자구CpG도갑기화시일충궁경암병변특이성개변,여HPV16감염가능존재밀절관계.
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 immunohistochemistry.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 HPV16 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.