The Association Of Promoter Methylation Of TAP1and TAP2Genes With The Cervical Lesion Pathogenesis In Uighur Women

Objective Transporter associated with antigen processing（TAP）is a member of antigenpresenting machinery （APM） family genes and plays an important role in humanleukocyte antigen classâ…  （HLA-Ⅰ）-mediated antigen presentation and immunesurveillance. Studies has shown that the dysfunction of APM family such asHLA-â… ,TAP1and TAP2was closely associated with the development of various tumors,especially the downregulation of gene transcription or the loss of protein expression maylead to the decreasing level of HLA-â…  mediated antigen presentation at the cell surfaceand result in the immune escape of tumor cells, but the underlying mechanism has notbeen intensively studied so far. In this study, we will investigate the association of cervicallesion pathoigenesis and carcinogenesis with the TAP1and TAP2gene expressionregulation, especially the intrinsic relationship between cervical cancer development anddysregulated gene expression as a mechanism at the gene promoter methylation level.Method （1） Genomic DNA was isolated from fresh cervical tissue specimens collectedfrom Uighur women with chronic cervicitis, cervical intraepithelial neoplasia（CINâ…¡/Ⅲ）,cervical squamous cell carcinoma （CSCC）. After designing and synthesizing PCR primersspecific to the CpG fragment corresponiding to target CpG islands by professionalsoftwares. The genomic DNA of cervical lesions was then analysed by PCR amplification followed by detection of single CpG site methylation level at the promoter region of TAP1and TAP2genes using Sequenom MassARRAY platform for the quantification of DNAmethylation.（2） After scanning the promoter and the proximal sequence at thetranscription start site by professional software and based on the genomic information ofTAP1and TAP2genes for potential sequences containing CpG islands, the PCR primersspecific to the CpG fragment were designed and synthesized, and target CpG islandmethylation of the genes in genomic DNA of SiHa cervical carcinoma cells was analyzedby bisulphate modified sequencing （bisulphate modification of the DNA, PCRamplification of the target fragment, cloning and sequencing）.（3） The transcription levelof TAP1ands TAP2genes in cervical lesions was analyzed by semi-quantitative RT-PCRusing mRNA-specific primers. Results （1） The target CpG fragment of TAP1genecontains23CpG sites. The analysis of the data resulted from the quantitative analysis ofsingle CpG site methylation by Sequenom MassARRAY platform, has shown that themethylation ratio （level） between two or three CpG sites from CpG₁, CpG₂.3, CpG₄,CpG₁6, CpG₁9, CpG₂0, CpG₂1, CpG₂2and CpG₂3associated with each other，but no association was found for the methylation ratio of other CpG sites. In addition tothis, there were significant differences in the methylation level of7CpG sites （describedabove） between CSCC, CIN or cervicitis, with the exception of CpG₁6、 CpG₁9. Theanalysis of methylation level of whole target CpG fragment indicated that the methylationlevel of TAP1was significantly higher in CSCC and CIN cases （0.0477±0.0387and0.0370±0.0261） than in cases of cervicitis （0.0345±0.0291, and P<0.05）. The target CpGfragment of TAP2contains8CpG sites, but no differences was found for any CpG sitemethylation between different groups of cervical lesions（P＞0.05）, inspite that themethylation ratio of CpG₃.4、CpG₅、CpG₇、CpG₈were associated with each other.（2） Four CpG islands （fragments） were analyzed in this study are located on-226～+104,+14～+421,+607～+970and+9152～+9422at the start site of transcription, respectively,contains23,31,22, and11CpG sites. The bisulphate sequencing of the4fragments above confirmed that5and6CpG sites respectively was methylated in the first CpG island（-226～+104） and third CpG island （+607～+970）, but no methylation was found for theremaining ones.（3） TAP1and TAP2mRNA expression level was decreased with thedevelopment of cervical lesion pathogenesis. The relative level of TAP1mRNAexpression in cases of CIN（0.483±0.196）and CSCC（0.167±0.067）and TAP2mRNAexpression in CSCC（0.374±0.135） significantly lower than in cervicitis（TAP1：0.960±0.261;TAP2：0.918±0.552）（P<0.05）. Conclusions （1） The hypermethylation ofcertain CpG sites （but not all） of TAP1gene at the promoter region is closely associatedwith the cervical lesion pathogenesis in Uighur women, and may become an importantmarker of cervical cancer.（2） TAP1gene promoter contains multiple CpG islands, buthypermethylation may occur only on certain CpG islands and sites.（3） Thedownregulation of TAP1and TAP2transcription is a marker of cervical carcinogenesis,and importantly, the downregulation of TAP1gene expression may become a marker ofcervical precancerous lesions. The TAP1gene promoter methylation and transcriptiondownregulation were correlated at high extent in cervical lesion development in Uighurwomen, and may explain the epigenetic regulation on gene expression.