ObjectiveTo evaluate the relationship and significant between low molecular-weight protein (LMP) methylation and expression,and Uyghur women cervical lesions with human papillomavirus (HPV) infection.MethodsGenetic 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.ResultsGene 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).ConclusionOur findings indicated that LMP7 methylation was significantly associated with cervical lesions and HPV infection.

OBJECTIVETo study the relationship between TAP (transporter associated with antigen processing) gene promoter regional methylation level and cervical lesions with HPV infection in Uyghur women.

METHODSA 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 methylation 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.

RESULTSEach 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 TAP1 positively correlated with HPV16 infection (χ(2) = 6.08, P = 0.039).

CONCLUSIONTAP1 methylation is a remarkable phenomenon occurring in a range of cervical lesions and significantly associated with cervical HPV infection.

ATP-Binding Cassette Sub-Family B Member 2 ; ATP-Binding Cassette Transporters ; genetics ; ATP-Binding Cassette, Sub-Family B, Member 3 ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Carcinoma, Squamous Cell ; genetics ; virology ; Cervical Intraepithelial Neoplasia ; genetics ; virology ; CpG Islands ; genetics ; DNA Methylation ; Female ; Human papillomavirus 16 ; Humans ; Middle Aged ; Papillomavirus Infections ; Promoter Regions, Genetic ; Uterine Cervical Neoplasms ; genetics ; virology ; Uterine Cervicitis ; genetics ; virology