The Association Of Cervical Cancer Development With The Gene Expression Control Of Antigen Processing Machinary In Uighur Women

Aim of the Study:The prevalence and mortality of cervical cancer in Uighur women has been very high and beyond the average level in China, known as a high incident cancer of Xinjiang. The infection with human papillomavirus (HPV) was believed to be the main cause of cervical cancer development, and indeed HPV was also detectable in Uighur women with cervical cancer at a rate comparable with other populations in and outside China. To promote the cervical cancer research based on clinical practice focusing on the treatment of Uighur women with cervical cancer, reveal the cervical cancer pathogenesis and seek for the nature of HPV induced pathogenesis and carcinogenesis as well as to establish the tumor specific molecular marker profile and clinical diagnostic standard have a great social-economic importance for the early diagnosis, preventive vaccination and clinical treatment of the cancer in Xinjiang.The formation and survival of a tumor cell is a sign of a successful immune escape and the failure of host in immune surveillance and elimination. The normal function of human leukocyte antigen class Ⅰ (HLA-Ⅰ) and antigen processing machinery (APM) is the prerequisite for the T cell-mediated immune surveillance. HLA-Ⅰ works together with the members of APM in the assembling, loading and presentation of endogenous antigenic peptide, mediates anti-tumor or anti-viral cellular and humoral immunity. Thus, the elimination of tumor cells by immune system or the tumor survival and development may greatly be depended on the normal or abnormal expression of APM and HLA-Ⅰ.In this study, we will focus on10candidate genes belong to the HLA-Ⅰ and APM family and clinical resources from the treatment of cervical cancer, analyze the relationship between cancer development and the alteration of candidate gene expression at three different levels of gene expression control including epigenomic modification (gene promoter methylation), transcription (mRNA) and protein expression. In the practical approach, we will start with the detection at protein and mRNA expression levels, as a first step to identify genes significantly different and representing the overall trend of changes, and abandon the genes without significance. In the second step, in accordance with the main topic of this study (epigenetic research), we will focus on genes that were tumor-specific down-regulated or loss of protein expression and analyze the aberrant methylation of CpG islands at promoter regions such as to design primers of candidate genes specific to promoter regions rich in CpG islands, to screen cervical cancer specific methylated genes by amplification of cervical cancer cell DNA, cloning and sequencing of target CpG fragments of corresponding genes.In the third step, we will analyze tissue DNA generated from clinical samples for the quantitative difference of methylation by mass spectrometry approach (Sequenom MassARRAY) and the dependence on HPV infection, to find out the molecular mechanism of the relationship between cervical carcinogenesis and HLA-I and APM expression regulation, provide with early diagnostic markers based on the alteration of HLA-I and APM gene expression, and to understand the cervical cancer pathogenesis related to the function of antigen presentation.Methods:(1) Paraffin-embedded and/or fresh tissue specimens of Uighur women with cervical disease were collected for the detection of protein and semi-quantitative mRNA expression level of TAP1、TAP2、LMP2、LMP7、calnexin、calreticulin、ERp57、ERAP1、 tapasin and HLA-Ⅰ genes by immunohistochemistry (IHC) and RT-PCR.(2) CpG island fragment specific primers were designed by scanning gene promoter region using specialized software based on genetic information obtained from the Genbank database online, and bisulphate treated DNA of cervical cancer cells was amplified with PCR followed by cloning into vector and sequencing to identify CpG sites related to gene promoter methylation.(3) By DNA Sequenom MassARRAY approach for quantitative detection of methylated DNA, we analyzed the cervical tissue DNA for CpG content of candidate genes provided by the analysis in "Part II" of this study using gene specific primer pairs to compare methylation level of target fragments and CpG sites among different tissues. The dependence of candidate gene methylation on HPV infection was analyzed by detection of HPV positivity and genotypes of tissue DNA by HPV typing chips. Results:(1) Immunohistochemical analysis showed that with the development of cervical lesions from normal uterine cervix to cervical intraepithelial neoplasia (CIN) or cervical squamous cell carcinoma (CSCC), the protein expression level of nine genes, including HLA-Ⅰ、TAP1、TAP2、LMP2、IMP7、ERAP1, Tapasn、CRT and ERp57, was altered from normal expression to partial loss or total loss of expression, with statistically significance except for calnexin (P<0.05). RT-PCR results suggested that the change in mRNA expression level of TAP1, TAP2, LMP2, LMP7, ERAP1, Tapasin, CRT and ERp57as well as the HLA-A, HLA-B and HLA-C genes coding for HLA-Ⅰ was also significant among cervical lesions, and correlated with the protein expression described above. From the analysis of the association of clinical prognostic parameters for cervical cancer with candidate gene expression, we found a reverse correlation of the downregulation of HLA-Ⅰ, TAP1, and LMP7and ERp57protein expression with tumor differentiation state (P<0.05) as well as the down regulation of HLA-Ⅰ, TAP1, LMP7, Tapasin, CRT and ERp57with lymphnode metastasis, both with a statistically significance (P<0.05). From the FIGO stage point of view, the ERp57protein expression was significantly lower in low stage cancer than in high stage. The downregulation of TAP1, TAP2, LMP7, ERAP1, Tapasin and ERp57protein expression was positively associated with HLA in CIN lesions, whereas TAP1, TAP2, LMP2, LMP7, ERAP1, ERp57and Tapasin was positively associated with HLA-Ⅰ in the cancer.(2) By sequencing of the CpG fragment methylation at gene promoter region, we identified the target CpG site methylation to various extents at promoter regions of TAP1, TAP2, LMP7, Tapasin and ERp57, with the ratio5/23,8/8,2/22,12/12and18/18representing methylated CpG sites in total CpG sites. However, no methylation was found for HLA-B, LMP2and ERAP1.(3) According to the data generated by Sequenom Mass ARRAY DNA detection, total methylation level of target fragments of TAP1, LMP7, and ERp57genes tissues was higher in cervical cancer than CIN and normal tissues with statistical significance (P<0.05), but no difference was found for TAP2and Tapasin. The single-site methylation level analysis showed that the single-site methylation was significant for almost all CpG sites of TAP1, LMP7and ERp57genes in cervical cancer tissues compared to CIN and the normal. Comparative analysis of target fragment methylation and the data of altered protein expression described in "part Ⅰ" of this study, we found a reverse correlation of TAP1, LMP7and ERp57gene methylation level with the protein expression. We detected13HPV genotypes in tissue DNA samples, including11high-risk and2low-risk types, with infection rate of52/78(69.8%). Comparative analysis of methylation level of target gene fragments between HPV positive and negative cases, we found a significant increase of promoter methylation of ERp57, TAP1and LMP7genes in HPV positive tissues.Conclusion:(1) The transcription downregulation and loss of protein expression of HLA-Ⅰ and antigen processing machinery (AMP) was closely associated with development of cervical cancer in Uighur women, and may become important candidates for the establishment of tumor molecular marker profile.(2) The target CpG fragments of TAP1, TAP2, LMP7, Tapasin and ERp57were CpG site methylated at various degrees in cervical cancer cells, may most probably the cause of the transcription downregulation or protein loss expression regulated at epigenetic level.(3) The promoter methylation of TAP1, LMP7and ERp57genes was a change specific to cervical carcinogenesis caused by HPV16infection, and an epigenetic regulation mechanism of protein expression too.