The Association Of Cervical Cancer Development In Uighur Women With The Methylation Of TFPI-2Gene At The Promoter Region

Objective: In previous reports, TFPI-2(tissue factor pathway inhibitor2) has beenknown as a candidate gene methylated in CpG islands at the promoter region specific tocervical squamous cell carcinoma, but the methylation level of TFPI-2gene promoter incervical cancer and precancerous lesions and its influence on gene expression was notfurther studied so far. In this project, we will study the role of promoter hypermethylationof TFPI-2gene in the development of cervical cancer and evaluate the potential of TFPI-2gene promoter hypermethylation as a molecular marker for early prognosis or diagnosis ofthe cancer, by characterization of the methylated CpG island sequence of the TFPI-2genein cervical carcinoma cells, methylation level of the gene at single CpG sites and theprotein expression level in cervical lesions.Methods:1. Using professional software, we scanned the TFPI-2gene for CpGislands at the promoter region, designed and synthesized PCR primers specific to the CpGfragment for the analysis by bisulphate modified sequencing method. The methylationlevel of the target CpG fragment in SiHa carcinoma cells was analysed by PCRamplification of genomic DNA after bisulphate modification followed by cloning andsequencing.2. We collected fresh cervical tissue specimens of Uighur women withchronic cervicitis, cervical intraepithelial neoplasia (CIN), cervical squamous cellcarcinoma (CSCC), and analyzed the genomic DNA to determine the single CpG sitemethylation level by setting up the CpG island at the promoter region of TFPI-2gene as atarget fragment, using Sequenom MassARRAY platform for the quantification of DNAmethylation.3. We analyzed the protein expression level of TFPI-2in paraffin-embeddedsections of cervical lesions by immunohistochemistry. Results:1. Target CpG fragment at the promoter region of TFPI-2gene analyzed inthis study contains9CpG sites. The results of bilsulphate sequencing showed that all CpGsites were fully methylated, without exception.2. According to the data resulted from thequantitative analysis of single CpG site methylation, the methylation level of whole targetCpG fragment of TFPI-2gene was significantly higher in CSCC tissues than in cervicitis(p<0.05), but no difference was found between CIN and CSCC or cervicitis. Furtherstatistical analysis showed that the methylation level (rate) between CSCC and cervicitiswas significantly different at only6single CpG sites (p<0.05).3. The TFPI-2proteinexpression level of was dramatically decreased with development of cervicitis to CINand CSCC, especially noticeable in the the loss of protein expression rate that wassignificantly highr in CSCC than in CIN or cervicitis (p<0.01).Conclusions: The CpG island of TFPI-2gene at the promoter region may mostprobably fully methylated in SiHa cervical carcinoma cells. The gene promoterhypermethylation or the loss of protein expression of TFPI-2may become a marker forcervical cancer development. The loss of TFPI-2protein expression may be an importantphenotype of the gene promoter methylation, and further reveal the mechanisms ofepigenetic regulation in cervical carcinogenesis.