Papillary Thyroid Carcinoma Specific Low Expression Genes TIMP3and MLH1Promoter Methylation Research

Objective:In the previous study by our group, the protein expression analysis in MGMT,Matrix Metalloproteinase Inhibitor3（TIMP3） and DNA Mismatch Repair Proteins （MLH1） low expression in Thyroid Carcinoma.they may will be Thyroid Cancer specific hypermethylation candidate genes. This study intends to elect TIMP3and MLH1genes, detection of two gene promoter region CpG island methylation status in papillary thyroid carcinoma, and to study its promoter CpG sites methylation levels of papillary thyroid carcinoma intrinsically linked. And evaluate the possibilities and value of TIMP3and MLH1genes promoter methylation as a molecular marker in PTC.Method:1collect tissue samples of PTC and nodular goiter from patients and extract genomic DNA.2.Searching for the TIMP3and MLH1gene promoter region sequence of CpG island methylation fragments using primer design software, bisulfite sequencing primer,BSP)cloning and sequencing of PCR amplified fragment by CpG Island, research, TIMP3and MLH1gene promoter methylation level in PTC.3to TIMP3and MLH1gene promoter CpG island fragment, relying Sequenom MassARRAY quantitative DNA methylation analysis platform, quantitative analysis of single CpG site methylated level in CpG island. Results:1Bisulfite sequencing showed hypermethylation percentage TIMP3gene cloning in thyroid carcinoma was70%, significantly higher than the nodular goiter0%（P<0.05）. TIMP3total12gene promoter CpG methylation sites appear in papillary thyroid carcinoma, including five CpG sites showed a high degree of specificity of papillary thyroid carcinoma. MLH1gene promoter in both basic thyroid tissue not detected methylation.2. Analysis of a single CpG methylation sites provided quantitative data Sequenom MassArray display TIMP3gene methylation rate in papillary thyroid carcinoma mean （0.28） and nodular goiter methylation rates mean （0.15） there was a significant difference （P<0.05）. MLH1gene methylation rate in papillary thyroid carcinoma average （0.20） and nodular goiter methylation rates mean （0.12） there are differences, but not statistically significant （P>0.05）. Single CpG sites methylation quantitative analysis of the test data provided by Sequenom MassArray seen,4CpG sites in promotor of TIMP3gene methylation rate in papillary thyroid carcinoma with nodular goiter mean significantly compared difference （P<0.05）. Conclusion:The promoter of MLH1gene in two kinds of thyroid tissues did not detect methylation, the low expression of MLH1in thyroid cancer may have nothing to do with methylation. TIMP3gene promoter in the degree of methylation in papillary thyroid carcinoma was significantly higher than nodular goiter that TIMP3gene promoter methylation and protein expression may be missing thyroid cancer molecular markers. According to BSP sequencing test results and MassArray quantitative methylation analysis that TIMP3gene CpG₁6and CpG₁7sites may be key sites of papillary thyroid carcinoma promoter methylation testing, is expected to become molecular markers for early diagnosis of thyroid cancer...