Methylation State Of TFPI-2Gene CpG Island In Thyroid Nodular Diseases And It’s Significances

ObjectivesThyroid carcinoma is a common malignant tumor. Papillary thyroid carcinoma accounts for60%of all thyroid carcinoma, and invasion and metastasis are the main reasons of treatment failure and leading to death of patient with thyroid carcinoma. Accordingly, it becomes increasingly concerned to looking for the related specific molecular markers as the invasion and metastasis of papillary thyroid cancer. Papillary thyroid carcinoma, nodular goiter and thyroid adenoma represent an enlarged thyroid gland containing circumscribed nodules within its substance. It is difficult to discriminate these which the clinical manifestation is similar.10-18%out of benign nodules have the possibility of malignant change. Deactivation of cancer suppressor genes is taken for important mechanism in the process of carcinogenesis, and CpG island methylation in the cancer suppressor gene promoter region is the most common change of deactivation. So, it has been the hot topic to search for the epigenetic molecular markers which used to diagnosis, estimating of invasion and metastasis and predicting malignant change, may be it can be used to guiding the treatment. Matrix metalloproteinase can degrade the proteins in extracellular matrix (ECM) and destroyed the barrier for protecting normal tissue. This is the most important reason of invasion and metastasis related to recovery. More and more studies have shown that tissue factor pathway inhibitor-2(TFPI-2) gene promoter was characterized by housekeeping gene, which expressed in most human tissues. But it was down-expression or no-expression in the tumors origining from above organizations, which often dued to promoter CpG island methylation. There for, TFPI-2gene was estimated as a cancer suppressor gene. The studies also showed that TFPI-2could inhibit the combination of transmembrane proteaseserine4(TMPRSS4), matrix metalloproteinases (MMPs) and the extracellular matrix (ECM), reduced the damaging effects of the TMPSS4and MMPs to the ECM, which inhibited tumor invasion and metastasis. In our study, the effects of the methylation status of TFPI-2gene promoter region on the metastasis of papillary thyroid carcinoma and differential diagnosis of papillary thyroid cancer, nodular goiter and thyroid adenoma were investigated, so as to provide molecular genetics target for the differential diagnosis of thyroid nodular diseases and the metastasis of papillary thyroid carcinoma, and in order to get the information about detecting the indicator of predicting malignant change earlier..This study includes the following three parts.Part1The expression of TFPI-2in thyroid nodular diseases and the relationships between TFPI-2andTMPRSS4, MMP-1in papillary thyroid carcinomaMethods1. The expressions of TFPI-2, TMPRSS4and MMP-1protein were detected by immunohistochemistry (IHC) in30cases of nodular goiter,20cases of thyroid adenoma and40cases of papillary thyroid cancer. The differences of the expression of TFPI-2in three thyriod nodular disease were analyzed, and the correlations between TFPI-2and TMPRSS4, MMP-1expressions in papillary thyroid carcinoma were analyzed. In papillary thyroid carcinoma, the relationships between TFPI-2, TMPRSS4, MMP-1expression and lymphatic metastasis in papillary thyroid carcinoma were analyzed.2. Statistical analysis:All the data were statistically analyzed by the SPSS14.0software. Chi-square test was used to analyze the enumeration data, and Spearman test was used to analyze the correlation. Statistical significance was determined at P＜0.05level.Results1. The results from IHC revealed that:the positive expression rate of TFPI-2protein was83.3%in nodular goiter,65.0%in thyroid adenoma and52.5%in thyroid carcinoma, respectively. There was significant difference among the groups, χ2=7.221, P=0.027; The expression of TFPI-2protein was significantly lower in papillary thyroid carcinoma with metastasis than that in papillary thyroid carcinoma without metastasis χ2=4.821, P=0.028; The expressions of TMPRSS4and MMP-1protein were significantly higher in papillary thyroid carcinoma with lymphatic metastasis than that in papillary thyroid carcinoma without lymphatic metastasis. The χ2value is4.835and6.852and the P value is0.028and0.009, respectively.2. The data from correlation analysis showed that there were significant negative correlation of expression between TFPI-2and TMPRSS4, MMP-1in papillary thyroid carcinoma. The correlation coefficient r is-0.383and-0.339, respectively, and the corresponding P value is0.015and0.032, respectively.Part2The methylation Status of TFPI-2gene promoter region and its effects on the expression of TFPI-2in thyroid nodular diseasesMethods1. The methylation status of TFPI-2gene promoter region was detected by methylation-specific PCR (MSP) in30cases of nodular goiter,20cases of thyroid adenoma and40cases of papillary thyroid carcinoma. The relationship between the methylation status of TFPI-2gene promoter region and the expression of TFPI-2in thyroid nodular diseases, and the effects caused by the methylation status of TFPI-2gene promoter region on TFPI-2and the relationship between the methylation status and lymphatic metastasis in papillary thyroid carcinoma was analyzed. 2. Statistical analysis:All the data were statistically analysed by the SPSS14.0software. Chi-square test was used to analyze the enumeration data, and Spearman test was used to analyze the correlation. Statistical significance was determined at P<0.05level.Results1. The results from MSP indicated that①the methylation rate of TFPI-2gene promoter region was16.7%in nodular goiter,25.0%in thyroid adenoma,50.0%in papillary thyroid carcinoma, respectively. There was significant difference among the groups.χ2=9.722, P=0.008.②The methylation rate of TFPI-2gene promoter region was88.9%in papillary thyroid carcinoma with lymphatic metastasis,18.2%in papillary thyroid carcinoma without lymphatic metastasis, respectively. There was significant difference between the groups.χ2=19.798, P=0.000.2. The data from correlation analysis suggested that there were significant negative correlation between the methylation status of TFPI-2gene promoter region and TFPI-2expression in nodular goiter, thyroid adenoma, and papillary thyroid carcinoma (P＜0.05or0.01). r value is-0.760,-0.545,-0.651,and P value is0.000、0.020、0.000, respectively.Part3The methylation Status of TFPI-2gene promoter region and its effects on the expression of TFPI-2, TMPRSS4and MMP-1in thyroid carcinoma cellsMethods1. Taken human papillary thyroid carcinoma TPC-1cells and human medullary thyroid carcinoma TT cells as study targets, the methylation status of TFPI-2gene promoter region was detected by MSP in TPC-1cells and TT cells in treatment with5-aza-dc group(treatment group) and treatment without5-aza-dc group (untreatment group).2. The expressions of TFPI-2, TMPRSS4and MMP1were investigated by Western blotting assay in TPC-1cells and TT cells in treatment and untreatment group. The relationships between the methylation status of TFPI-2gene promoter region and TFPI-2, TMPRSS4and MMP1expression in TPC-1cells and TT cells in two groups were analyzed.3. Statistical analysis:All the data were statistically analysed by the SPSS14.0software. The measurement data were presented as x±s.Independent-Samples T Test was used to analyze the measurement data. Statistical significance was determined at P＜0.05level.Results1. The results from MSP displayed that the promoter region of TFPI-2gene in thyroid carcinoma cell TPC-1and TT cells was hyper-methylation state, whereas it was demethylation after treated with5-aza-dc.2. The results from Western blotting showed that the expression of TFPI-2was significantly higher in thyroid carcinoma TPC-1cells and TT cells in treatment group than in untreatment. t value was32.704,18.432, P value was0.000,0.000, respectively, the expressions of TMPRSS4and MMP1were significantly lower in thyroid carcinoma TPC-1cells and TT cells in treatment group than untreatment group (P＜0.01).Conclusions:1. Hypermethylation of TFPI-2gene promoter region might be the important mechanism of inactivation of TFPI-2gene in papillary thyroid carcinoma.2. Methylation status of TFPI-2gene promoter region and the expression of TFPI-2might be used as important molecular markers in the differential diagnosis of papillary thyroid carcinoma, nodular goiter and thyroid adenoma.3. There is close relationship between the methylation status of TFPI-2gene promoter region, subsequent the expression of TFPI-2, TMPRSS4, MMP-1and the lymphatic metastasis of papillary thyroid carcinoma.4. The role of TFPI-2in the lymphatic metastasis of papillary thyroid carcinoma might be associated lower expression of TFPI-2and higher expression of MMP-1 and TMPRSS4caused by hypermethylation of TFPI-2gene promoter region.5. TFPI-2hypermethylation and low expression might be an indicative factor of malignant change of benign nodule.The innovation in present study:Incidence rate of thyroid carcinoma has increased and part of benign nodules occurs malignant change. Mechanisms of invasion in thyroid cancer remain poorly understood. But the above problem haven’t been concerned. In present study, took tissue from nodular goiter, thyroid adenoma, papillary thyroid carcinoma and thyroid carcinoma cell lines as the materials, aimed to investigate the effects of the methylation status of TFPI-2gene promoter region on the metastasis of papillary thyroid carcinoma and differential diagnosis of papillary thyroid cancer, nodular goiter and thyroid adenoma, so as to provide an epigenetics molecular for the differential diagnosis of thyroid nodular diseases and the metastasis of papillary thyroid carcinoma. Provide a clue to predicting malignant change of benign nodule.