The clinical significance of Tumor suppressor gene methylation expression in nodular thyroid disease Feng Wei, Zhaoxia Wang, Yun Wu Department of Endocrinology,The First Affiliated Hospital of Baotou Medical College,Inner Mongolia University of Science and Technology, China

Nodular thyroid disease pathogenesis 1. Otherfactors Immune factors factors Genetic Environmental iodine intake abnormalities, infections, stress Radiation exposure. mutations,Activation, tumor suppressor genes Immune dysfunction, autoantibody formation Thyroid cell degeneration, inflammation Nodular thyroid disease Pathogenesis is very complex Relationship with Genetic and environmental factors and thyroid nodular disease closely. It is a multi-factor, multi-gene complex process in many ways,

Gene silencing The tumor suppressor gene inactivation Tumor formation DNA methylation Other factor Background DNA methylation and other factors together lead to gene silencing, causing the inactivation of tumor suppressor genes resulting in tumorigenesis

Background Detected circulating DNA in the blood of patients with malignant to genetic diagnosis in recent years has become a hot topic. the blood circulating DNA may become a new molecular markers of tumor diagnosis disease staging and prognosis effective.

Background 4 tumor suppressor genes DAPK- - death-associated protein kinase is a tumor suppressor gene, a calcium / calmodulin- dependent serine / threonine protein kinase, Is a kind of actin filaments associated with pro-apoptotic proteins involved in the induction of apoptosis, inhibition of tumor progression and metastasis RARβ2 receptor hormone nuclear receptor superfamily members, and a variety of biological effects mediated by retinoic acid, A variety of biological effects mediated by retinoic acid, plays a very important role in the regulation of cell differentiation and proliferation process RASSF1A through the RAS mediated signaling channel to play a variety of biological effects, and can promote apoptosis, Cell cycle arrest, and play an important role in tumorigenesis and development. PTEN Can inhibit the growth of tumor cells, Promote apoptosis Involved in the regulation of the cell cycle and the inhibition of tumor metastasis The above four genes in a variety of advanced tumors including thyroid cancer, there are varying degrees of mutation or missing.

Objective: This study detected the tumor suppressor gene (PTEN, RASSF1A, DAPK, RARβ2 ) DNA methylation and mRNA expression status in the peripheral blood of patients with nodular thyroid disease. meantime,discuss its clinical significance. For the benign and malignant thyroid nodules early identification of molecular diagnosis is based.

subjects: GROUP NUMBER SEX AGE NODULE SIZE CALCIFICATION ECT FNACACCORD pathology M F cm 1cm YES NO COLD WARM YES NO Adenoma Nodular Thyroid cancer GD Hashimoto control Table 1 experimental groups general clinical conditions

PCR products were identified Extract the DNA template Modified DNA methylation Methylation specific PCR MSP electrophoresis results Calculated for each group gene methylation rate MSP Experimental methods in peripheral blood the rate of Each group gene methylation Conclusion Relationship between the rate of clinical data with gene methylation Collecting Blood samples

PCR products were identified Mononuclear cell separation PBMC Total RNA extraction CDNA was synthesized (M-MLV) Reverse transcriptase polymerase chain reaction RT-PCR electrophoresis Each group gene mRNA positive rate RT-PCR Experimental methods Gene mRNA positive expression rate Conclusion Relationship between the rate of clinical data with gene mRNA expression Collecting Blood samples

Result 1 Four genes MSP The high methylation status of DAPK, RARβ 2 PTEN, RASSF1A 4 genes in the peripheral blood in thyroid cancer adenoma nodular goiter groups, thyroid cancer> adenoma> nodular goiter. RARβ2 gene hypermethylation was found in Hashimoto's thyroiditis nodules

Result 1 Four genes MSP electrophoresis m :the DM bpDNA Marker; 1: cancer; 2: nodular goiter; 3: normal control group; M: methylation bands 98bp, U: non-methylation of bands, 106bp, (50bp at the primer- dimers RARβ2 PTEN RASSF1A DAPK M:DM bpDNA Marker, 1 : Acancer patient; Sample 2: thyroid adenoma; samples: normal control group; M methylation bands, 149bp, U unmethylated bands 151bp (50bp at the primer-dimers)

Result 1 Adenoma Nodules Thyroid cancer GD Hashimoto Control RASSF1A PTEN 45% 40% 35% 30% 25% 20% 15% 10% 5% 0 DAPK RARβ2 Figure 1 The methylation rate distribution of PTEN RASSF1A DAPK RARβ2 in the experimental group

Result 2 Four genes mRNAexpression DAPK, RARβ2, PTEN, RASSF1A gene mRNA expression in the peripheral blood of thyroid cancer and adenoma reduce or even missing, And was negatively correlated with 4 genes methylation. Tip 4 genes promoter methylation may be one of the reasons causing gene expression to reduce or missing.

Result 2 The four genes mRNA expression electrophoresis DAPK RARβ2 PTEN RASSF1A M DM bpDNA marker; 1,2: nodular goiter; 3,4: adenoma; 5: Hashimoto's thyroiditis; 6,7: thyroid cancer; RT-PCR product of 351bp; product of β-actin 550bp; M DM bpDNA marker; 1,2: nodular goiter; 3,4: adenoma;5,6,7: thyroid cancer; RT-PCR product of 335bp; product of β-actin 550bp;

Result 2 Adenoma Nodules Thyroid cancer GD Hashimoto Control RASSF1APTE N 90% 80% 70% 60% 50% 40% 30% 20% 10% 0 DAPK RARβ2 Figure 2 The expression rate distribution of PTEN RASSF1A DAPK RARβ2 mRNA in the experimental group

Result 3 Clinical significance DAPK, RARβ2 PTEN and RASSF1A gene promoter methylation and mRNA expression are realated with lymph node metastasis in thyroid cancer patients.No relationship between nodule size, calcification and ECT.

Result 4 Clinical significance PTEN, RASSF1A methylation and loss of expression play an important role in the elderly thyroid cancer. PTEN, RASSFIA genes may be a prognostic indicator of thyroid cancer. Female thyroid cancer more likely to be found in the RASSF1A gene promoter region methylation

Discussion: Tumor suppressor genes regulate cell growth and differentiation and has the potential to inhibit tumor growth function. When they functionally inactivated or when genes deletion, mutation can lead to tumorigenesis.

Discussion: The test results found that the group of thyroid cancer and thyroid adenoma group occurred 4 genes hypermethylation and mRNA transcripts reduced or missed.

Discussion: The experiment result found a negative correlation between the methylation and mRNA expression of the 4 genes.

It showed that the methylation of the 4 genes promoter region CPG can inhibit the mRNA expression,resulting in functional inactivation and thus participate in the incidence of thyroid cancer and thyroid adenoma.

Discussion: Be alert of the thyroid cancer when hypermethylation occured in benign thyroid adenoma. The reason maybe to discover that methylation can be detected in the peripheral blood of patients with thyroid adenoma during the tumor growth and metabolic process,when the tissue or cells injure,necroses and release DNA into the blood.

Discussion: HT is related with PTC. Some study proposes that the HT may be one of high risk factors for thyroid cancer. The above study may suggest that RARβ2 gene promoter hypermethylation may contribute to Chronic lymphocytic thyroiditis Development of thyroid cancer.

Discussions: The methylation rate of PTEN RASSF1A RARβ2 has nothing to do with the nodule size, calcification and ECT.There is no statistically significant difference. Methylation is related with the age lymph node metastasis in thyroid cancer.

Conclusions: The determination of MSP DNA and mRNA in peripheral blood of the pations with nodular thyroid play an important role in early diagnosis about the cancer.

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