The Methylation Of DKK3Gene And Its Regulation In Papillary Thyroid Carcinoma

Background and ObjectiveThyroid carcinoma is the most common endocrine malignancy in the world, and papillary thyroid carcinoma (PTC) accounts for over75%of all thyroid malignancies. The incidence of PTC has been rising rapidly in recent decades worldwide. Previous studies have showed that multiple genetic alterations involved in thyroid carcinogenesis, but its molecular is poorly understood. However, the changes of oncogenes and tumor suppressor genes silenced could not completely explain the activation/inactivation by the classical regulatory control. Recent data suggest that tumor suppressor genes were silenced by epigenetic mechanism, DNA methylation is the most important mechanism in epigenetic. Because DNA methylation is reversible, some demethylation drug could reverse the CpG methylation of tumor suooressor gene, recover its function for tumor suppress. There are two demethylation drug at present,5-azaC and5-azaCdR.5-azaC as one of demethylation drug, it could combine the Dnmts, restoring the function for tumor surpressor gene and inhibiting the growth of tumor cells. It has been recently shown that DKK3as a new tumor suppressor gene. The Dickkopf (Dkk) family consists of four members DKK1, DKK2, DKK3and DKK4. DKK3is rather unique in homology analysis of the DNA sequence, expression profiles and biological functions, it combines with the receptor of Wnt, restaining the Wnt pathway and resulting in tumor production. It has been recently shown that methylation of DKK3gene contributes to carcinogenesis and serves as a potential biomarker for the diagnosis in several human malignancies. DKK3is expressed in many normal human tissues and is commonly down-regulated in human cancers such as lung cancer, bladder cancer, breast tumor, esophagus tumor and gastrointestinal tumors. The role of DKK3gene and its epigenetic alterations remain unknown in thyroid cancer. We used Methylation-specific PCR and RT-PCR to examine the methylation of the DKK3promoter and its mRNA expression in49samples of PTC and their matched adjacent non-cancerous epithelium (NCE), the results were compared with the clinicopathological data, evaluating DKK3as a putative tumor suppressor gene in PTC. MTT assay was used to examine the growth of PTC cell line after treatment with5-azacytidine (5-azaC) and accompanied by measurement of DKK3gene promoter methylation and mRNA expression under this condition, evaluating the clinical implications of promoter hypermethylation of the DKK3gene in PTC.Material and Method1. MSP and RT-PCR were used respectively to examine the CpG methylation of the DKK3promoter and its mRNA expression in49samples of PTC and their matched adjacent non-carceious epithelium (NCE), analyed the clinicopathological data.2. MTT method was applied to detect the growth of TPC-1after treated by5-azaC with2、5、10、20、50、100、200、400μmol/L. DKK3gene DNA, mRNA were determined by MSP and RT-PCR after TPC-1treated by5-azaC with5、10、20、50μmol/L.3. Statistical analyses were performed using SPSS software version10.0χ2test, t test, and one-way ANOVA were used to analyze the significance. P<0.05was considered statistically significant.Results 1. In PTC, the frequency of DKK3gene promoter methylation was38.8%(19/49), significantly higher than that in NCE (P<0.05). The positive rate of DKK3gene mRNA expression was44.9%(22/49) in PTC, lower than that in NCE (P<0.01). In PTC, the hypermethylation of DKK3gene and expression of its mRNA had correlation with pathology stage, TNM stage, lymph node metastasis respectively (P<0.05).2. There was distinct correlation between methylation of DKK3gene promoter and expression of its mRNA in PTC(P<0.05)3.5-azaC could reduce TPC-1cells viability in a dose and time-dependent manner(P<0.05). After TPC-1cells treated by5、10、20、50μmol/L of5-azaC for48h, the relative expression of DKK3mRNA is0.208±0.017、0.365±0.013、0.489±0.017、0.582±0.011respectively, and the expression of DKK3mRNA was gradually increased in a dose-dependent manner (F=370.356, P<0.05). Meanwhile, DKK3gene was demethylated partly after treated with5-azaC (Fm=315.188,Fum=195.257;P<0.05)Conclusions1. Methylation of the DKK3promoter might be one of the mechanisms resulting in transcription silence, and might play an important role in carcinogenesis and progression of PTC2.5-azaC might effectively cause the demethylation of DKK3gene CpG-rich promoter regions, activate its expression, and inhibit the growth of TPC-1cells.