TET1 Induces DNA Hydroxymethylation To Regulate Prognosis Of Papillary Thyroid Carcinoma Patients

Background and ObjectivePapillary Thyroid Carcinoma(PTC)is the most common endocrine malignancy caused by thyroid follicular epithelial cells.In recent years,the morbidity of thyroid cancer increased rapidly.The National Cancer Center issued a report in 2018 indicating that thyroid cancer has risen to the fourth place among women in China.Thyroid cancer can be classified into Papillary Thyroid Carcinoma,Follicular Carcinoma,Medullary Carcinoma and Undifferentiated Carcinoma according to the histogenesis and morphology.PTC is the most common endocrine malignancy,accounting up to 85% of the total thyroid cancer.Although PTCs generally tend to be biologically indolent,some patients harbor aggressive clinical characteristics such as distant lymph node metastasis and recurrence.Therefore,it still imposes a great burden on people.Literatures showed that lymph node metastasis exists in about 80% PTC patients.Various studies on the development and pathogenesis of PTC demonstrate that epigenetic alteration plays an important role in such cancer.DNA methylation which occurs at the fifth position of cytosine(5-mC)has been identified as one of the most researched epigenetic modifications in mammals up to date,DNA methylation plays an essential part in the regulation of cellular differentiation and development.In recent years,DNA hydroxymethylation becomes one of the most researched in investigation epigenetic.Several studies indicated 5-hmC is the intermediate of DNA demethylation and the decrease of global 5-hmC has been considered as a biomarker of malignant transformation which has been verified through a wide range of human solid cancers and has been shown to be closely related to TET protein families(TET).TET can dynamically regulate the level of DNA methylation through oxidizing 5-methylcytosine(5-mC)into 5-hydroxymethylcytosine(5-hmC).However,as far as we know,global DNA methylation and hydroxymethylation have not been studied in PTC yet.Therefore,we conducted this investigation to explore the status of global DNA methylation and hydroxymethylation in PTC tissues and cancer-free thyroid tissues.In this study,we identified for the first time that the content levels of 5-mC,5-hmC and TET in tissue of Papillary Thyroid Carcinoma(PTC)are both reduced,the impact of TET1-3 on content of 5-mC and 5-hmC were also evaluated.These may contribute to the diagnosis of this type of cancer.Methods1.Collecting 24 pairs of tissues including papillary thyroid cancer tissues and cancer-free thyroid tissues from the Department of Thyroid Surgery,First Affiliated Hospital of Zhengzhou University from January 2018 to May 2018 and then preserved them in liquid nitrogen.2.DNA and RNA were extracted from papillary thyroid cancer tissues and cancer free tissues using centrifugal column method and Trizol method,respectively.Their concentration and absorbance at 260 and 280 nm were measured through ultramicro-spectrophotometer.3.The expression level of TET1-3 in papillary thyroid cancer tissues and cancer free tissues were detected using qRT-PCR,and the correlation between the expression level and the clinicopathological characteristics of papillary thyroid carcinoma patients were analyzed according to these data.4.The expression level of 5-mC in papillary thyroid cancer tissues and cancer free tissues were detected using ELISA method.5.The expression level of 5-hmC in papillary thyroid cancer tissues and cancer free tissues were detected though ELISA method.6.The correlation between the expression level of TET1-3,5-mC and 5-hmC were analyzed though the SPSS 22.0.7.According to the expression level of TET1,patients with papillary thyroid carcinoma were divided into high-expression group and low-expression group.The correlation between the expression level of TET1 and the clinicopathological characteristics of patients was analyzed using SPSS 22.0.Results1.In ELISA assays,we observed that the level of 5-mC in papillary thyroid cancer was significantly lower than that in cancer free tissues(3.45% in cancer-free tissues and 1.38% in cancer tissues,p=0.001),moreover the level of 5-hmC was significantly decreased either(0.019% in cancer-free tissues and 0.011% in cancer tissues,p=0.02).2.In the qRT-PCR analysis of the TET,data indicated that the expressions of TET protein family in papillary thyroid papillary cancer tissues were down-regulated(TET1 P<0.01;TET2 P=0.04;TET3 P<0.01).3.Analyses were performed to identify whether there are any correlations among 5-mC,5-hmC and these three TET proteins in PTC tissues.The expression level of 5-hmC was confirmed to be a slightly positive association with TET1(r=0.42,p=0.043)but no relevance to TET2(r=-0.23,p=0.277)or TET3(r=0.20,p=0.354).The expression level of 5-mC showed no relevance to TET proteins(TET1: r=0.35,p=0.096;TET2: r=0.001,p=0.995;TET3: r=0.12,p=0.379).Results for the comparison between TET1 and clinicopathologic characteristics of papillary thyroid carcinoma demonstrated that the downregulation of TET1 was correlated with lymph node metastasis(p=0.046)and TNM staging of thyroid cancer(p=0.002).Conclusion1.5-hmC is a biomarker for Papillary Thyroid Carcinoma,TET1 induces the expression level of 5-hmC in Papillary Thyroid Carcinoma to regulate the development of PTC.2.The decrease of TET1 was a negative prognostic factor for Papillary Thyroid Carcinoma patients,which was related to lymph node metastasis and TNM staging.3.5-hmC and TET1 can be used as feasible targets for diagnosis and treatment of Papillary Thyroid Carcinoma.