Papillary Thyroid Carcinoma Is Regulated By TET3 Through 5hmC And BRAF Modification

Thyroid cancer accounts for 7%to 15%of thyroid nodules and is the most common endocrine tumor.The thyroid papillary carcinoma occupies an increasing proportion,because the huge number of patients is still increasing,we need to develop effective diagnosis and treatment of thyroid cancer strategy.Routine clinical pathology assessment is currently the basis of risk stratification for patients with thyroid cancer.The main clinical pathologic factors include age,sex,tumor size,extrathyroidal extension,lymph node metastasis,distant metastasis and advanced stage.But the traditional clinical and pathological factors can not be effective and accurate identification of invasive thyroid cancer.At present,the best predictors and diagnostic guidelines for thyroid papillary carcinoma are still controversial.Therefore,great progress has made in the field of molecular biology.BRAF mutations are the common somatic mutations in thyroid cancer and occur in about 45%of thyroid papillary carcinomas.BRAF can activate MEK to cause downstream MAPK pathway effects.However,the function BRAF gen may not only change by genetic changes.In recent years,more and more studies have focused on epigenetic modifications,which can elicit genetic changes in gene expression or cell phenotype without changing the DNA sequence.DNA methylation is the most widely studied epigenetic modification which occurs in the Cp G site,whereas Cp G islands are not methylated,and most gene promoters are located in this region,suggesting that there is a correlation between methylation and tumor.5-methylcytosine is a common Cp G modification in the genome,which is usually associated with gene silencing.In this study,we collected the date of 50 patients and analysis the clinical and pathological characteristics.The BRAF sequencing results were analyzed and the result suggest that BRAF^V600E mutation is associated with extrathyroidal invasion and lymph node metastasis.In the meantime,the immunohistochemistry result of 5hm C found that 5hm C level is a clinical index with higher specificity and sensitivity,suggested 5hm C could serve as a clinical predictive indicator.In the thyroid tissue,5hm C level of genomic DNA was significantly increased in Dot-blot,and the real-time PCR and Western-blot indicated the expression of TET3 was elevated in RNA and protein levels.In vitro experiments,we blocked the expression of TET3 in TPC-1 and B-CPAP cells by small interfering RNA.Immunofluorescence assay showed that the decrease of TET3 expression is associated with the methylation level.CCK-8,Transwell and Wound Healing test showed that the decrease of TET3 is associated with low cell proliferation,invasion and migration ability.Since TPC-1 and B-CPAP were BRAF wild-type and BRAF mutant cell lines,and there was no significant difference between these groups,we concluded that TET3 can affect cell function by demethylation and is not affected by BRAF mutation.The CRISPR/Cas system is the immune system of prokaryotes which can give prokaryotes resistance to exogenous genetic material,after transfect the synthesis of guide RNA and Cas9 nuclease complex into the cells,the target DNA can be cut at the designated genome site and finnaly achieve gene knockout or introduction.This method can be simple and efficient targeting of specific genomic DNA sequence,which can let people find new gene function quickly,develop new cellular and animal disease models,and make progress in cancer treatment.In this study,CRISPR/Cas9 was used in thyroid papillary carcinoma to explore the mechanism of hydroxymethylase TET affecting thyroid papillary carcinoma cells.First,we established stable TET3 knockout TPC-1 cell line by CRISPR/Cas9,and then overexpress the wild type BRAF gene.Compared with the control group,the TET3 knockout group has lower demethylation level,invasion,proliferation and migration ability,but after overexpress BRAF gene in the TET3 knockout group,we can observe the restore of the ability of invasion,proliferation and migration.The experiment was repeated in BRAF mutant cell line B-CPAP and the same results were observed.The Ch IP-q PCR analysis revealed that TET3 could bind to the promoter region of the BRAF genes and regulate the expression of BRAF.Indicated that TET3is involved in the development of tumors depending on the activity of BRAF gene.