Screening And Functional Identification Of TET1 Mutations In Colon Cancer

DNA hydroxymethylation is another important epigenetic modification found after DNA methylation,which plays an important role in gene expression regulation and chromosome remodeling.TET1?ten-eleven-translocation 1,TET1?gene as a member of the TET family of proteins that regulate the formation of hydroxymethylation of DNA can rely on Fe²⁺and?-ketoglutarate to catalyze5-methylcytosine to form 5-hydroxymethyl-cytosine?5hmC?and further oxidized to form 5-formylcytosine?5fC?and 5-carboxylcycosine?5ca C?in epigenetics Important position.TET protein is closely related to the occurrence of tumor.TET2 and TET3are widely regarded as a tumor suppressor gene in TET protein family,and TET1 is controversial between tumor suppressor gene and oncogene.Gene mutation is an important reason for tumorigenesis,TET2 mutation is currently found much more,and the study is relatively more thorough.In myeloproliferative tumors,systemic mast cell hyperplasia,chronic myeloid leukemia and myelodysplastic syndrome have numerous mutations of TET2,and it has been demonstrated that partial mutations can affect the function of TET2,some of which have reported that TET2 mutants have been used as a oncogene and have used TET2 mutations to predict the occurrence of cancer.However,TET1 mutations have been reported very few,the study of TET1 in the tumor mutation in the analysis of TET1 is an oncogene or tumor suppressor gene will have a critical significance.In this study,13 cases of colon cancer tissue were extracted DNA,followed by the design the amplification primers of TET1 coding region then sequencing after amplified.Through the sequencing of these 13 cases of colorectal cancer,we found two missense mutations,corresponding to the mutations in the mRNA and the changes were 3784 A?G and 6749 G?A,corresponding to the position on the protein and and the changes were I123I?M and 2082 E?K.We used site-specific mutagenesis to mutate TET1 plasmids and transfected the wild-type and mutant TET1overexpressing plasmids into HEK293 cells,the expression of 5hmC was detected by Dot blot after extraction of the DNA,the expression of TET1 protein was detected by Western blot after extraction of the protein.Our results showed that single mutations of 6749 G?A and double mutations of3784 A?G and 6749 G?A were able to significantly reduce the ability of TET1 to catalyze 5mC formation of 5 hm C,And there was no significant difference between double mutations and single mutations.In this paper,the mutation of TET1 gene in colon cancer was analyzed and two mutations were identified to help elucidate the function of TET1 and its significance in tumorigenesis.