Screening And Identification Of TET Mutations In Esophageal Cancer

TET protein is an important hydroxymethylated enzyme,including TET1 and TET2 and TET3,could catalyze 5-methyl-cytosine(5mC)to form5-hydroxymethyl-cytosine(5hmC).TET protein plays an important role in the occurrence and development of tumors and other diseases.Currently,TET2 and TET3 are widely considered to be a tumor suppressor gene,but the role of TET1 is controversial.Gene mutation is an important cause of tumorigenesis,there are multiple gene mutations have been found about TET protein family in the tumor,especially TET2 mutation frequency is higher,reports of TET1 mutations are rare,and so far,only one case of TET3 mutation has been reported.Studying the mutations of the TET family in the tumor is important for analyzing the mechanism by which TET affects the occurrence of the tumor.DNA was extracted from 25 cases of esophageal cancer tissues and 25 cases of paracancer tissues in the clinical samples,and the TET mutation sites were screened to verify the selected mutation sites in esophageal cancer cell lines.Clinical samples of25 cases of cancer and 25 cases of esophageal tissue adjacent tissue RNA extraction,using the fluorescent quantitative PCR technique to detect TET expression in esophageal cancer,further combining with TCGA database of TET1 relevance to esophageal cancer clinical data analysis.Site specific mutation technique is used to analyse the TET1 plasmid fixed point mutation,wild-type and mutant TET1 plasmids were transfected into HEK293 cells.,by dot blot to detect the expression of 5hmC,by Western blot detection TET1 protein expression.Wild-type and mutant TET1 plasmids were transfected into EC109 cells,and the effects of this mutation on the proliferation,migration and invasion of EC109 cells were studied by MTT,scratch and Transwell experiments.Through research we found A high frequency of TET1 missense mutation,corresponding mutation position and changes in mRNA were 3874 A?G,respectively corresponding to the position and changes in protein were 1123I?M,and there are also the mutation in the esophageal cancer cell line.Rt-pcr results showed low expression of TET1,2 and 3 in esophageal cancer compared with paracancerous tissue.TET1 expression was lower in esophageal carcinoma cell lines than in normal esophageal cells.Dot blot and Western blot showed that the 1123 mutation had no significant effect on the hydroxymethylated function and protein expression of TET1,however,this mutation has certain effects on cell proliferation and invasion.The inhibition effect of transfection of TET1 and 1123 mutant plasmids on the proliferation and invasion ability of EC109 cells was significantly different.Rt-pcr results suggested that the decreased proliferation and invasion ability of EC109 cells induced by transfection of 1123 mutant plasmids might be related to the down-regulation of GLUT1 and MTA2 genes,providing new ideas for the early identification of esophageal cancer.