The Establishment And Optimization Of The Detection Method Of TET Protein Oxidation Activity And The Effect Of TET Protein On Cell Proliferation And Apoptosis

Objective Genome methylation level is closely related to cell proliferation,apoptosis and tumor occurrence and development.Ten-eleven translocation(TET)participates in DNA demethylation by oxidizing 5 methylcytosine(5m C).Therefore,finding a method to specifically regulate TET protease activity in vitro or in vivo has an important guiding role in the study of cell proliferation and tumorigenesis.In this study,for the screening of TET protein specific activators or inhibitors,the detection method of TET protein oxidation activity in vitro was optimized,that is,high resolution melting curve(HRM).In order to verify the specific activator or inhibitor of TET protein at the cellular level,an overexpressing cell line of TET protein was established.At the same time,by using the established TET protein overexpression cell line,the effect of TET protein on cell cycle,proliferation and apoptosis was studied.Methods For the screening of TET protein-specific activators or inhibitors,we transformed the p CDF-His-m Tet1 CD plasmid into E.coli B21,collected and purified the TET1 protein expressed in E.coli B21,and used Western Blot and DNA Dot Blot to detect the oxidative activity of purified TET protein and TET protein,respectively.At the same time,we tested the effects of different DNA length,DNA amount,fluorescent dye SYBR Green I concentration,and buffer conditions on HRM.In order to verify the specific activator or inhibitor of TET protein at the cellular level,we constructed a TET overexpression plasmid in vitro,and then introduced it into cells by cell transfection method,using antibiotic screening method to obtain the stable expression of TET protein overexpression cells.Besides,Western Blot and DNA Dot Blot methods were used to verify the expression of TET1 protein and the oxidation activity of TET protein in the stable cell line.In addition,we examined the effects of TET1 protein on cell cycle,apoptosis and proliferation by flow cytometry and cell counting.Results Western Blot and DNA Dot Blot results showed that TET1 protein with oxidative activity was obtained from E.coli B21.The results of HRM optimization showed that a satisfactory melting curve could be obtained under the condition of using 50 ng of DNA with a length of 100 bp in Buffer(25 m M TAPS-HCl,2 m M Mg Cl2,50 m M KCl)with 10 × SYBR.The HEK293 T cell line with inducible TET protein overexpression was successfully obtained.Verification of Western Blot and DNA Dot Blot for HEK293T(Tet1CD)cell line confirmed the presence of inducible and oxidative TET1 protein in HEK293T(Tet1CD)cells.Flow cytometry results showed that TET1 CD overexpression promoted apoptosis(P <0.05)and increased the proportion of cells at G2 stage(P <0.05).Cell count results showed that TET1 CD overexpression inhibited cell proliferation(P <0.0001).Conclusion: The optimized conditions of HRM and the purified TET1 protein with oxidative activity laid a solid foundation for the screening of TET protein-specific activators and inhibitors.In addition,TET1 protein may ultimately inhibit cell proliferation by promoting apoptosis and blocking the cell cycle,and its effect on cell proliferation provides another idea for highly efficient cell culture in vitro.