The Influence Of TET2 On The Growth Of CML Cells

Objective: This study aims to investigate the effect of TET2 on the growth of CML cells.Methods:(1) The bone marrow nucleated cells of CML patients and healthy donors were obtained with gradient centrifuge using Ficoll. PCR and directly sequencying were used to sequence exon3 to 11 of TET2 gene. Subsequently, Sanger sequencing of PCR products were performed in CML-CP and CML-BC patients;(2) Using Immunomagnetic beads to purify CD34+ cells, then qRT-PCR was used to measure mRNA expression of TET2 in bone marrow mononuclear cells(BMMNCs) and CD34+ cells of CML-CP patients and healthy controls;(3) Small hairpin RNAs(shRNAs) against TET2 were delivered with lentiviral vectors to K562 cells, then qRT-PCR, Western blot, and Dot-blot were used to check transfection efficiency;(4) Lentiviral vectors were constructed to knock down TET2 and then transduced into K562 cells, then control and TET2 silenced cells were assessed for their cell proliferation and colony formation;(5) The sensitivity of TET2 silenced cells in the treatment of different concentration of Imatinib mesylate(IM) were measured and compared to the control cells.Results: 1. No TET2 mutation was detected in CML-CP and CML–BC patients; 2.The mRNA expression of TET2 from bone marrow mononuclear cells(BMMNCs) and CD34+ cells in CML-CP were significantly lower than those normal donors(P < 0.05); 3.Two independent shRNA sequences against TET2 were validated to suppress the transcript and protein expression of TET2 as high as 50% and the levels of 5-hmc were dramatically reduced in genomic DNA when compared with the control group; 4. Cell proliferation and colony-forming cell capacities were significantly enhanced by TET2 silence compared to control cells(P < 0.05); 5. We also found that targeting TET2 suppressed the sensitivity of K562 cells in the treatment of IM(P < 0.05).Conclusion: 1. In this study, TET2 mutation are not detected in CML-CP and CML-BC patients; 2. TET2 has significantly lower expressed transcript in bone marrownucleated cells and CD34+ cells from CML-CP patients compared with healthy donors; 3.Successfully constructed silence of TET2 in K562 cell lines by lentiviral-mediated RNA interference. 4. Targeting TET2 could enhance K562 cells proliferation and colony formation; 5. We found that the sensitized IM response could be suppressed by TET2 knockdown.