K562 Cells Induced During The Differentiation Of Wt1 Gene Expression And Its Isomer Ratio Changes

Objective To study the expression of WT1 gene and its isoforms during the differentiation of leukemia cell line K562 by fluorescence quantitative RT-PCR and discuss the relationship between WT1 gene and hematogenic cell differentiation , further elucidate the function of WT1 gene during cell proliferation and differentiation.Methods 1. Fluorescence RT-PCR detect system was formed .2.K562 cells were grown in 10ng/ml TPA (12 - tetradecanoylphorbol - 13 - acetate) to induce the differentiation. . Cells viability were verified by NBT reduction test and cells immunophenotype.3. Expression of WT1 gene and 17aa+, KTS+ isoforms were determined by fluorescence quantitative RT-PCR during differentiation of leukemia cell line K562,usingβ-actin as a internal standard. The relative ratio of the four splice variants WT1(+/+),WT1(+/-),WT1(-/+),WT1(-/-) were calculated.Results 1. Three standard curves were formed by positive standard and the coefficient of correlation were all above 0.995.2. During the differentiation of K562 cell, the NBT reduction rate and the CD9 positive rate both increased gradually . 3. There were two high expression levels of WT1gene during the differentiation of K562 cell. As the time of TPA treating was prolonged, the ratio of 17aa+,KTS+ isoforms decreased gradually. The ratio of WT1 (+/+) decreased gradually, 0 hour was 0.40±0.19,and 96hour was 0.15±0.07. While the ratio of WT1(-/-) increased, 0 hour was 0.15±0.11, and 96hour was 0.38±0.12. The other two isoforms ratios did not change significantly.Conclusion 1. TPA can induce K562 cell to differentiate to megakaryocyte.2. During the differentiation of K562 cell induced by TPA, the expression of total WT1 decreased first, but increased again after 72 hours. 3. During the differentiation of K562 cell induced by TPA, the relative ratio of WT1(+/+) decreased gradually, while ratio of WT1(-/-) increased.4. There were two high expression levels of WT1gene during the differentiation of K562 cell, and the relative ratio of the four splice variants changed. Before the differentiation, the majority was WT1(+/+), but after the differentiation, it turned to be WT1(-/-). It indicates that WT1gene may stimulate or inhibit cell differentiation by regulating its four splice variants WT1(+/+),WT1(+/-),WT1(-/+),WT1(-/-).