| Objective:To clarify the expression of Wilms'tumor 1 (WT1) gene in leukemic cells and its subcellular localization, observe the biological effects of its inhibitor-Curcumin and the related mechanisms. Meanwhile, RNA interference (RNAi) was used to downregulate WT1, and to explore the mechanistic roles of WT1 in regulating proliferation of leukemic cells with functional assays.Methods:Realtime-PCR and Western blot were used to detect expression of WT1 mRNA and protein. The immunofluorescence and Western blot were used to clarify the subcellular localization of WT1. The genome-wide transcriptional targets of WT1 were analyzed using Chromatin ImmunoPrecipitation-DNA Selection and Ligation (ChlP-DSL) approach. MTT method was used to detect cell proliferation and then proliferation curve and inhibition rate were analyzed; cell cycle, apoptosis, and differentiation were analyzed by flow cytometry. Lentiviral vectors pLKO.1-puro containing the WT1-shRNA or the control-shRNA were constructed and transfected into leukemic cells to downregulate WT1 expression, and to delineate its biological effects and possible mechanisms.Results:WT1 was expressed in most leukemic cells, except U937. WT1 protein localizes in both cytoplasm and nucleus, predorminantly in cytoplasm. Since WT1 was highly expressed in K562, a cell line derived from CML blast phase and resistant to many drugs, the following studies were focused on K562 cells:1. Curcumin could inhibit cell proliferation in a time and dose dependent manner, it also showed synergistic effects on proliferation inhibition with Glevec or chemotherapeutic drug-VP16. Meanwhile, Curcumin induced cell differentiation and cell cycle arrest at G2/M phase.2. Using RNAi technique, WT1 could be downregulated by 70%-80%at mRNA and protein levels. Cell proliferation and colony formation were obviously inhibited, meanwhile RNAi was also found to enhance the sensibility of chemotherapeutic drugs. Cell cycle progression analysis delineated that the ratio of G0/G1 phase increased while the cells in S phase decreased distinctively after WT1 RNAi. ChlP-DSL experiment identified a cohort of genes whose promoters are targeted by WT1, These genes were then classified into cellular signalling pathways using MAS software including Wnt/β-catenin pathway, MAPK signaling pathway, apoptosis pathway, cell cycle and so on. Therefore, we focused on the Wnt/p-catenin signal pathway. Just as supposed,β-catenin, an important gene in the canonical pathway, was also downregulated following WT1 RNAi, and interestingly, the target genes of P-catenin which participate in cell proliferation and cell cycle regulation, such as CCND1 and MYC were also downregulated distinctively. From above mentioned, we suppose that the biolgical effects of WT1 RNAi may be the results of inactivation of Wnt/β-catenin pathway, which was regulated by WT1 on transcription levels.Conclusion:WT1 was highly expressed in many leukemic cells. The inhibitor of WT1, Curcumin, showed great anti-leukemic effects, such as proliferation inhibition, cell cycle arrest, synergistic function with other drugs, all the above implicated that Curcumin would show bright perspective in treatment of hematological tumors, especially in CML. Down-regulation of WT1 by RNAi, cell proliferation and colony formation were inhibited, and cell cycle arrested at G0/G1 phase. In a word, as an important oncogene, WT1 takes part in leukemogenesis and progression possibly through regulating the Wnt/β-catenin pathway.
|
PDF Full Text Request