The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms＇ tumor 1 suppressor gene （WT1） and CML28 abnormally expressed in malignant diseases of the hematopoietic system and...The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms＇ tumor 1 suppressor gene （WT1） and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the rela- tionship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA （siRNA） against WT1 and CML28 in leukemic cell line K562 to examine the interac- tion between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was de- tected by using RQ-PCR and Western blotting. K562 cells transfected with WTI-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by us- ing immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide im- portant clues for further study on the role of CML28 and WT1 in leukemic cells.展开更多
Objective To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm’ tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. Methods The H...Objective To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm’ tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. Methods The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively. Results The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC<sub>50</sub>) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner. Conclusions Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G<sub>0</sub>/G<sub>1</sub> phase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.展开更多
文摘The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms＇ tumor 1 suppressor gene （WT1） and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the rela- tionship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA （siRNA） against WT1 and CML28 in leukemic cell line K562 to examine the interac- tion between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was de- tected by using RQ-PCR and Western blotting. K562 cells transfected with WTI-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by us- ing immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide im- portant clues for further study on the role of CML28 and WT1 in leukemic cells.
基金Supported by National Natural Science Foundation of China(No.81403223)Zhejiang Provincial Natural Science Foundation of China(No.LQ14H290003)Science and Technology Foundation of Zhejiang Province(No.2015C33173)
文摘Objective To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm’ tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. Methods The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively. Results The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC<sub>50</sub>) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner. Conclusions Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G<sub>0</sub>/G<sub>1</sub> phase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.
