| ObjectiveWnt signal pathway plays a critical role in the process of embryonic development, Improper activation of Wnt singal pathway may be involved in tumorigenesis. It has been reported in many studies that Wnt5a is a good marker for invasive and prognosis of tumor, While the expression of Wnt5a is variant in different kinds of tumor. So the exact mechanism of Wnt5a in tumors is not clear .especially, the relationship between wnt signal pathway and tumorigenesis of leukemia was rarely reported. In our prophase study , the expression of Wnt5a gene in myeloid leukemia was decreased or lost, and the conditioned medium of Wnt5a can inhibit the malignant proliferation of K562 cells .in this study, we planned to observe if the decreased expression of Wnt5a was a universal phenomenon in leukemia and its molecule mechanism, so as to furtherly testify if the lost expression of Wnt5a was related to the leukemia .this study found that the decreased expression of Wnt5a in cases of leukemia and leukemia cell lines for the first time, and explored the reasons for the lack of Wnt5a expression, and layed a solid foundation for in-depth research of Wnt5a gene in the occurrence of leukemia . Our studies may provide new tumor marker for gene diagnosis and provide new therapy target for leukemia.Methods and materials1. RT-PCR was performed to determined the levels of wnt5a expression in bone marrow samples from 27 health adults and CD34+ cell from 10 umbilical cord blood and leukemia cell lines K562 ,Jurkat,HL-60,U937.2. RT-PCR was performed to determined the levels of expression of wnt5a in bone marrow samples from 68 patients with leukemia and 35 patients with leukemia-CR.3. Methylation-specific PCR(MSP) was performed to determined the status of methylation of Wnt5a gene promoter in bone marrow samples from health adults and patients with leukemia and leukemia cell lines K562 ,Jurkat,HL-60,U9374. 4. After exposing the leukemia cells to the demethylation reagent 5-aza-2'-deoxylcytidine(Adc)-, the change of levels of Wnt5a expression and the status of methylation of Wnt5a gene promoter was observed.5. After exposing the leukemia cells to the demethylation reagent 5-aza-2'-deoxylcytidine(Adc)- for 4 days, the proliferation of cells was evaluated by counting with trypan blue staining. The total cell counting and statistical analyse were carried out and a growth curve was plotted.6. Flow cytometry was performed to assay the cell cycle of K562 cells after being exposed to Adc for 4 d.Result1. Wnt5a mRNA were expressed in 24 bone marrow samples of healthy adults, and the positive rate was 88.9% (24/27); Wnt5a mRNA were expressed in all of the samples of CD34 + umbilical cord blood cells; Wnt5a mRNA were not expressed in leukemia cell lines K562, HL-60, U937 and was weakly expressed in Jurkat cell line.2. Wnt5a mRNA were expressed in 7 bone marrow samples from patient with leukemia, and the positive rate was 10.1% (7/68),;Wnt5a mRNA were all expressed in 23 bone marrow samples from patients with leukemia-CR ,and the positive rate was 65.7% (23/35).3. Methylation of Wnt5a gene promoter were not found in bone marrow samples from healthy adults using the methylation-specific PCR; methylation were found in the leukemia cell lines K562, HL-60, U937, and partial methylation was found in the leukemia cell line Jurkat; Loss of methylation was found in bone marrow samples patients leukemia -CR.4. after being exposed to demethylation reagent (Adc) ,the expression of Wnt5a was increased and methylation was lost in the leukemia cell lines K562, Jurkat, HL-60, U937 .5. after being exposed to demethylation reagent (Adc) for 4 days .The growth of K562 cell was inhibited , but the vitality was not affected.6. The number of K562 cells exposed to Adc at G2 phase was 3 times more than control .Conclusion:1. Wnt5a mRNA was expressed in 88.9% (24/27)of bone marrow samples from healthy adults ,in 100% (10/10)of CD34+ cell in umbilical cord blood samples , in 13.2% (9/68)of bone marrow samples from patients with leukemia, in 65.7%(23/35)of bone marrow samples from in patient with leukemia-CR (5 times more than that in samples from bone marrow of the patients with leukemia). It is implicated that the loss of Wnt5a expression may be related to the tumorigenesis of leukemia.2. The methylation of Wnt5a gene promoter were found in some patients with leukemia and leukemia cell lines (K562,Jurkat,HL-60,U937),and were not found in bone marrow samples from healthy adults and patient with leukemia-CR. It is implicated that the methylation of Wnt5a gene promoter may be one of the reasons for the decline of Wnt5a expression.3. After being exposed to demethylation reagent, the demethylation of Wnt5a promoter were found in leukemia cell lines K562,Jurkat,HL-60,U937 ,and the expression of Wnt5a was increased. Compared with the control, the growth of leukemia cell line K562 exposed to demethylation reagent was significantly inhibited, and the number of cells in G2 phase rised significantly. It is implicated that Adc can block cell cycle of K562 cells in G2 phase and inhibit cell proliferation.Our studies demostrate that: the loss expression of Wnt5a was a universal phenomenon in leukemia, and the expression of Wnt5a was recovered in the patient with leukemia-CR. The silent expression caused by methylation of Wnt5a gene promoter was the mechanisms of the loss expression of Wnt5a in leukemia, Wnt5a played a role of anti-oncogene in leukemia, and the loss expression of Wnt5a was one of the mechanisms of leukemia genesis.
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