Study On The Relationship Between Methylation Status Of Wnt5a Promoter And Etiopathogenesis Of Myeloblastic Leukemia

ObjectiveWnt family contains several members and shows a highly regulated pattern of expression and plays distinct roles during development and tissue homoeostasis. Wnt5a, a member of the Wnt family, has been recently found to be correlated with tumorigenesis. The effects of Wnt-5a in kinds of tumors are significantly different. The incidence of AML maintains a pretty high level in China. There are infrequent reports about the relationship between Wnt5a and AML at home and abroad. Our earlier studies of the gene demostrate that the low expression of Wnt5a is universal in leukemia, and the expression of Wnt5a was recovered in the patient with leukemia-CR. Wnt5a may play a role of anti-oncogene in leukemia, and the loss expression of Wnt5a is one of the mechanisms of leukemia genesis. However, the mechanisms of expression silence are not clear. This study was aimed to detect the methylation status of Wnt5a promoter in the acute myeloblastic leukemia patients'and CR patients'bone marrow, and take research on the relationship between aberrant methylation status of Wnt5a gene promoter and mechanism of myeloblastic leukemia, in order to find a new tumor marker and a potential therapeutic target for AML.Methods1. The methylation status of Wnt5a promoter was analyzed by methylation-specific PCR(MSP) and verified in both 30 AML patients and 31 AML-CR patients.2. The methylation status of Wnt5a promoter was analyzed by MSP and verified in 17 CML patients in chronic phase and 3 CML patients in blast crisis.3. The expression of Wnt5a gene was detected with RT-PCR in primary AML cells and marrow stromal cells.4. The methylation status of Wnt5a promoter in primary AML cells and marrow stromal cells was analyzed by MSP. 5. MTT assays were performed to detect the endogenous Wnt5a effect on the proliferation of U937 and the proper treatment time and dosage.6. RT-PCR was performed to detect the expression of Wnt5a while MSP was performed to detect the status of methylation of Wnt5a gene promoter before and after treating with Adc.7. Flow cytometry was performed to assay the cell cycle of U937 cells after being exposed to Adc for 2d.Results1. Methylation of Wnt5a gene promoter were found in bone marrow of samples from 30 AML patients at 73.3% by the methylation-specific PCR; methylation of Wnt5a gene promoter were found in 31 AML-CR patients at 9.7% by the methylation-specific PCR.2. Methylation of Wnt5a gene promoter were not found in bone marrow of samples from either 17 CML-CP patients or 3 CML-BC patients.3. The bone marrow stromal cells and primary AML cells were isolated by gradient centrifugation and purified by anchoring culture. Wnt5a mRNA were expressed in stromal cells but not in AML cells analysed by RT-PCR.4. Methylation of Wnt5a gene promoter were not found in bone marrow stromal cells, and methylation status were partly found in AML cells.5. The modest concentration to treat U937 cells with Adc were 3μmol/L～5μmol/L for 48 or 72 hours separately and the abilities of proliferation were measured by MTT test.6. The Adc up-regulates the expression of Wnt5a mRNA and demathylated status of Wnt5a gene promoter after treating with modest range. And there was a linear relationship between expression levels and Adc concentration over a certain range.7. The number of U937 cells exposed to Adc at G2 phase was higher than the control.Conclusion:1. MSP analysis showed that CpG islands of Wnt5a promoter is hypermethylated in AML patients in acute phase(73.3%), which is rather higher than that in AML-CR patients(9.7%). Our previous study showed loss of Wnt5a expression may be related to the tumorigenesis of leukemia. This study is identical to this view at epigenetics level. It shows that hypermethylation of CpG islands of Wnt5a promoter induces the decline of Wnt5a expression and results in AML. 2. The positive frequency of methylation of Wnt5a promoter is lower after clinical treatment. It implied that aberrant DNA methylation of Wnt5a promoter may be related to the malign hyperplasia of leukemia cells in bone marrow. This could be used as a clinical index for appraising the therapeutic efficacy and prognosis.3. The aberrant DNA methylation of Wnt5a promoter were not found in either 17 CML-CP patients or 3 CML-BC patients. We have found that the Wnt5a mRNA expression decreased in CML patients and K562 cells, which is similar to that in AML. However, the contrary results in methylation study suggest a different regulating mechanism which calls for further research.4. We initially established the methods of bone marrow stromal cell and primary leukemia cell culture. Wnt5a was found high expression in mRNA level but unmethylation in epigenetics in bone marrow stromal cells of AML patients. It suggests the aberrant DNA methylation of Wnt5a promoter specifically associated with the tumor cells.5. After being exposed to demethylation reagent Adc, the demethylation of Wnt5a promoter were found in leukemia cell lines U937, and the expression of Wnt5a was increased. Compared with the control, the growth of leukemia cell line U937 exposed to demethylation reagent was significantly inhibited, and the number of cells in G2 phase rised. It is implicated that Acd can block cell cycle of U937 cells in G2 phase and inhibit cell proliferation.Our studies demostrate that:the positive frequency of the methylation of Wnt5a promoter increased in AML acute phase and decreased in AML-CR phase.The molecular mechanisms of Wnt5a gene silencing in AML is the aberrant DNA methylation of Wnt5a promoter. This rule is not applicable to CML. It is proved that Wnt5a plays a anti-oncogene role in leukemia and the loss of expression of Wnt5a was(is) one of the mechanisms of leukemia genesis in our experiments and previous studies. There is a relation among the aberrant methylation of Wnt5a promoter, the low expression of mRNA and the pathogenesis of acute myeloid leukemia.