Correlation Between Epigenetic Alteration Of WNT Pathway Inhibiting Gene And Acute Leukemia And The Possible Effects That Genistein Might Have On The Histone Modification In WNT Pathway Inhibiting Gene

Objective: Our study chosen antagonist gene of Wnt pathways as the target genes to explore the changes of Epigenetic and the correlation between the expression of Wnt pathways antagonism gene and histone modification.Then,the Genistein was used to interfere cell lines with high methylation genes in Wnt pathways in order to find such as the effect of transcription regulation,influence and the mechanism of histone modification.These could provide a new way for the treating of leukemia.Methods: Firstly,real-time qPCR method was used to detect the mRNA expression level of Wnt pathway antagonist gene(Wnt5a,HDPR1,DKK1,DKK3)in acute leukemia patients and normal people.By using the Pyrophosphate sequencing and methylation specific PCR(MSP)to test the statues of Wnt5 a gene promoter region methylation.With the help of Western Blot method,the Wnt5 a protein and Histone H4 lysine 20-Monmethylation(H4K20me1)total expression level in acute myelogenous leukemia and normals were compared.The correlation between Wnt5 a protein and H4K20me1 in leukemia;Secondly,the acute lymphocytic leukemia cell line Jurkat and acute myeloid leukemia cell line U937,which were all belong to the high methylation deactivation of Wnt pathway antagonist gene(Wnt5a,HDPR1,DKK1,DKK3)were chosen to the basic objects.Then the Genistein was added to two kinds of cells.The cell proliferation,apoptosis,cycle changes,gene expression of Wnt pathways antagonism(Wnt5a,HDPR1),H4K20me1 methyltransferase(set8),Wnt pathways downstream target genes AXIN2 and c-myc were detected.BSP technology and Pyrophosphate sequencing was used to analyze the changes of Wnt5 a promoter region methylation in control group and dosing groups.Moreover,enrichment degree of H4K20me1,and H3K9 ac in Wnt5 a,were inspected by Chromatin Immunoprecipitation –quantitative Polymerase Chain Reaction(CHIP-qPCR)technology,which was located in promoter region and coding region respectively.Then the Genistein influenced the genome protein modification on WNT signaling pathways downstream target gene expression throughout which way was deeply studied.In the end of this part,the relationship with the Wnt5 a and genome protein H4K20me1 and possible mechanism of changes in the apoptosis and cell cycle caused by Genistein were analyzed;Thirdly,we designed and synthesized a lentivirus carrier that was high expressed in Wnt5 a gene.Then the Jurkat and Kasumi cells that was low expression of Wnt5 a gene were carried out the transfection experiment.The growth curve,the proliferation activity,colony-forming level,cell cycle and apoptosis cells were tested.With the help of Western Blot method,the changes of key effect protein β-catenin,Wnt pathways downstream target genes c-myc,cell cycle related proteins(G2/M phase)and apoptosis related proteins were detected.Lastly,this study clarified the mechanism of Wnt5 a genes in anti-proliferation and inducing the apoptosis in leukemia cells.Results: 1.Wnt pathways antagonist gene(Wnt5a HDPR1,DKK1,DKK3)was widely low expressed in acute myeloid leukemia.However it showed differently in lymphocytic leukemia.The Wnt5 a showed a lower expression in acute T lymphocyte leukemia.The DKK3 in myeloid and acute leukemia were generally lower.The expression of HDPR1 among the acute lymphoblastic leukemia and normal people showed no significant differences.And the DKK1 in acute lymphoblastic leukemia was significantly higher than normal.2.The extensive of Wnt5 a gene methylation in acute myeloid leukemia patients leaded to the decreased expression of Wnt5 a gene.3.The total protein content of H4K20me1 in acute myeloid leukemia was higher than the normal people.Moreover,the expression of Wnt5 a was positively correlated to expression of H4K20me1.Some evidences provided that the higher expression of Wnt5 a were related to the enrichment of H4K20me1,which was located in Wnt5 a in promoter and coding region.While the expression of H4K20me1 had no relevance with Wnt5 a in normal people.4.By the intervention of Genistein,the proliferation of Jurkat and U937 cells were inhibited in vitro.The Genistein could block the cell cycle process and induct apoptosis because of activating the H4K20me1 methyltransferase(set8)and improving the enrichment of H4K20me1 in the Wnt5 a promoter region.Through above ways,the suppressor genes of Wnt pathway were activated and the Wnt/β-catenin signaling pathways were inhibited.In this study,the state of genome protein modification on the activation of suppressor genes played a key role of Wnt pathway.The purpose of regulation of gene expression could be achieved by changing the genome protein H4K20me1 modified state without reversing the methylation status.5.In the Jurkat and Kasumi cell line that was lower expression of Wnt5 a gene,overexpression of Wnt5 a gene could inhibit cell proliferation,promote apoptosis,block the cell cycle in G2/M phase.This condition was the same with the Genistein inducing.Hence,Genistein was considered to show the antitumor effect by raising the Wnt5 a expression.In the light of the role of Wnt5 a playing in significant anti-tumor effect,it might be an important target for gene therapy.Conclusions: The antagonists gene methylation of Wnt pathway generally exists in patients with acute leukemia,which is particularly significant in patients with AML,while as for ALL,it was not the same in different patients and types;The Wnt5 a gene expression in patients is positively correlated with gene promoter region and the coding region of H4K20me1 enrichment.H4K20me1 by means of promoting the transcription initiation and transcription elongation increase the expressive level of Wnt5a;the mechanism of intervention with Genistein in leukemia cells enhancing the expression of tumor suppressor gene Wnt5 a is that the involvement of enrichment of H4K20me1,which was located in Wnt5 a in promoter and coding region,and there was nothing to do with the methylation status.Wnt5 a gene exert the similar function as Genistein in cell cycle arrest,apoptosis,anti-proliferation.Wnt5 a gene is able to inhibit CDK1 kinase activity and induces upregulation of P21,activation of non classical Wnt pathway,which resulting in downregulation of β-catenin and inhibition of Wnt classic pathway downstream gene transcription.