| Objective:Select the abnormal hyper-methylation suppressor gene(WIF-1 ? DDK3 ?HDPR1)of the WNT signaling pathway in the cell strain NB4 of the acute promyelocytic leukemia as the target, detecting the epigenetics of DNA methylation and histone methylation modification, to explore the epigenetic mechanism of arsenic trioxide on the treatment of acute promyelocytic leukemia. By detect the different expression of DNA methyltransferase, Histone methyltransferase, histone demethylase in the NB4 cell lines after the affectation of arsenic trioxide,to analysis the potential mechanism that Arsenic trioxide regulatory DNA and histone methylation; and detect the different expression of the target gene ?-catenin ?Survivin,cyclin-D1 and C-myc, to further explore the affection in the downstream of WNT signaling pathways, than to make a relatively complete explain of the epigenetic mechanism of arsenic trioxide on the treatment of acute promyelocytic leukemia.Method:1.CCK-8 assay was used to observe the affection of survival rate in the NB4 cell line of acute promyelocytic leukemia after As203 pretreatment; the apoptosis rate and cell cycle proportion of the NB4 cell lines pretreated by As203 was detected by flow cytometry; Bisulfite sequencing polymerase chain reaction(BSP) combine with the TA cloning sequencing method was applied to quantify the methylation promoter region of the suppressor gene WIF-1,DDK3 and HDPR1 in the NB4 cell lines, and evaluate the advantage and disadvantage of BSP compared with MSP and pyrosequencing.2.Chromatin immunoprecipitation assay was applied to detect the monomethylation, dimethylation or trimethylation of H3K9, which is the promoter region of histone WIF-1,DDK3 and HDPR1 pretreated by the As203, q RT-PCR to determine DNA methyltransferase(DNMT1,DNMT3 A and DNMT3B),histone H3K9methyltransferase(SUV39a,G9a) and Histone H3K9 demethylase(JMJD2A,JMJD2 B and JMJD2 C and JMJD2D),to further investigate the affection of As203 on the histone H3K9 and DNA methylation.3.q RT PCR was applied to detect the expression of the suppressor gene WIF-1,DDK3, HDPR1 of the WNT/?-catenin signaling pathway, as well as the important regulator protein ?-catenin and the m RNA of the target gene(Survivin,cyclin-D1,C-myc)in the downstream of the WNT signaling pathway, to make a relatively complete explain of the epigenetic mechanism of arsenic trioxide on the treatment of acute promyelocytic leukemia.Result:1. Having been pretreated by different As203 concentration, the inhibition rate of NB4 cell proliferation has raised as well as the concentration and duration increased,and the apoptotic rate as well. The early apoptosis was mainly found in the first 24 h and 48 h in the G2/M phase cell-cycle, while the moderate or late apoptosis was mainly found in the G0/G1 phase in the 72 h.2. Bisulfite sequencing polymerase chain reaction(BSP) combine with the TA cloning sequencing method has found that the methylation of gene WIF-1, DDK3,HDPR1 reduce progressively along with the duration of As203 concentration, the methylation decreased significantly. After compared with the MSP and pyrosequencing, the BSP demonstrate a significantly advantage in detecting the methylation. With simple and high repeatability, and it is capable to detect the Cp G methylation sites of the target gene accurately, which got a high sensitivity and specificity to analysis the suppressor gene methylation of the WNT signaling pathways.3.The relative expression of HDPR1 m RNA shows up-regulation trend while transmethylase DNMTl, DNMT3 A and DNMT3 B is opposite as the increase of the As203 concentration and duration detected by q RT-PCR.4. As the increase of the As203 concentration and duration, histone H3K9me3 in the promoter region of WIF-1, DDK3 and HDPR1 all have a varying degrees of decline, and HDPR1 is the most distinct; while H3K9me1, 2 have no change by CHIP. Meanwhile, the relative expression of SUV39 a and G9 a m RNA occur down-regulation trend, and G9 a m RNA is the most apparent,while the relative expression of JMJD2 D m RNA up regulates, and JMJD2 A, JMJD2 B,JMJD2C show no effect by q RT-PCR.5.The expression of ?-catenin significantly decreased, survivin, c-myc and cyclin-D1 as well, which have a statistical significance compared with control group by q RT-PCR as the increase of the As203 concentration and duration.Conclusion:1.As203 can significantly inhibit the growth and proliferation of NB4 cell of acute promyelocytic leukemia, and arrest cell cycle progression in G2/M phase as the increase of the As203 concentration and duration.2.As203 can reverse the hypermethylation of the DNA and down-regulate the methylation of H3K9 me3 in the promoter region of WIF-1,DKK3 and HDPR1 in NB4 cell of acute promyelocytic leukemia, so as to recovery the expression of the tumor inhibitor gene WIF-1,DKK3 and HDPR.3.BSP has a higher detection rate of methylation, meanwhile, it is simple and easily-duplicative, and it can also accurately detect the methylation of Cp G sites in each target gene. With whose high sensitivity and specificity in the analysis of the methylation of the suppressor gene of the Wnt signaling pathway, it can be widely used to select different types of samples according to the analysis of the methylation of gene promoter.4.As203 may suppress the expression of histone methyltransferase SUV39 and a G9 a and DNA methyltransferase DNMT3A?DNMT3B and DNMTl to reverse the hypermethylation of DNA and histone.5.As203 may further down regulate the methylation of DNA and histone on the basis that As203 directly suppresses the hypermethylation of DNA and histone.6.As203 can also down regulate the expression of histone methyltransferase SUV39h1 to reduce the development of the complex of PML-RARa/SUV39h1,inhibit the methylation of H3K9, promote the transcriptional expression of gene and differentiation of hemocyte.7.As203 can down regulate the expression of DNA methyltransferase DNMTl and histone methyltransferase a G9 a to reduce the development of the complex of DNMT1- UHRF1-G9 a, suppress the methylation of H3K9 and promote the transcriptional expression of gene.8.The expression of c-myc and cyclin-D1 in cell NB4 absolutely decreases along with the change of drug concentration and duration of As203,which may have a relationship of the repression of Wnt/?-catenin signaling pathway. |
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