The Mechanism Investigation On Epigenetic Pathway In Myelodysplastic Syndrome Regulated By Curcumin

Part Ⅰ. Curcumin regulates histone methylation status in MDS derived SKM-1cells by miR-101via targeting EZH2Aim:. This study was aimed to investigate the inhibitory effect of natural medicine curcumin on myelodysplastic syndrome (MDS) derived SKM-1cells. The potential epigenetic targets as well as their action mechanism were studied to find possible new targets in MDS treatment.Methods:Sanger sequencing was applied to find the hot gene mutations in MDS derived SKM-1cells and energy function was used to calculate the Gibbs free energy (△△G) of mutated protein. Effects of curcumin on cell proliferation, cell cycle and apoptosis were studied by3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, DNA staining with PI and apoptosis assay via flow cytometry. The activity of caspase3was calculated by caspase activation assay and the protein expression of caspase3, cleaved caspase3were measured by western blot (WB). The epigenetic drugs decitabine was also used as controls. The histone methyltransferase EZH2and ASXL1, the histone deacetylase HDAC8, the DNA methyltransferase DNMT3a were measured by WB or immunofluorescence technique. The protein expression of histone H3K4me3, histone H3K9me3and histone H3K27me3, acetyl-histone H3, acetyl-histone H4and HOXA9were also determined by WB. miR-101-3p, ezh2mRNA and hoxa9mRNA expression was measured by Real-time RT-PCR. Dual luciferase reporter gene analysis was used to verify the target gene of miR-101. The EZH2-shRNA and control vector were constructed by lentivirus infection. The down streaming correlated gene expression was also detected by WB Results:(1)Heterozygous Y646C mutation in EZH2gene and G652S mutation in ASXL1gene can be found in SKM-1cells. The gibbs free energy for mutated EZH2was1.46kcal/mol.(2) Curcumin had a dose-and time-dependent anti-proliferative effect on SKM-1cells. The IC50at24hours was22.24μM±0.22μM; the IC50at48hours was12.88μM±0.53μM. After incubation with50μM curcumin for48h, the results showed a significant increase of G1phase cells and decrease of S phase cells (p<0.05). The elevated caspase3activity (p<0.01) and elevated cleaved caspase-3protein expressin (p<0.05) can be observed in dose-dependent manner.(3)Curcumin down regulated the expression of EZH2, HDAC8, ASXL1and DNMT3a (p<0.05); while decitabie can only down regulate expression of DNMT3a unless it was used together with curcumin.(4) Curcumin can down regulate the expression of histone H3K4me3and H3K27me3, up-regulate the expression of acetyl-histone H4(p<0.05), release histone H3K27me3from nucleus into cytoplasm and finally inhibits the mRNA and protein expression of HOXA9.(5) Dual luiferase reporter gene analysis verified EZH2was a direct target of miR-101and curcumin can reduce the transcription and expression of EZH2via targeting miR-101. The shEZH2can down regulate expression of DNMT3a, H3K4-me3, H3K27-me3and HOXA9compared with vector control (p<0.01).Conclusion:Y646C mutation of EZH2can disrupt its protein stability. Curcumin can induce caspase-3dependent and cell cycle dependent apoptosis. It regulates the EZH2and other epigenetic regulators via targeting miR-101to break down the balance of histone methylation and histone acetylation and hence inhibit expression of HOXA9. Part Ⅱ. Establishment of SKM-1cell NOD/SCID mouse model and investigation of anti-tumor effect on myelodysplastic syndrome in vivo regulated by curcuminAim:The study was aimed to establish the myelodysplastic syndrome derived SKM-1cell NOD/SCID mouse model and investigate the anti-tumor effect of curcumin on myelodysplastic syndrome in vivoMethods:SKM-1cells were inoculated into NOD/SCID mice by intravenous injection. The mice were killed if any symptoms including the body weight loss (>20%), severe somnolence, irritability occured. The human CD45+cells were monitored via flow cytometry. Peripheral blood and bone marrow smear was applied to judge the dysplasia. Complete blood count was monitored to observe if any trilineage cytopenia occurred. Curcumin was injected intraperitoneally with controls. The overall survival time, dynamic body weight, complete blood smear and human CD45+cells were recorded accordingly. microPET scan was performed to monitor the tumor burden and metastasis. The mechanism of anti-tumor effects of curcumin on this mouse model was also investigated.Results:The mouse developed transient leukopenia and anemia at the end of stage in this xenotransplant model. The peripheral blood and bone marrow smear showed dysplasia on erythrocytes, granulocytes and moncytes. Curcumin can improve the overall survival by attenuating the tumor mass via down-regulating EZH2expression and hence down regulation of H3K27me3and H3K4me3.Conclusion:SKM-1cell NOD/SCID mouse model can develop the MDS like disease transiently at the end of onset stage. Micro PET imaging scan can be used to monitor the tumor burden. Curcumin, as a natural medicine, plays an anti-tumor effect by attenuating EZH2expression and hence reducing the expression of histone H3K27me3and H3K4me3...