| BackgroundAcute leukemia (AL) is a malignant disease of clonal hematopoietic stem cell abnormalities. Variety of factors led to malignantly proliferative blood cells, which enter the bloodstream and infiltrate into tissues and organs, causing anemia, bleeding, infection, and a series of clinical manifestations. Related studies showed that the physical, chemical, viral, and genetic factors are likely to be related to the incidence of leukemia. Conversion of proto-oncogenes, distortion of tumor suppressor genes and inhibition of apoptosis may play major roles in leukemogenesis.Wnt pathway plays an important role in the regulation of cell proliferation, movement and differentiation during embryonic development. Abnormal activation of Wnt pathway can promote tumor cell growth, proliferation, inhibition of apoptosis. And abnormal activation of the Wnt pathway can be found in90%of colorectal cancer. It also plays an important role in the development of malignant hematopoietic diseases, including contributing to malignant B lymphoid cell proliferation in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), and accelerating the chronic phase to accelerated phase and blast phase transition in chronic myeloid leukemia (CML). Homeobox genes (Hoxgenes), widely exist in vivo, and play an important role in the organisms development and differentiation, in which tail type homeobox gene CDX2(caudal type homebox transcription factor2, CDx2) is one of the most important genes, and is important to control the longitudinal axis differentiation and development. CDX2can be expressed in the intestinal epithelium and pancreas, and no CDX2expression in breast, prostate, kidney, liver, bone marrow and peripheral blood. And studies showed that CDX2is highly expressed in leukemia cells of90%acute monocytic leukemia patients. Scholl et al proved that abnormal expression of CDX2is prevalent in hematologic malignancies in studies of patients with acute leukemia. Studies have shown that the gene expression of CDX2is regulated by RA, Wnt and Fgf pathway. Abnormal activation of the Wnt pathway can lead to increased expression of CDX2gene. In recent years, the study has found that tumor suppressor gene SFRPs of SFRP12,4,5promoter CpG island structure in the Wnt pathway upstream, and frequent CpG island methylation causes gene silencing can activate the Wnt signaling. The pathway thus contributing to the leukemia cells resistant to apoptosis. Decitabine is a demethylation drug, and plays an unique role in inhibition of methylation. Decitabine has a dual mechanism of action, low doses can activate to silence gene expression and promote cell differentiation; high-dose is cytotoxic. This indicates that decitabine can activate to the tumor suppressor gene upstream of Wnt pathway by gene demethylation, close the Wnt pathway to some extent, and make CDX2gene expression down.ObjectiveThis study was to observe K.562cell proliferation, apoptosis and CDX2gene and protein expression with decitabine and to research the relationship between decitabine and CDX2in leukemic cells. And it’s also to provide experimental basis to decitabine sa a new anti-leukemia drug.Method1.WST-1cell proliferation inhibition rate was detected:the final concentration of0.02μmol/L,0.2μmol/L,1molμL,5μmol/L,10μmol/L,20μmol/L of decitabine of K562cells, respectively,8h,16h,24h,32h, WST-1assay and calculate the rate of cell proliferation, proliferation inhibition rate measured according to decitabine role in the best time and the best concentration. 2.Cell apoptosis observed by Flow cytometry:the final concentration to1μmol/L decitabine acts on K562cells for16h, flow cytometry AnnexinV FITC/PI double staining to detect apoptosis rate.3.RT-PCR method to detect the expression of CDX2mRNA:the final concentration to0.02μmol/L,0.2μmol/L,1μmol/L2μmol/L5μmol/L,20μmol/L decitabine role in K562cells16h,RT-PCR assay the impact to CDX2mRNA expression of K562cells.4.Western blot assay CDX2protein expression:the final concentration to1μmol/L decitabine role in K562cells16h,CDX2protein expression is detected in K562cells by Western blot assay.5.Statistical analysis:The experimental data application SPSS17.0statistical software for statistical analysis, all measurement data using one-way ANOVA, statistical data were used (x±s) expressed. To test the level a=0.05to P<0.05was considered statistically significant.Results1WST-1The results show:the final concentration of0.02μ mol/L,0.2μ mol/L,1μ mol/L,5μ mol/L,10μ mol/L,20μ mol/L of decitabine dosing for16hours, K562cells are subject to different degrees of inhibition, inhibition rates were (29.6±0.35)%,(36.5±0.94)%,(54.4±1.11)%,(70.3±2.12)%,(81.1±2.65)%,(88.2±3.13)%, with the control group (1.5±0.09)%, the differences were statistically significant (P<0.01); different concentrations of decitabine between any two groups, the difference was statistically significant (P<0.05). Final concentration of1u mol/L of decitabine dosing8hours,16hours,24hours,32hours later, K562cell proliferation inhibition rate was (31.3±1.51)%,(54.4±1.11)%,(68.0±1.23)%,(68.8±1.28)%, compared with control group, the differences were statistically significant (P<0.01); and the role of8hours and16hours a statistically significant difference (P<0.01), but16hours24,32hours and proliferation inhibition rate was no significant difference (P>0.05), and therefore determine the effect of16hours for the role of decitabine optimal point in time. Software process°analysis showed that decitabine in16hours IC50was0.784μ mol/ L, close to1μ mol/L, it is determined as the concentration of decitabine optimal concentration for subsequent cell apoptosis and expression of CDX2analysis of the change.2Flow cytometry results showed that:when1u mol/L of decitabine of K.562cells after16h, AnnexinV FITC/PI double staining showed that apoptosis rate:(29.6±1.2)%, while the control group (0.76+0.08)%, the difference was statistically significant (P<0.01).3RT-PCR and Western blot results showed that:a final concentration of0.02μ mol/L,0.2μ mol/L,1μ mol/L,5u mol/L,10μ mol/L,20μ mol/L of decitabine of K562cells after16hours, CDX2mRNA expression levels were0.84±0.028,0.74±0.034,0.63±0.020,0.51±0.023,0.41±0.011, compared with the control group,0.94±0.019, the difference was statistically significant (P <0.01); different concentrations decitabine between CDX2mRNA expression levels were significantly different (P<0.05). Final concentration of1u mol/L decitabine treatment of K562cells after16h, CDX2protein relative expression levels of0.90±0.046,1.20±0.057with the control group, the difference was statistically significant (P<0.01).Conclusion1.Decitabine can effectively inhibit the proliferation of K562cells, and its inhibition has a time-and-concentration dependent manner.2.Decitabine can induce apoptosis of K562cells.3.Decitabine significantly down-regulate expression of CDX2mRNA and protein in K562cells. |
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