Effects Of Decitabine On The Gamma-globin Gene Of Major Beta-thalassemia Erythroid Cells Cultured In Vitro

Objective To investigate the effects of decitabine on the gamma-globin of major beta-thalassemia erythroid cells cultured in vitro, provide a new way for beta-thalassemia therapy.Methods1. K562cells were treated with decitabine for24h,48h and72h. Decitabine at concentrations of0to300nmol/L. Quantitative reverse transcription polymerase chain reactions (Q-RT-PCR) was applied to detect the expression of gamma globin gene. The survivals of K562cells were examined by Count Cell Kit-8(CCK8). The Annexin V+/PI-cell were detected by Flow cytometry (FCM). The gamma-globin gene expression of the best effect induced was the purpose of decitabine screening. 2. The expression levels of α, β, γ-globin gene, the levels of hemoglobin and apoptosis in decitabine group and control group were carefully analyzed by quantitative real-time PCR (Q-RT-PCR), high performance liquid chromatography (HPLC), FCM, respectively. The effects of decitabine on the gamma-globin were investigated in of major beta-thalassemia erythroid cells cultured in vitro.Results1. Gamma-globin gene expressed concentration and time-dependent in K562cells induced by decitabine. In48h, the expression level of gamma-globin gene was significantly higher when compared with72h and24h(P<0.01).The expression level of gamma-globin gene did not differ significantly between300nmol/L group and200nmol/L group (P=0.743). Both these groups, the expression levels of gamma-globin gene were significantly higher when compared with100nmol/L group and50nmol/L group (P<0.05). K562cell growth inhibition, and increase in apoptosis, with concentration and time dependence. Concentration of200nmol/L and300nmol/L had good effects, but the more rates of survival and apoptosis in K562cells were examined in300nmol/L group than200nmol/L group (P<0.05).200nmol/L was selected as the most suitable to decitabine effect concentration.2. Decitabine effective induced the expression of y-globin gene in major beta-thalassemia erythroid cells cultured in vitro. The mRNA expression of γ-globin gene was significantly higher in decitabine group14.19±1.96than in control group11.15±1.53(P<0.01). Simultaneously, the disequilibrium between α-and β-, γ-globin gene expression was partly modified by decitabine. The ratios of (α/β+γ) in decitabine group and control group, were1.33±0.16and1.65±0.17, respectively (P<0.01). Thus, treatment of the cells with decitabine was also associated with increased levels of fetal hemoglobin (HbF).ConclusionThe induction of fetal globin gene expression and HbF production by decitabine were proved by this examine in major beta-thalassemia erythroid cells cultured in vitro. Clinical application is expected in the future for thalassemia therapeutics.