Effect Of SiRNA On The Alpha-Globin Of Major Beta-Thalassemia Erythroid Cells Cultured In Vitro

β-thalassemia is caused byβ-globin gene mutations that either reduce or abolish production ofβ-globin chain leads to an imbalance in the production ofα- and nonα-globin chain.As a result,the relative excessαchains precipitate to the erythrocyte membrane,forming inclusion bodies and causing intramedullary destruction of the erythroid precursors,ineffective erythropoiesis and oxidative membrane damage associate with apoptosis of red blood cells. Gene therapy forβ-thalassemia has been focused onβ-globin engineering. Down-regulate properly endogeneticα-globin gene expression,reduce the production ofα-globin chain may be helpful in improving the symptoms ofβ-thalassemia.RNA interference is referred to as the recently discovered process of post-transcriptional gene silencing that is initiated by double-stranded RNA molecules known as small interfering RNA.As RNAi is capable of down-regulating target gene expression,it has been used extensively to study gene function,infectious viruses,etc.Also,RNAi can be a new option for gene therapy.We evaluated the effects of small interfering RNA(siRNA)targeting alpha-globin gene on the expression level of alpha-globin chain mRNA in major beta-thalassemic erythroid cells cultured in vitro.The study was composed by two parts.In partⅠ,the effective siRNA and optimal concentration were screened by 3 siRNAs targeting alpha-globin gene transferred into the normal adult donor originated erythroid cells cultured in vitro with liposome-induced gene transfection.In partⅡ,the effects of liposomal transfection of siRNA on the alpha-globin of major beta-thalassemic erythroid cells cultured in vitro were observed.Our study focused on the modification of alpha-globin chain production of beta-thalassemic erythroid cells on gene level,the melioration of the unbalance of erythroid cells globin,and the exploration of the new idea of RNA interference for beta-thalassemia.PartⅠEffect of small interfering RNA on the Alpha-globin gene expression in erythroid cells cultured in vitroObjective To evaluate the effects of small interfering RNA(siRNA)targeting alpha-globin gene on the expression level of alpha-globin chain mRNA in erythroid cells cultured in vitro.Methods Three kinds of siRNA for alpha-globin gene were designed and chemical synthesized,and transferred into the normal adult donor originated erythroid cells cultured in vitro with liposome-induced gene transfection.After transfection,at the time of 24h,48h and 72h,the transfection efficiencies were inspected by flow cytometry,the levels of mRNA in alpha-globin gene were detected by real-time quantitation PCR.The effectiveness of siRNA and optimal concentration of siRNA were screened.Results1.Liposomal could effectively transfect the FCM-labeled siRNA into erythroid cells cultured in vitro.The transfection efficiencies were 61.2%,44.3%,33.7 %at 24h,48h and 72h respectively.The most effective one was that at 24h.2.The effective siRNA of inhibition on alpha-globin gene expression was screened.We designed 3 kinds of siRNA that targeting alpha-globin gene and transferred into erythroid cells cultured in vitro.Results of RT-PCR indicated that all of them could down-regulated alpha-chain mRNA expression.The most effective one was theα2 siRNA,relative levels of mRNA expression were 0.56±0.02,0.41±0.02,0.40±0.02,respectively,at time of 24h,48h and 72h after transfection.There was sinificantly statistical difference between that of 24h and 48h,24h and 72h,respectively(P＜0.05),but no statistical difference between 48h and 72h(P＞0.05)3.siRNA down-regulated alpha-globin gene mRNA expression were dosedependent and time-dependent in a certain range.The inhibition efficiency was higher as the siRNA concentration increased.The relative expression levels of alpha-chain mRNA in groups of 120 nmol/L,160 nmol/L and 200 nmol/L were lower than those in groups of 80 nmol/L and 40nmol/L(P＜0.05=,but there were no statistical difference between the groups of 80 nmol/L and 40nmol/L,and among the groups of 200 nmol/L,160 nmol/L, 120 nmol/L(P＞0.05).The relative expression levels in groups of 120 nmol/L,160 nmol/L and 200 nmol/L at the time of 48h and 72h were lower than those at the time of 24h(P＜0.05),but there were no statistical difference at the time of 48h and 72h(P＞0.05) 4.There were significantly statistical difference among different concentrations of siRNA in trypan blue exclusion test(P＜0.05).The trypan blue exclusion rates was decreased as the concentration increased and time lasts. Conclusion The siRNAs targeting alpha-globin gene were transferred into erythroid cells cultured in vitro with liposome-induced gene transfection can down-regulate the alpha-chain mRNA expression level 48h after transfection. The effective siRNA isα2 siRNA and the optimal concentration is 120nmol/L.PartⅡEffect of siRNA on the alpha-globin of major beta-thalassemia erythroid cells cultured in vitroObjective To study effects of liposomal transfection of siRNA on the alpha-globin of major beta-thalassemic erythroid cells cultured in vitro,explore the gene therapeutic possibility of siRNA for beta-thalassemia.Methods The expression levels of alpha-,beta- and gamma-globin of normal controls and major beta-thalassemia patients were examined by real-time quantitation PCR.α2 siRNA targeting alpha-globin gene was transferred into major beta-thalassemic erythroid cells cultured in vitro by liposomal with a concentration of 120nmol/L.48h after transfection,real-time quantitation PCR was used to detect the expression levels of alpha-,beta- and gamma-globin mRNA of erythroid cells in siRNA group and control group to observe the effect of siRNA on relative globin gene expression.Hemoglobin levels were measured by HPLC 96h after transfection.Alpha-globin chain precipitates in erythroid cells were observed by transmission electron microscope before and after the treatment of siRNA.Results1.Compared with normal control,there was an obvious unbalance among alpha-,beta- and gamma-globin gene expression levels in major beta-thalassemia patients.The ratio ofα/βandγ/β+γgene in major beta-thalassemia patients and normal control were 31.74±3.45 and 1.184±0.22,respectively(P＜0.01),γ/β+γgene in major beta-thalassemia patients and normal control were 0.93±0.05 and 0.06±0.01,respectively(P＜0.01).α2 siRNA targeting alpha-globin gene transfected by liposomal can partially inhibited the expression of alpha-globin gene in major beta-thalassemic erythroid cells cultured in vitro.The expression of alpha-globin gene mRNA in siRNA group and control were 0.56±0.04 and 0.85±0.03,respectively(P＜0.01).At the same time,siRNA modified the disequilibrium ration ofα/βandα/β+γgene,the ratio ofα/βin siRNA group and control were 10.5±0.98 and 15.9±1.71,respectively(P＜0.01),the ratio ofα/β+γin siRNA group and control were 1.34±0.04 and 2.31±0.08,respectively(P＜0.01)2.After the treatment of siRNA,the levels of HbA and HbF increased in major beta-thalassemic erythroid cells.As the alpha-globin gene down-regulated by siRNA,the level of HbA in siRNA group and control were 54.46±4.12 and 49.21±3.89,respectively.HbF in siRNA group and control were 33.67±3.92 and 30.75±2.63,respectively.3.Observed by transmission electron microscope,the precipitates of alpha-globin chains in major beta-thalassemic erythroid cells were lessened, especially in early erythroblast stage,after the treatment of siRNA.Conclusion a2 siRNA targeting alpha-globin gene transfected by liposomal can partially inhibited the expression of alpha-globin gene in major beta-thalassemic erythroid cells cultured in vitro,modify the disequilibrium ration ofα/βandα/β+γgene,reduce the precipitates of alpha-globin chains in erythroid cells.Theoretically,siRNA can lessen the damage of relative excess alpha-globin chains to beta-thalassemic erythroid cells,and prolong the life of red blood cells,reduce hemolysis,and improve the symptom of major beta-thalassemia patients.