RNAi抑制α珠蛋白和BCL11A基因表达对重型地贫红系细胞珠蛋白基因mRNA表达的影响

Effect of combined blocking of alpha- globin and BCL11A genes by RNAi on mRNA expression of globin genes in erythroid progenitor cells from patients with thalassemia major

目的探索应用α珠蛋白和BCL11A基因siRNA纠正β地中海贫血(β-地贫)红系前体细胞α/(β+γ)失衡的可行性。方法分两组,对照组为随机序列siRNA,实验组为α珠蛋白siRNA组、BCL11A siRNA组及2个基因(α珠蛋白基因siRNA和BCL11A基因siRNA)联合处理组。4组样本均来自6例β-地贫患者,分离其外周血CD34+细胞并培养成红系前体细胞,分别转入相应的siRNA,用qRT-PCR法检测各组α、β、γ珠蛋白基因mRNA表达水平,计算阻断后各基因的mRNA(处理前mRNA/处理后mRNA×100%)及α/(β+γ)mRNA比值,并进行组间比较。结果联合处理组与α珠蛋白siRNA组相比较,α珠蛋白基因mRNA表达水平差异无统计学意义[(75.7%±5.3)%vs(68.3±7.2)%,P=0.071];联合处理组与BCL11A siRNA组相比较,BCL11A基因mRNA表达水平差异有统计学意义[(71.1±5.8)%vs(56.8±6.1)%,P=0.047],但γ珠蛋白基因mRNA表达水平差异无统计学意义[(150.1±8.7)%vs(161.3±12.7)%,P=0.105];联合处理组与对照组相比较,α/(β+γ)mRNA比例差异有统计学意义(3.978±0.487 vs 6.141±0.958,P=0.011)。结论通过联合应用BCL11A和α珠蛋白双基因siRNA改善β地贫患者的α/(β+γ)失衡是可行的,需要进一步优化条件,提高联合阻断的效果。

Objective To explore the feasibility of combined interference of alpha- globin and BCL11 A genes with siRNA in correcting alpha /（ beta ＋ gamma） imbalance in erythroid progenitor cells from patients with beta- thalassemia. Methods One control group（ random sequence siRNA） and three experimental groups（ including α- globin-siRNA group,BCL11A- siRNA group and α- globin ＋ BCL11A- siRNA group） were set up. Four samples were collected from 6 patients with β- thalassemia. Erythroid progenitor cells were cultured from peripheral CD34 ＋ cells in 6 patients. Corresponding siRNAs were transfected. Expressions of globins and BCL11 A mRNA were detected by qRT- PCR.Blocking effect was calculated as mRNA expression ratio（ level of mRNA after blocking / level of mRNA before blocking ×100%） and compared among groups. Ratio of alpha /（ beta ＋ gamma） mRNA was also calculated and compared among groups. Results Comparing to the α- globin- siRNA group,the mRNA level of α- globin was not statistically significantly different in the α- globin ＋ BCL11A- siRNA group [（ 75. 7 ± 5. 3） % vs（ 68. 3 ± 7. 2） %,P = 0. 071]. The difference in BCL11 A mRNA expression was significant between the combined treatment group and the BCL11A- siRNA group[（ 71. 1 ± 5. 8） % vs（ 56. 8 ± 6. 1） %,P = 0. 005],but the difference in γ- globin mRNA expression was not significant [（ 150. 1 ± 8. 7） % vs（ 161. 3 ± 12. 7） %,P = 0. 105]. Meanwhile,the difference in alpha /（ beta ＋ gamma）mRNA was significant between the combined treatment group and the control group（ 3. 98 ± 0. 49 vs 6. 14 ± 0. 96,P =0. 011）. Conclusion The combined use of BCL11 A and alpha- globin siRNAs improves alpha /（ beta ＋ gamma） imbalance in patients with beta- thalassemia. Further study should be dedicated to optimization of working condition of the two siRNAs for better combined blockade.