Ranjbaran Reza, Nikogoftar Zarif Mahin, Sharifzadeh Sedigheh, Golafshan Habibollah, Pourfathollah Ali Akbar
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Cell J. 2018 Oct;20(3):318-325. doi: 10.22074/cellj.2018.5181. Epub 2018 May 15.
Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies. Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G→A substitution in HBG promoter in prevention of transcriptional silencing of the γ-globin.
In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively.
Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. γ to β-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells (EPCs) were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in β/γ-globin gene expression in transfected EPCs at the experimental end point (day 28) whereas, due to the absence of β-globin gene expression following K562 differentiation, this rate was not evaluable.
Our results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit γ-globin gene silencing during erythroid differentiation.
在β - 血红蛋白病中，胎儿血红蛋白（HbF）增加被认为是一种具有临床益处的现象。受胎儿血红蛋白遗传性持续存在的启发，预防γ - 珠蛋白基因沉默可能是上调这些患者HbF表达的合适策略。因此，我们的目的是评估在HBG启动子中诱导 - 117 G→A替换在预防γ - 珠蛋白转录沉默方面的潜在可行性。
在本实验研究中，将人外周血来源的造血干细胞（HSCs）和K562细胞系分化为红系细胞。使用细胞形态学参数和CD235a表达的流式细胞术分析来检测红系成熟情况。将合成的嵌合塑型体转染到分化细胞中。通过流式细胞术评估嵌合塑型体导入靶细胞的效率。限制性片段长度多态性和DNA测序验证了寡核苷酸定向诱变。分别通过等位基因特异性定量聚合酶链反应（AS - qPCR）和定量PCR对基因转换频率和珠蛋白基因表达进行定量。
表达CD235a的细胞增加以及对红系成熟不同阶段的观察结果证实了HSCs和K562细胞中的红系分化。估计γ到β - 珠蛋白基因转换发生在HSC分化的第18 - 21天。流式细胞术分析表明，超过70%的红系祖细胞（EPCs）被嵌合塑型体转染。在EPCs和K562细胞中，最高基因转换效率分别为7.2%和11.1%。在实验终点（第28天），诱导突变导致转染的EPCs中β/γ - 珠蛋白基因表达下降1.97倍，而由于K562分化后不存在β - 珠蛋白基因表达，该比率无法评估。
我们的结果表明嵌合塑型术在诱导EPCs和K562细胞中感兴趣的突变方面是有效的。我们还证明，单核苷酸启动子变体能够在红系分化过程中显著抑制γ - 珠蛋白基因沉默。
