Okhovat Mohammad Ali, Ziari Katayoun, Ranjbaran Reza, Nikouyan Negin
Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Department of Pathology, Be'sat Hospital, AJA University of Medical Sciences, Tehran, Iran.
PLoS One. 2018 Feb 1;13(2):e0189267. doi: 10.1371/journal.pone.0189267. eCollection 2018.
Alpha-hemoglobin stabilizing protein (AHSP) is a molecular chaperone that can reduce the damage caused by excess free α-globin to erythroid cells in patients with impaired β-globin chain synthesis. We assessed the effect of sodium phenylbutyrate and sodium valproate, two histone deacetylase inhibitors (HDIs) that are being studied for the treatment of hemoglobinopathies, on the expression of AHSP, BCL11A (all isoforms), γ-globin genes (HBG1/2), and some related transcription factors including GATA1, NFE2, EKLF, KLF4, and STAT3. For this purpose, the K562 cell line was cultured for 2, 4, and 6 days in the presence and absence of sodium phenylbutyrate and sodium valproate. Relative real-time qRT-PCR analysis of mRNA levels was performed to determine the effects of the two compounds on gene expression. Expression of all target mRNAs increased significantly (p < 0.05), except for the expression of BCL11A, which was down-regulated (p < 0.05) in the cells treated with both compounds relative to the levels measured for untreated cells. The findings indicated that sodium valproate had a more considerable effect than sodium phenylbutyrate (p < 0.0005) on BCL11A repression and the up-regulation of other studied genes. γ-Globin and AHSP gene expression continuously increased during the culture period in the treated cells, with the highest gene expression observed for 1 mM sodium valproate after 6 days. Both compounds repressed the expression of BCL11A (-XL, -L, -S) and up-regulated GATA1, NFE2, EKLF, KLF4, STAT3, AHSP, and γ-globin genes expression. Moreover, sodium valproate showed a stronger effect on repressing BCL11A and escalating the expression of other target genes. The findings of this in vitro experiment could be considered in selecting drugs for clinical use in patients with β-hemoglobinopathies.
α-血红蛋白稳定蛋白（AHSP）是一种分子伴侣，可减少β-珠蛋白链合成受损患者中过量游离α-珠蛋白对红系细胞造成的损伤。我们评估了两种正在研究用于治疗血红蛋白病的组蛋白脱乙酰酶抑制剂（HDIs）——苯丁酸钠和丙戊酸钠，对AHSP、BCL11A（所有异构体）、γ-珠蛋白基因（HBG1/2）以及一些相关转录因子（包括GATA1、NFE2、EKLF、KLF4和STAT3）表达的影响。为此，在有和没有苯丁酸钠和丙戊酸钠的情况下，将K562细胞系培养2、4和6天。进行mRNA水平的相对实时定量逆转录聚合酶链反应分析，以确定这两种化合物对基因表达的影响。除BCL11A的表达外，所有靶mRNA的表达均显著增加（p < 0.05），相对于未处理细胞测量的水平，在用两种化合物处理的细胞中BCL11A的表达下调（p < 0.05）。研究结果表明，丙戊酸钠在抑制BCL11A和上调其他研究基因方面比苯丁酸钠具有更显著的作用（p < 0.0005）。在处理的细胞培养期间，γ-珠蛋白和AHSP基因表达持续增加，在6天后观察到1 mM丙戊酸钠的基因表达最高。两种化合物均抑制BCL11A（-XL、-L、-S）的表达，并上调GATA1、NFE2、EKLF、KLF4、STAT3、AHSP和γ-珠蛋白基因的表达。此外，丙戊酸钠在抑制BCL11A和提高其他靶基因表达方面表现出更强的作用。该体外实验的结果可用于为β-血红蛋白病患者选择临床用药时参考。
