Li Biaoru, Zhu Xingguo, Ward Christina M, Starlard-Davenport Athena, Takezaki Mayuko, Berry Amber, Ward Alexander, Wilder Caroline, Neunert Cindy, Kutlar Abdullah, Pace Betty S
Department of Pediatrics, Augusta University, Augusta, GA, USA.
Department of Biochemistry and Molecular Biology, Boston University, Boston, MA, USA.
Exp Hematol. 2019 Feb;70:85-96.e5. doi: 10.1016/j.exphem.2018.11.002. Epub 2018 Nov 6.
Inherited genetic modifiers and pharmacologic agents that enhance fetal hemoglobin (HbF) expression reverse the clinical severity of sickle cell disease (SCD). Recent efforts to develop novel strategies of HbF induction include discovery of molecular targets that regulate γ-globin gene transcription and translation. The purpose of this study was to perform genome-wide microRNA (miRNA) analysis to identify genes associated with HbF expression in patients with SCD. We isolated RNA from purified reticulocytes for microarray-based miRNA expression profiling. Using samples from patients with contrasting HbF levels, we observed an eightfold upregulation of miR-144-3p (miR-144) and miR-144-5p in the low-HbF group compared with those with high HbF. Additional analysis by reverse transcription quantitative polymerase chain reaction confirmed individual miR-144 expression levels of subjects in the two groups. Subsequent functional studies in normal and sickle erythroid progenitors showed NRF2 gene silencing by miR-144 and concomitant repression of γ-globin transcription; by contrast, treatment with miR-144 antagomir reversed its silencing effects in a dose-dependent manner. Because NRF2 regulates reactive oxygen species levels, additional studies investigated mechanisms of HbF regulation using a hemin-induced oxidative stress model. Treatment of KU812 cells with hemin produced an increase in NRF2 expression and HbF induction that reversed with miR-144 pretreatment. Chromatin immunoprecipitation assay confirmed NRF2 binding to the γ-globin antioxidant response element, which was inhibited by miR-144 mimic treatment. The genome-wide miRNA microarray and primary erythroid progenitor data support a miR-144/NRF2-mediated mechanism of γ-globin gene regulation in SCD.
增强胎儿血红蛋白（HbF）表达的遗传性基因修饰因子和药物可逆转镰状细胞病（SCD）的临床严重程度。近期开发新型HbF诱导策略的努力包括发现调节γ-珠蛋白基因转录和翻译的分子靶点。本研究的目的是进行全基因组微小RNA（miRNA）分析，以鉴定与SCD患者HbF表达相关的基因。我们从纯化的网织红细胞中分离RNA，用于基于微阵列的miRNA表达谱分析。使用来自HbF水平不同的患者样本，我们观察到低HbF组中miR-144-3p（miR-144）和miR-144-5p相较于高HbF组上调了8倍。通过逆转录定量聚合酶链反应进行的额外分析证实了两组受试者个体的miR-144表达水平。随后在正常和镰状红细胞祖细胞中进行的功能研究表明，miR-144可使NRF2基因沉默，并伴随γ-珠蛋白转录的抑制；相比之下，用miR-144拮抗剂处理可呈剂量依赖性逆转其沉默作用。由于NRF2调节活性氧水平，额外的研究使用血红素诱导的氧化应激模型研究了HbF调节机制。用血红素处理KU812细胞可使NRF2表达增加和HbF诱导，而miR-144预处理可使其逆转。染色质免疫沉淀试验证实NRF2与γ-珠蛋白抗氧化反应元件结合，而miR-144模拟物处理可抑制这种结合。全基因组miRNA微阵列和原代红细胞祖细胞数据支持SCD中miR-144/NRF2介导的γ-珠蛋白基因调节机制。
