Does HbF induction by hydroxycarbamide work through MIR210 in sickle cell anaemia patients?

Sickle cell disease (SCD) is characterized by chronic haemolytic anaemia as a result of a single point mutation in the HBB (b-globin) gene. Hydroxycarbamide (HC, also termed hydroxyurea) is efficacious for sickle cell anaemia (SCA) patients. We previously reported significant improvement in the clinical condition of 98% of SCA patients after HC therapy (Italia et al, 2009). The efficacy of HC is due to its ability to increase fetal haemoglobin (HbF). However, HbF induction by HC is highly variable among patients, and the mechanism of HbF induction is not yet clear, although direct transcriptional effects and altered cell cycle kinetics have been proposed (Lou et al, 2009). Understanding the developmental progression of globin gene expression, reactivation of HBG1/HBG2 (c-globin genes) expression and HbF production in the adult is an important therapeutic strategy for SCA (Rogers et al, 2008). MicroRNAs (miRNAs) are potent molecular regulators that modulate the expression of protein-coding genes by inhibiting mRNA translation. They have been described to have a possible role in globin switching and can modulate transcriptional erythroid-specific regulators (Fornari et al, 2013). Thus, we hypothesized that miRNA regulation may be involved in HC-mediated HbF induction. Kosaka et al (2008) analysed 324 human miRNAs to investigate their role in erythroid maturation; interestingly MIR210 was one of the miRNAs that was significantly elevated when UT-7 cells were stimulated with erythropoietin and MIR210 expression was elevated during erythroid maturation in vitro. The authors suggested that MIR210 might play a regulatory role in erythroid maturation. Hence, we studied MIR210 expression patterns in SCA patients treated with HC to determine whether MIR210 is involved in the induction of HbF by HC. We collected peripheral blood samples from 10 SCA patients after obtaining informed consent, at baseline and then 3 months after HC therapy (10 mg/kg/d). The control group comprised of 10 normal individuals. HbF levels were measured by high performance liquid chromatography (VARIANT haemoglobin Testing System; Bio-rad Laboratory, Hercules, CA, USA). The mean HbF levels increased from 16 87 2 98% at baseline to 30 19 2 37% in SCA patients after 3 months of HC therapy (P < 0 01) (Fig 1A). CD71 (also termed TFRC) is the receptor for the ironbinding protein transferrin and is present on reticulocytes and erythroid progenitors. Hence, CD71 erythroid cells were enriched after CD45 leucocyte depletion using magneticactivated cell sorting (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany). This ‘double-MACS’ procedure yielded approximate recovery rates of 28–45% for each step. The average recovery of CD71 cells was 2 3 9 10 cells per ml from normal healthy controls, 5 32 9 10 and 3 527 9 10 cells per ml from SCA patients at baseline and after 3 months of HC therapy, respectively. The purity of CD71 populations was >87% and contained mainly erythroid progenitors, primarily reticulocytes and nucleated erythrocytes. We further extracted total RNA and miRNA (Ambion mirVana kit; Life Technologies, Carlsbad, CA, USA). MIR210 (HGNC: 31587) expression was determined by quantitative polymerase chain reaction using MIR451A (HGNC: 32053) as the reference gene for normalization (Walker et al, 2011) and Taqman probes (part 4427975; Applied Biosystems, Carlsbad, CA, USA). The baseline study identified a 4 01 1 41-fold increase in MIR210 expression in the untreated SCA patients compared to healthy normal controls. MIR210 expression was significantly up-regulated in the patients after HC therapy with a fold increase of 7 49 1 031 (P < 0 05) when compared to baseline, suggesting a role of MIR210 in HbF induction. Figure 1B shows the relative quantification of MIR210 in the SCA patients after HC therapy. Analysis of HbF levels and MIR210 expression in paired samples (baseline and 3 months after HC therapy) for each patient by Spearman’s correlation coefficient indicated a significant association between HbF and MIR210 expression in response to HC therapy (P < 0 0001) (Fig 2A). Thus, the increase in MIR210 expression was associated with HC-mediated induction of HbF. We also analysed HBG2 (HGNC: 4832; c-globin gene) expression in all 3 groups by TaqMan hybridization probe (HBG2; HB HS00361131_g1; Applied Biosystems, Carlsbad, CA, USA) using human 18S rRNA (HS99999901_s1; Applied