Modulation of MicroRNA Expression In Sickle Reticulocytes Is Associated with Hydroxyurea Treatment and Fetal Hemoglobin Induction

Hydroxyurea has both laboratory and clinical efficacy for children, adolescents, and adults with Sickle Cell Anemia (SCA), and its benefits are primarily due to its ability to increase fetal hemoglobin (HbF). However, HbF induction by hydroxyurea is highly variable among patients, and its mechanism of HbF reactivation remains unclear. MicroRNAs (miRNA) are small non-coding RNAs that can regulate gene expression by inhibiting transcription or translation of targeted proteins. Recently, miRNAs have been implicated in cellular regulation and differentiation including hematopoiesis and hemoglobin switching. In an effort to elucidate the mechanisms behind hydroxyurea-mediated HbF induction, we tested the hypothesis that hydroxyurea modulates miRNA expression in sickle reticulocytes in vivo and this modulation is associated with changes in HbF levels.

As part of the prospective Hydroxyurea Study of Long-term Effects (HUSTLE, NCT00305175), total RNA was purified from CD71+ reticulocytes isolated from the peripheral blood of SCA patients, either prior to hydroxyurea treatment or after reaching stable maximum tolerated dose (MTD); non-SCA adults were included as controls. Initially, differential miRNA expression associated with disease and/or hydroxyurea exposure was determined by microarray and then confirmed by real-time PCR in a cross-sectional analysis of hydroxyurea-treated (n=13) and untreated (n=22) SCA patients, plus controls (n=8). Subsequently to identify hydroxyurea-mediated changes in miRNA expression and its association with HbF induction, miRNA levels were measured by real-time PCR in paired samples collected from patient reticulocytes at baseline and at hydroxyurea MTD (n=41). Statistical correlation to HbF levels used Spearman correlation coefficient and mixed model analysis.

Microarray analysis identified 108 miRNAs expressed in CD71+ reticulocytes, 10 of which were significantly different in SCA patients with or without hydroxyurea exposure and normal controls. Real-time PCR confirmed that miRNA expression of human miRNA (hsa-mir) 29a, 130b, 215, and 494 were upregulated, while hsa-mir-223 was downregulated, in untreated SCA patients compared to non SCA controls. Additional analysis of 41 paired samples showed that 3 miRNAs, hsa-mir 148a, 151-3p, and 494, were significantly upregulated with hydroxyurea treatment (Table 1). At MTD, HbF levels increased an average of 17% from a mean of 8.9 ± 6.1% at baseline (range 0.0–22.9%) to a mean of 25.9 ± 9.0% at MTD (range 9.4–55.9%).Significant associations between hsa-mir 26b miRNA expression and HbF levels were identified, both at baseline (r_s= -.34; p=0.03) and at MTD (r_s= -.32; p=0.04). Using mixed model analysis, change in hsa-mir 151-3p expression was significantly associated with the change in HbF (p=0.047) from baseline to MTD.

We identified specific miRNAs that are significantly associated with sickle cell anemia, hydroxyurea treatment, and hydroxyurea-mediated HbF induction. These studies suggest that miRNA regulation, specifically hsa-mir 26b and 151-3p, may be involved in hydroxyurea-mediated HbF induction in patients with sickle cell anemia. Future studies to identify the relevant protein targets may lead to a better understanding of hydroxyurea's mechanisms of action and patient response to the drug including the observed inter-patient variability in HbF response.

Results from real-time PCR shows differential miRNA expression in non-SCA controls compared to untreated SCA patients from cross-sectional analysis, and upregulation of 3 miRNAs in SCA patients at baseline compared to MTD from paired-sample analysis. Negative fold change indicates a downregulation.

Off Label Use: Hydroxyurea used to treat sickle cell anemia in children.

Off Label Use: Hydroxyurea used to treat sickle cell anemia in children.