MIR-144-mediated NRF2 gene silencing inhibits fetal hemoglobin expression in sickle cell disease

Abstract

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.

MIR29B mediates epigenetic mechanisms of HBG gene activation

“…While these data in KU812 cells were supportive of the ability of MIR29B to regulate HBG , we next performed studies under physiological conditions, using primary erythroid progenitors generated from normal CD34 + stem cells. As previously reported by our group erythroid progenitors were generated in a two‐phase culture system (Li et al , ). Transfection of erythroid progenitors with 50 and 100 nmol/l MIR29B produced a 7‐ to 8‐fold increase in MIR29B expression (Fig A), while combining 100 nmol/l MIR29B with 0·5 μmol/l Dec did not increase levels further.…”

“…While in vitro studies provide evidence for the ability of MIR29B to induce HbF, to gain evidence for its physiological relevance in SCD, we performed genome‐wide miRNA expression profiling as recently reported (Li et al , ). After obtaining informed consent, blood samples were collected from individuals with SCD ( n = 12) not on HC therapy, ranging from 4 to 20 years old.…”

“…Reticulocytes isolated from the peripheral blood were used for miRNA analysis on the miRCURY LNA microRNA Array (Exiqon, Woburn, MA, USA). Raw data were quantile normalized using a model‐based background correction algorithm as recently published by our group (Li et al , ). The miRNA genes differentially expressed between the high and low HbF groups were identified (Li et al , ) and raw data deposited in the National Center for Biotechnology Information Gene Expression Omnibus database, accession number GSE111356.…”

“…All data were analysed by a two‐tailed Student's t ‐test and P < 0·05 was considered statistically significant. For the miRNA microarray analysis, two study groups consisting of SCD patients with HbF < 8·6% (low HbF; n = 6) and HbF > 8·6% (high HbF; n = 6) were analysed as previously reported (Li et al , ).…”

“…In previously published work, we demonstrated that MIR29B , inhibits de novo synthesis of the DNMT enzymes DNMT3A and DNMT3B, in breast cancer cells and restores control of aberrantly expressed tumour suppressor genes involved in cell cycle control, including TP73, CDH1, RASSF1, CCNA1 , and CDKN1C (Starlard‐Davenport et al , ). Recently, we identified MIR144 as a repressor of NFE2L2 and HbF expression in patients with SCD (Li et al , ) in a genome‐wide microarray‐based miRNA screen of RNA isolated from reticulocytes. Herein, we demonstrated that MIR29B mediates HBG activation and induces HbF in KU812 cells and human primary erythroid progenitors.…”