MicroRNA29B induces fetal hemoglobin via inhibition of the HBG repressor protein MYB in vitro and in humanized sickle cell mice
Authors: Gu Qingqing , Palani Chithra D. , Smith Alana , Li Biaori , Amos-Abanyie Ernestine Kubi , Ogu Ugochi , Lu Lu , Pace Betty S. , Starlard-Davenport Athena 
Published: November 2022

Introduction

Therapeutic strategies aimed at reactivatingHBGgene transcription and fetal hemoglobin (HbF) synthesis remain the most effective strategy to ameliorate the clinical symptoms of sickle cell disease (SCD). We previously identified microRNA29B (MIR29B) as a novel HbF inducerviatargeting enzymes involved in DNA methylation. We provided further evidence that the introduction of MIR29B into KU812 leukemia cells significantly reduced MYB protein expression. Therefore, the aim of this study was to determine the extent to which MIR29B mediates HbF inductionviatargetingMYBin KU812 leukemia cells and human primary erythroid progenitors and to investigate the role of MIR29B in HbF inductionin vivoin the humanized Townes SCD mouse model.

Materials and methods

Human KU812 were cultured and normal CD34 cells (n= 3) were differentiated using a two-phase erythropoiesis culturing system and transfected with MIR29B (50 and 100 nM) mimic or Scrambled (Scr) controlin vitro. A luciferase reporter plasmid overexpressing MYB was transfected into KU812 cells. Luciferase activity was quantified after 48 h. Gene expression was determined by quantitative real-time PCR.In vivostudies were conducted using Townes SCD mice (6 per group) treated with MIR29B (2, 3, and 4 mg/kg/day) or Scr control by 28-day continuous infusion using subcutaneous mini osmotic pumps. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes at weeks 0, 2, and 4. Flow cytometry was used to measure the percentage of HbF-positive cells.

Results

In silicoanalysis predicted complementary base-pairing between MIR29B and the 3′-untranslated region (UTR) ofMYB. Overexpression of MIR29B significantly reducedMYBmRNA and protein expression in KU812 cells and erythroid progenitors. Using a luciferase reporter vector that contained the full-lengthMYB3′-UTR, we observed a significant reduction in luciferase activity among KU812 cells that co-expressed MIR29B and the full-lengthMYB3′-UTR as compared to cells that only expressedMYB3′-UTR. We confirmed the inhibitory effect of a plasmid engineered to overexpressMYBonHBGactivation and HbF induction in both KU812 cells and human primary erythroid progenitors. Co-expression of MIR29B andMYBin both cell types further demonstrated the inhibitory effect of MIR29B onMYBexpression, resulting inHBGreactivation by real-time PCR, Western blot, and flow cytometry analysis. Finally, we confirmed the ability of MIR29B to reduce sickling and induce HbF by decreasing expression ofMYBandDNMT3gene expression in the humanized Townes sickle cell mouse model.

Discussion

Our findings support the ability of MIR29B to induce HbFin vivoin Townes sickle cell mice. This is the first study to provide evidence of the ability of MIR29B to modulateHBGtranscription byMYBgene silencingin vivo. Our research highlights a novel MIR-based epigenetic approach to induce HbF supporting the discovery of new drugs to expand treatment options for SCD.