Data_Sheet_1_MicroRNA29B induces fetal hemoglobin via inhibition of the HBG repressor protein MYB in vitro and in humanized sickle cell mice.docx

IntroductionTherapeutic strategies aimed at reactivating HBG gene 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 inducer via targeting 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 induction via targeting MYB in KU812 leukemia cells and human primary erythroid progenitors and to investigate the role of MIR29B in HbF induction in vivo in the humanized Townes SCD mouse model.
Materials and methodsHuman 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) control in 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 vivo studies 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.
ResultsIn silico analysis predicted complementary base-pairing between MIR29B and the 3′-untranslated region (UTR) of MYB. Overexpression of MIR29B significantly reduced MYB mRNA and protein expression in KU812 cells and erythroid progenitors. Using a luciferase reporter vector that contained the full-length MYB 3′-UTR, we observed a significant reduction in luciferase activity among KU812 cells that co-expressed MIR29B and the full-length MYB 3′-UTR as compared to cells that only expressed MYB 3′-UTR. We confirmed the inhibitory effect of a plasmid engineered to overexpress MYB on HBG activation and HbF induction in both KU812 cells and human primary erythroid progenitors. Co-expression of MIR29B and MYB in both cell types further demonstrated the inhibitory effect of MIR29B on MYB expression, resulting in HBG reactivation 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 of MYB and DNMT3 gene expression in the humanized Townes sickle cell mouse model.
DiscussionOur findings support the ability of MIR29B to induce HbF in vivo in Townes sickle cell mice. This is the first study to provide evidence of the ability of MIR29B to modulate HBG transcription by MYB gene silencing in 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.

引言
旨在重新激活HBG基因转录与胎儿血红蛋白（fetal hemoglobin, HbF）合成的治疗策略，仍是改善镰状细胞病（sickle cell disease, SCD）临床症状的最有效手段。本团队此前通过靶向参与DNA甲基化的酶，将微小RNA29B（microRNA29B, MIR29B）鉴定为一种新型HbF诱导剂，并进一步证实将MIR29B导入KU812白血病细胞后，可显著降低MYB蛋白的表达水平。因此，本研究旨在明确MIR29B在KU812白血病细胞与人原代红系祖细胞中，通过靶向MYB介导HbF诱导的程度，并探究MIR29B在人源化Townes镰状细胞病小鼠模型体内诱导HbF的作用。

材料与方法
体外培养人KU812细胞，采用双相红细胞生成培养体系对正常CD34细胞（n=3）进行分化，并转染MIR29B模拟物（浓度分别为50 nM与100 nM）或阴性对照（Scrambled, Scr）。将过表达MYB的荧光素酶报告质粒转染至KU812细胞，48小时后检测荧光素酶活性。采用实时定量PCR检测基因表达水平。
体内实验采用Townes镰状细胞病小鼠（每组6只），通过皮下微型渗透泵持续输注28天，分别给予MIR29B（剂量为2、3、4 mg/kg/天）或阴性对照Scr。分别于第0、2、4周采集血液样本，进行全血细胞计数（complete blood count, CBC）加分类计数及网织红细胞检测；采用流式细胞术检测HbF阳性细胞的占比。

结果
生物信息学分析预测，MIR29B与MYB的3'非翻译区（3′-untranslated region, UTR）存在互补碱基配对。过表达MIR29B可显著降低KU812细胞与红系祖细胞中MYB的mRNA及蛋白表达水平。采用包含MYB全长3'UTR的荧光素酶报告载体，与仅表达MYB 3'UTR的细胞相比，共转染MIR29B与MYB全长3'UTR的KU812细胞的荧光素酶活性显著降低。本团队证实，在KU812细胞与人原代红系祖细胞中，过表达MYB的质粒可抑制HBG激活与HbF诱导。在两种细胞中共转染MIR29B与MYB后，通过实时PCR、蛋白质印迹（Western blot）及流式细胞术分析，进一步证实MIR29B对MYB表达的抑制作用可导致HBG重新激活。最后，在人源化Townes镰状细胞病小鼠模型中，本团队证实MIR29B可通过降低MYB与DNMT3基因的表达，减少红细胞镰变并诱导HbF生成。

讨论
本研究结果证实，MIR29B可在Townes镰状细胞病小鼠体内诱导HbF生成。本研究首次证实，MIR29B可通过沉默MYB基因在体内调控HBG转录。本研究提出了一种基于微小RNA的新型表观遗传策略以诱导HbF生成，为开发治疗镰状细胞病的新型药物、拓展治疗选择提供了支持。