miRNA-144/451 Regulates Cell Surface TfR1 Expression in Normal and β-Thalassemic Erythroblasts

β-thalassemia is a common inherited blood disorder caused by mutations in the β-globin gene (HBB) leading to a reduction of the corresponding protein. Consequently, excess free α-globin forms toxic intracellular inclusions causing hemolysis of circulating erythrocytes, maturation arrest and apoptosis of erythroid precursors (ineffective erythropoiesis). We showed recently that loss of the bi-cistronic erythroid microRNA locus miR-144/451 improves IE and hemolysis in the Hbb th3/+ mouse model for β-thalassemia through several potential mechanisms (PMID: 37339583). Loss of miR-451 caused de-repression of the LKB1 cofactor Cab39 leading to activation of AMPK, suppression of mTORC1 and enhanced ULK1-mediated autophagy of free α-globin. We also showed that miR-144/451−/− β-thalassemic erythroblasts exhibited 57% reduction in non-heme iron, activation of the eIF2α kinase heme-regulated inhibitor (HRI) and up to 45% reduction in cell surface expression of TfR1/CD71, the major iron importer into the cell. Thus, miR-144/451−/− may ameliorate β-thalassemia in part by inducing iron restriction (PMID 30401707). Hence, we sought to understand how miR-144/451 regulates cell surface TfR1/CD71 expression in normal and β-thalassemic erythroblasts. In developmental stage-matched (EryB, CD71high/FSClow) splenic erythroblasts from WT (Hbb +/+) or β-thalassemia (Hbb Th3/+) mice, germline loss of miR-144/451 caused 16 ± 4 % (p<0.05) or 55± 9 % (p<0.001) reductions in cell surface TfR1/CD71, respectively, as measured by immuno-flow cytometry. Western blot (WB) analysis of Hbb +/+ or Hbb Th3/+ Ter119+ splenic erythroblasts showed 32 ± 14 % (p<0.05) or 67 ± 23% (p<0.01) reductions of TfR1 protein, respectively. RT-PCR analysis of the same cells showed no significant differences in the levels of mature or nascent (intron-containing) TfR1 mRNAs upon loss of miR-144/451. These findings suggest that miR-144/451 regulates the expression of TfR1 translationally or post-translationally. Compared to splenic erythroblasts, loss of miR-144/451 had a smaller effect on TfR1/CD71 expression in bone marrow erythroblasts. These findings suggest that that miR-144/451 regulates TfR1 expression preferentially during stress erythropoiesis, which occurs in the spleen of mice. To investigate whether TfR1 expression is regulated by MIR-144/451 during human erythropoiesis, we used Cas9 to disrupt the miRNA locus in healthy donor (HBB+/+), peripheral blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as in β-thalassemia (HBB -/- ) bone marrow derived CD34+ HSPCs followed by in vitro erythroid differentiation. Immuno-flow cytometry showed that MIR-144/451 disruption in developmental stage-matched CD49d+/Band3+ or CD49d-/Band3+ human HBB+/+ erythroblasts caused 61 ± 7 % (p<0.001) or 41 ± 13 % (p<0.01) reductions in cell surface TfR1/CD71, respectively. Similar findings were observed in HBB-/- erythroblasts. Thus, the regulation of TfR1 expression by miR-144/451 during erythropoiesis is conserved between mice and humans. We are currently investigating further the mechanisms by which miR-144 and/or miR-451 promote TfR1 expression at the cell surface, including the identification of relevant microRNA target mRNAs.

β-地中海贫血是一种常见的遗传性血液疾病，由β-珠蛋白基因（HBB）突变引起，导致相应蛋白质减少。因此，过量的游离α-珠蛋白形成有毒的细胞内包涵体，导致循环红细胞溶血、红细胞前体成熟停滞和凋亡（无效红细胞生成）。我们最近发现，双顺反子红细胞微小RNA位点miR - 144/451的缺失通过几种潜在机制改善了Hbb th3/+ β-地中海贫血小鼠模型中的无效红细胞生成和溶血（PMID：37339583）。miR - 451的缺失导致LKB1辅因子Cab39去抑制，从而激活AMPK，抑制mTORC1并增强ULK1介导的游离α-珠蛋白自噬。我们还发现miR - 144/451 - / - β-地中海贫血成红细胞的非血红素铁减少57%，eIF2α激酶血红素调节抑制剂（HRI）激活，细胞表面主要的铁输入蛋白转铁蛋白受体1/CD71（TfR1/CD71）的表达降低多达45%。因此，miR - 144/451 - / - 可能部分通过诱导铁限制来改善β-地中海贫血（PMID 30401707）。因此，我们试图了解miR - 144/451如何调节正常和β-地中海贫血成红细胞中细胞表面TfR1/CD71的表达。 在来自野生型（Hbb +/+）或β-地中海贫血（Hbb Th3/+）小鼠发育阶段匹配的（EryB，CD71高/FSClow）脾脏成红细胞中，通过免疫流式细胞术测量，miR - 144/451的种系缺失分别导致细胞表面TfR1/CD71减少16 ± 4%（p<0.05）或55 ± 9%（p<0.001）。对Hbb +/+或Hbb Th3/+ Ter119+脾脏成红细胞的蛋白质印迹（WB）分析显示，TfR1蛋白分别减少32 ± 14%（p<0.05）或67 ± 23%（p<0.01）。对相同细胞的逆转录 - 聚合酶链反应（RT - PCR）分析显示，miR - 144/451缺失后，成熟或新生（含内含子）TfR1 mRNA水平无显著差异。这些发现表明miR - 144/451在翻译水平或翻译后水平调节TfR1的表达。与脾脏成红细胞相比，miR - 144/451的缺失对骨髓成红细胞中TfR1/CD71表达的影响较小。这些发现表明miR - 144/451在应激性红细胞生成过程中优先调节TfR1的表达，应激性红细胞生成发生在小鼠的脾脏中。 为了研究在人类红细胞生成过程中TfR1的表达是否受MIR - 144/451调节，我们使用Cas9破坏健康供体（HBB +/+）、外周血动员的CD34+造血干细胞和祖细胞（HSPCs）以及β-地中海贫血（HBB - / - ）骨髓来源的CD34+ HSPCs中的微小RNA位点，然后进行体外红细胞分化。免疫流式细胞术显示，在发育阶段匹配的CD49d +/Band3+或CD49d -/Band3+人类HBB +/+成红细胞中，MIR - 144/451的破坏分别导致细胞表面TfR1/CD71减少61 ± 7%（p<0.001）或41 ± 13%（p<0.01）。在HBB - / - 成红细胞中也观察到了类似的结果。因此，在红细胞生成过程中miR - 144/451对TfR1表达的调节在小鼠和人类中是保守的。我们目前正在进一步研究miR - 144和/或miR - 451促进细胞表面TfR1表达的机制，包括鉴定相关微小RNA的靶mRNA。