miRNA-144/451 Regulates Cell Surface TfR1 Expression in Normal and β-Thalassemic Erythroblasts
Authors: Christakopoulos Georgios E. , Fernandez Alfonso G. , Yao Yu , Telange Rahul , Michael Dudley W. , Ganz Tomas , Nemeth Elizabeta , Weiss Mitchell J 
Published: December 2024

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 Hbbth3/+ 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 (HbbTh3/+) mice, germline loss of miR-144/451 caused 16 ± 4 % (p&lt;0.05) or 55± 9 % (p&lt;0.001) reductions in cell surface TfR1/CD71, respectively, as measured by immuno-flow cytometry. Western blot (WB) analysis of Hbb+/+ or HbbTh3/+ Ter119+ splenic erythroblasts showed 32 ± 14 % (p&lt;0.05) or 67 ± 23% (p&lt;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&lt;0.001) or 41 ± 13 % (p&lt;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.