| Objective:?-thalassemia(BT)is a common hereditary disease and has become a "global disease burden" with the increase of its incidence worldwide.Due to the dramatic reduction or complete absence of ?-globin chain synthesis caused by ?-globin gene mutation,the excess?-globin chain precipitates on the cell membrane and thus leads to the death of erythroid cells and ineffective erythropoiesis(IE),which usually results in severe hemolytic anemia,splenomegaly,and growth retardation.Although there are many therapeutics for?-thalassemia,including blood transfusion,iron removal,splenectomy,drugs for induction of y-globin production,hematopoietic stem cell transplantation,and gene therapy,all the available therapeutic strategies for ?-thalassemia have significant limitations.Long-term blood transfusion causes iron overload in patients.Excessive iron deposits in heart,liver,and other organs,resulting in organ failure.The drugs for iron removal are not only expensive,but also leads to adverse reactions such as granulocytopenia,skin infection,allergy,hearing and vision impairment.Splenectomy increases the risk of venous thrombosis.The drugs that induce production of ?-globin,such as hydroxyurea,do not reduce blood the frequency of blood transfusions.Hematopoietic stem cell transplantation is a treatment that can cure?-thalassemia,but with high cost,difficult matching,graft rejection,graft-versus-host disease,and other treatment-related toxicity.Gene therapy is considered as an alternative treatment for patients with ?-thalassemia,but the techniques involved require complex and expensive resources,and the viral vector integrated into the genome may cause genotoxicity.Therefore,exploring a safe and effective method for the treatment of ?-thalassemia has important scientific significance for the treatment of human ?-thalassemiamicroRNA(miRNA/miR)is a class of non-coding small RNA molecules,with only 18?25 nucleotides in length,and play important roles in regulating gene expression.MiR-144 and miR-451 are the most abundant miRNAs in erythrocytes and are the protectors of erythrocytes during normal erythropoiesis.We have previously reported that miR-144/451 knockout mice develop microcellular hemolytic anemia due to the increased sensitivity of erythrocytes to oxidative stress,which is caused by decreased reactive oxygen species(ROS)scavenging ability.The erythroid precursors from fetal liver,bone marrow,and spleen of miR-144/451 knockout mice commit to more apoptosis when the mice are under stress conditions such as 5-fluorouracil(5-FU)induced clearance of erythroid progenitor cells,phenylhydrazine(PHZ)-induced rapid destruction of mature red blood cells,and acute blood loss by bleeding.Therefore,miRNAs may become a tool for the diagnosis and treatment of many diseases,especially erythroid diseases.Methods:Using animals as human disease models,we have extensively studied the regulation of red blood cell development by miRNAs.During normal and stress hematopoiesis,miR-144/451 plays important roles in anti oxidation,cell survival and erythroid maturation The preliminary studies from our research group found that the expression of miR-144/451 increased significantly in erythroid cells from ?-thalassemia mice and the anemia in?-thalassemia mice was improved after knockouting miR-144/451.Furthermore,the expression of nuclear factor erythroid 2-related factor 2(Nrf2)and its regulated antioxidant enzymes superoxide dismutase(SOD)were increased in ?-thalassemia mice deficient of miR-144/451.Based on these findings,this project has further confirmed the phenotypes and investigated the underlying molecular mechanism.?.We have also examined the expression of miR-144/451 in ?-thalassemia,iron deficiency anemia(IDA),cancer related anemia(CRA),aplastic anemia(AA),myelodysplastic syndrome(MDS),and chronic hemorrhagic anemia(CHA)by quantitative real-time PCR(qRT-PCR).?.Expression levels of miR-144/451 in spleen of mKO/th3+/-mice by qRT-PCR;spleen archtecture and erythroid cells by histopathology and immunohistochemistry;percentages of erythroid cells in bone marrow and spleen with CD44 and Ter 119 staining by flow cytometry.?.Half-life of erythrocytes from the peripheral blood of the ?-thalassemia mice defienciet of miR-144/451 by flow cytometry.?.Confirmation of the effect of miR-144/451 deletion on the increase of ROS and iron overload in ?-thalassemia.The levels of reactive oxygen species(ROS)in erythrocytes and mitochondrial superoxide indicator(MitoSOX)before and after hydrogen peroxide(H2O2)treatment were detected using flow cytometor.Serum iron levels were detected by spectrophotometer.Serum ferritin levels were detected by Enzyme-Linked ImmunoSorbent Assay,ELISA).Iron in bone marrow,spleen,and liver was examined by Prussian blue staining.Detection of Phen-Green-SK(PGSK)labeled cellar iron by flow cytometry.?.Confirmation of the effect of miR-144/451 depeltion on the anemic changes by another thalassemia mouse model generated by ? IVS-2-654 mutation.?.Nrf2 knockout mice and SOD transgenic mice are used as in vivo models to study the potential mechanism of miR-144/451's role in ?-thalassemia.?.The role of miR-144/451 in ferroptosis pathway of ?-thalassemia mice through a series of iron-related experiments including biochemical indexes and intracellular iron,and ferroptosis-related experiments including transmission electron microscope.Nrf2 is not only the main regulator of antioxidant,but also the negative regulator of ferroptosis.Results:?.The expression of miR-144/451 is significantly increased in chronic compensatory anemia diseases,especially in ?-thalassemia.?.mKO/th3+/-mice showing the improved indexes of complete blood count from peripheral blood,decreased reticulocytes,long erythrocyte half-life,and less compensatory erythropoiesis in spleens.?.miR-144/451 deletion significantly reduces the levels of ROS in erythrocytes and improve iron overload in ?-thalassemia mice.?.The differential expression of antioxidant gene Nrf2 is screened by transcriptome analysis of nucleated erythroid cells in the bone marrow of th3+/-and mKO/th3+/-mice.High expression of Nrf2 in mKO/th3+/-mice is confirmed by qRT-PCR and Western blot.?.No significant improvement in erythroid phenotypes of th3+/-mice by crossing SOD transgenic mice overexpressing SOD compared with that of th3+/-mice.Conclusions:?.miR-144/451 in ?-thalassemia mice increased significantly;?.Deletion of miR-144/451 alleviates anemia in ?-thalassemia mice;?.The anemia improvement of ?-thalassemia mice after miR-144/451 deletion is SOD independent.This project not only enables us to better understand the role of miR-144/451 in erythrocyte development,but also provides theoretical and practical basis for molecular targeted therapy in patients with ?-thalassemia. |
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