Preliminary Study Of The Role Of GATA1-regulated MiR-144/451 In Anemia

miR-144 and miR-451 are a bi-cistronic miRNA gene loci and located in 17 Chr qll.2 in human genome.A large number of experiments in vivo and in vitro have found that miR-144/451 playes vital role in red cell maturation and synthesis of globin.Several studies find that there are altered expression of miR-144/451 in ?-thalassemia,polycythemia vera,sickle cell disease and other diseases of the blood system.Study has showed that expression of miR-451 is up-regulated in the precursor of RBC in human(3-thalassemia.So we wonder if miR-144/451 also plays an important role in other types of anemia.To test this hypothesis,wide type mice(wt),miR-144/451 knockout mice(miR-144/451-/-,mko)are selected as the research objects.And constructed Iron deficiency anemia?chronic blood loss and phenylhydrazine cause hemolytic anemia mouse model.And combined with clinical samples,such as IDA anemia,tumor anemia patient samples and regeneration barrier anemia patient samples to analyzed the result.Mainly for the following experiments:1)construct Iron deficiency animal model,chronic blood loss anemia model,and phenylhydrazine hemolytic anemia animal models;2)Detect the expresstion of miR-144/451 in all kinds of anemia model of the peripheral blood,bone marrow and in embryonic mice liver;3)Study the molecular mechanism and its clinical significance of increased expression of miR-144/451 in anemia animal models.This study aims to clarify the relationship between miR-144/451 and other types of anemia besides ?-thalassemia,and the molecular mechanism.This research is divided into the following 2 parts:Partl:The construction of anemia mice model and the expression of miR-144/451.Objective:Through physical and chemical methods to establish iron-deficiency anemia?chronic blood loss and phenylhydrazine cause hemolytic anemia mouse model,observe the mental state of anemia mice,cases of skin mucous membrane,and detection the expression of miR-144/451 in anemia condition.Methods:1)Feeding the C57BL/6 mice with Iron deficiency food with is purchased from Trophic Animal Feed High-Tech Co,Ltd,China.Then Collection the peripheral blood in the 4-5 weeks after raising iron deficiency feed.Record the change of the relevant indicators,detect the degree of anemia in mice.We extracted the total RNAs from bone marrow and peripheral blood.Then qRT-PCR was carried out to detect the expression of miR-144/451 in different stages of erythroid development.2)C57BL/6 mice by intraperitoneal injection phenylhydrazine solution and the concentration is 65 mg/kg.Acquire the peripheral blood prior the injection and the day of 1,2,3,4,5,6,7 after injection.Record the change of the relevant indicators,detect the degree of anemia in mice.We extracted the total RNAs from bone marrow and peripheral blood.Then qRT-PCR was carried out to detect the expression of miR-144/451 in different stages of erythroid development.3)Bloodletting for the C57BL/6 mice every two days and six times(every time 200?l).Then acquire the peripheral blood and detect the degree of anemia in mice,record the change of the relevant indicators.qRT-PCR was carried out to detect the expression of miR-144/451 in different stages of erythroid development.Result:Anemia symptoms appeared in mice,the skin and mucosa is pale,peripheral blood erythrocyte.hemoglobin content and erythrocyte number deposited reduced,increased reticulocyte percentage.performance for anemia mice.Extracted the total RNAs from peripheral blood.Then qRT-PCR was carried out to detect the expression of miR-144/451 and the expression of miR-144/451 is increased.Conclusion:Anemia mice model was established,the expression of miR-144/451 is increased.Maybe it participate in the occurrence of anemia.Part2:The function of miR144/451 in the process of anemia.Objective:To deal with the wt mice and miR-144/451 knockout in the same condition,and abserve the anaemic condition.Investigate the function of miR-144/451 in the process of anemia.Methods:To deal with the wt mice and miR-144/451 knockout use the methods of establish mice anemia model.Parameters of peripheral blood were detected in 2 different type of mice to definite anemia in these mice.Wright-Giemsa staining of peripheral blood smear were carried out to observe morphology of red blood cells,the number of erythrocytes.Flow cytometry were used to test differentiation and maturation degree of erythrocytes in peripheral blood and bone marrow.Morphological changes of Spleen in mice were detected to identify the situation of anemia mice.Nuclear red blood cells of bone marrow in 4 different types mice groups were sorted by microbeads.Western Blot and qRT-PCR were applied to gauge the expression levels of GATA1.qRT-PCR and were measured to detect the expression levels of oxidative stress enzymes activity and relative activity including SOD,Catalase,GPX1,NQO1 and Nrf2.Results:1)Wright-Giemsa staining of peripheral blood smear prompted that the change of morphology of RBC in peripheral blood was significantly serious in mko mice.The size of erythrocytes was more nonuniform and the number of targeted red blood cells increased.We also found the number of reticulocyte was increased significantly in mko mice;2)Percentage of reticulocyte increased in peripheral blood and bone marrow via flow cytometry.The maturity of erythrocytes in peripheral blood was decreased in mko mice in contrast with wt mice;3)Spleen morphological results suggested that the size and weight of spleen significantly enlargement in mko mice compared with wt mice.4)The expression of GATA1 is increased in anemia mice.Conclusion:The experimental results clearly identified that the anemia degree in mko mice was more serious than wt mice deal with by the same motheds.The occurrence of anemia caused by exogenous stimuli is regulate by other mechanisms in mice.