The Effect Of Protein 4.1R In Erythroid Progenitor Cells Proliferation And Its Expression In AML

BackgroundErythropoiesis is a multi-stage process.This process mainly includes three stages:early development stage,terminal differentiation stage and reticulocyte maturation stage.In the early development stage of erythropoiesis,hematopoietic stem cells differentiate into erythroid progenitor cells,and then the erythroid progenitor cells go through the terminal differentiation stage to form reticulocytes,and finally develop into mature red blood cell.The abnormal expression of regulator of cell proliferation in the early stage of erythropoiesis may lead to the abnormal development of erythropoiesis and may lead to the occurrence of hematological malignancies such as acute myeloid leukemia(AML).To find and study the regulator of cell proliferation in the early development of erythropoiesis can increase understanding of the mechanism of the regulation of cell proliferation in erythropoiesis and it is of great significance for the diagnosis and treatment of hematologic malignancies.Protein 4.1R is a membrane skeleton protein first found in mature red blood cells,which can maintain the stability of red blood cell membrane and participate in the regulation of cell proliferation,cell activation and so on.Previous studies on protein4.1R have focused on its function of mature red blood cells,whether it can regulate the cell proliferation in the early stage of erythropoiesis is still unclear.In this study,we used protein 4.1R knockout mice to explore the effect of protein4.1R on the proliferation of erythropoietic progenitor cells in erythropoiesis and used the public database to explore the expression of protein 4.1R gene in AML.This study is helpful to understand the possible role of protein 4.1R in erythropoiesis and pathogenesis of AML.Methods1.Peripheral blood smears of protein 4.1R knockout mice and wild type mice were made to compare the proportion of blood cells.Flow cytometry analyzed the proportion of terminal stage cells of erythroid differentiation and erythroid progenitor cells of protein 4.1R knockout mice and wild-type mice.2.Colony formation assay was performed to compare the number of BFU-E colonies and CFU-E colonies of protein 4.1R knockout mice and wild type mice.3.The expression of EPO m RNA in protein 4.1R knockout mice and wild type mice kidney cells were detected by q RT-PCR and expression of EPOR on CFU-E cell surface in protein 4.1R knockout mice and wild type mice was analyzed by flow cytometry.4.HPA database was used to investigate the expression of protein 4.1R gene in normal human bone marrow and blood.GEPIA was used to analyze the differential expression of protein 4.1R gene between AML and normal specimen and the expression of protein 4.1 gene in different clinical phenotypes of AML.UCSC Xena was used to analyze the effect of protein 4.1R gene expression on overall survival(OS)of AML patients.5.Co-expression analysis of protein 4.1R gene by R studio,GO and KEGG pathway enrichment analysis of co-expression genes.Using String and Cytoscape to construct protein-protein interaction network.GEPIA was used to analyze the differential expression of co-expression genes between AML and normal specimen,the correlation between co-expression genes and protein 4.1R gene.6.Oncomine database was used to validate the differential expression of protein4.1R gene between AML and normal samples.Results1.The proportion of immature red blood cells of peripheral blood in protein 4.1R knockout mice is higher than those in wild type mice.Moreover,the proportion of terminal stage cells of erythroid differentiation and CFU-E cells in bone marrow of protein 4.1R knockout mice is higher than that of wild type mice,the proportion of BFU-E cells in bone marrow of protein 4.1R knockout mice is not significantly different from that of wild type mice.2.The number of CFU-E colonies in bone marrow of protein 4.1R knockout mice is higher than that of wild type mice,and there is no significant difference between BFU-E colonies in bone marrow of protein 4.1R knockout mice and wild type mice.3.The relative expression of EPO m RNA in kidney cells of protein 4.1R knockout mice is higher than that of wild type mice,and the expression of EPOR on CFU-E cell membrane in bone marrow cells of protein 4.1R knockout mice is higher than that of wild type mice.4.According to the analysis of HPA database,protein 4.1R gene has higher expression level in bone marrow and blood.By analyzing GEPIA database,we found that the expression level of protein 4.1R gene in AML is lower than that in normal specimen.The expression level of protein 4.1R gene in different FAB subtypes of AML patients is uneven.The analysis of protein 4.1R gene related overall survival rate found that the low expression of protein 4.1R gene is associated with poor prognosis of AML.5.Co-expression genes were found to be associated with cell proliferation by GO and KEGG pathway enrichment analysis.SKP2,COPS2,CDC27,BUB1,CDC27 and others co-expression genes related to cell proliferation were screened.These co-expression genes are strongly correlated with protein 4.1R gene.The expression level of SKP2,COPS2,CDC27,CUL1,NASP and BUB1 in AML are lower than that in normal specimen,while the expression of FBXW8 and USP13 in AML are higher than that in normal specimen.6.Through the analysis of oncomine database,we found that there are three groups of dataset support protein 4.1R gene expression is down regulated in AML.Conclusion1.The absence of protein 4.1R induce the hyperproliferation of CFU-E by enhanceing the expression of EPOR on CFU-E cell surface and expression of EPO.2.The expression level of protein 4.1R in AML is lower than that in normal specimen.The low expression level of protein 4.1R gene is correlated with poor prognosis of AML.Protein 4.1R gene and its co-expression gene may participate in pathogenesis of AML through participating cell proliferation.