Exploration Of The Role Of Apoptosis Gene RTN3 In Human Erythropoiesis

PurposesErythrocytes are the most abundant hematopoietic cells in mammals.Erythropoiesis is the process during which hematopoietic stem cells(HSCs)proliferate,differentiate and ultimately produce enucleate reticulocytes.HSCs generate erythroid progenitor cells,terminal erythroid differentiation begins with morphologically recognizable proerythroblasts,which subsequently undergo sequential mitoses to become basophilic,polychromatic,and orthochromatic erythroblasts.Then orthochromatic erythroblasts expel their nuclei to generate reticulocytes.Finally,reticulocytes become mature red cells.Cell apoptosis occurs in the process,which be called eryptosis.Studies have shown that in pathological and physiological conditions,the occurrence of apoptosis has effects on the production of mature red cells.The apoptotic gene RTN3 is a key gene that mediates three major apoptotic pathways in eukaryotic cells.Through the study of RTN3,we can explore its role in erythropoiesis.MethodsTo understand the role of RTN3,we firstly constructed RTN3 knockdown and overexpression vectors.K562 and HEL cells were transduced with lentivirus to knock down or overexpress RTN3.Quantitative real-time PCR and western blot were conducted to check knockdown or overexpress efficiency.Next,RTN3 knockdown and overexpression of K562 and HEL cells were induced with hemin,which could promote the cells differentiate into erythroid lineage.Cell proliferation,differentiation,apoptosis were detected.Finally,CD34~+cells that were isolated from human cord blood were transduced with lentivirus to knock down or overexpress RTN3,and then were cultured in vitro to differentiate into erythroblasts.The differentiation,apoptosis,cell cycle and enucleation of late stage erythroid cells were detected by flow cytometry.ResultsAfter knocking down RTN3,the cell proliferation significantly reduced in erythropoiesis.RTN3 knockdown significantly impaired enucleation of late stage erythroid cells.In the terminal differentiation of erythroid cells,knockdown of RTN3caused cell cycle arrest,which was blocked in S period.Apoptotic and differentiation were no effect.After overexpressing RTN3,the cell proliferation significantly reduced companied by increased apoptosis,cell apoptosis mediated by RTN3 was partly caspase3-dependent apoptosis,but the ability of cell denucleate and differentiate were no effect.Conclusionwhen the expression level of RTN3 was maintained in a certain range,the normal proliferative activity of the cells can be maintained.RTN3 had a maintenance effect on the enucleation of late stage erythroid cells.Cell apoptosis mediated by RTN3 was partly caspase3-dependent apoptosis.In addition,RTN3 also involved in important biological events,such as cell differentiation and cell cycle.