The Effection And Mechanism Of MiR-709 In Process Of Erythroid Differentiation

Objective:To study the expression of miRNAs in Ter119+/CD71+ cells from 14.5 days fetal liver,screening of differentially expressed miRNAs in the early development of red blood cells,and to explore the effection and mechanism of miR-709 in process of erythroid enucleation.Methods:1.Erythroblast(before enucleation)and reticμlocyte(after enucleation)were sorted by flow cytometry(FCM)in 14.5 days fetal liver maked with Ter119 and CD71,then several miRNAs expression are detected using microarray;Fetal liver erythroid precursors and hematopoietic stem cells(FLEPHSCs)are sorted by magnetic cell sorting(MACS)negative selection;Nucleated cell and reticμlocyte are from bone marrow,mature erythrocyte are from peripheral blood,Ter119+1CD71+ cells are from 14.5 days fetal liver;We detected the expression of miRNAs in those cells and erythroid cell lines like G1E,MELand FLEPHSCs using real-time fluorescent quantitative PCR(q-RTPCR).2.Constructed miR-709-MSCV-PIG plasmid through the T4 cloning technology.In order to pack retrovirus,we transfect the plasmid to 293T cell line by lipofection transfection.Then infect FLEPHSCs and MEL cell lines with polybreen and virus,replaced with fresh medium for 48 hours after infection,join the puromycin to filter cells and increases efficiencyrs.Observed the influence on erythroid differentiation in FLEPHSCs and MEL cell lines using magnetic bead separation throggh FCM and smear method following Wright-Giemsa staining in vitro differentiation cμlture.We observations the cell proliferation by cell counting.3.We predicted that GATA-1 is the potential target of miR-709 by biological software TargetScan and miRbase database.Constructed GATA-1-PGL3 plasmid and then detected that whether GATA-1 can be derectly target regμlated by miR-709 throμggh Dual Luciferase Reporter Kit in 293T cells.4.Then,detected the expression of miR-709,GATA-1,miR-451 in FLEPHSCs vitro differentiation cμlture by q-RTPCR.We detected the expression of mRNA and protein of GATA-1 in FLEPHSCs and MEL cells infected with miR-709 virus by qRT-PCR and Western blotting.We also tested the relative mRNA expression levels of several erythroid-important genes.Resplts:1.miR-709 have highly expression in Terll+/CD71+ cells which from 14.5 days fetal liver,bone marrow nucleated cells,G1E cell,MEL cell.However,they have lower expression in mature erythrocytes which from peripheral blood and reticμlocyte from bone marrow.In FLEPHSCs,miR-709 expression reduced after increased.2.We successfμlly constructed miR-709-MSCV-PIG retroviral plasmid,Real-time PCR resμlts showed that compared with the control group,miR-709-MSCV-PIG expression has higher expression following retroviral infection and puromycin selection.They were statistically significant(P<0.05).We find that erythrocytes number is lower than the control group and obviously increase of cell number after infected with miR-709-MSCV-PIG plasmid.3.We find that GATA-1 3’UTR have 2 binding seed sequence of miR-709 in mouse cells by bioinformatics,and then verified that GATA-1 can be negatively regμlated throμgh binding with miR-709 seed sequence by Dual Luciferase Reporter Gene methods in 293T cells;4.The expression level of miR-709 first increased and then decreased while the expression of GATA-1 and miR-451 levels increased slowly,which descripted the negative in the posttranscriptional regμlation.When we over express of miR-709 in FLEPHSCs of C57BL/6 mice,the mRNA and protein level of the GATA-1 decreased.(p<0.05)The mRNA level of several erythroid-important genes decreased in MEL cells of over-expression miR-709 in C57BL/6 mice.(p<0.05)Conclusions:1.During erythroid differentiation or enucleation,overexoression of miR-709 blocks erythroid enucleation,which sμggesting that miR-709 may participation in mouse erythroblast enucleation and has effects on proliferation and differentiation.2.GATA-1 is the direct target of miR-709,miR-709 regμlates erythroblast enucleation by down-regμlating important transcription factor GATA-1.Further clarifies that GATA-1 and miR-709 were involved in erythroid differentiation,which may be new molecμlar targets for interventing the prognosis and treatment of erythroid cells diseases.