Targeting Down-regulation Of Bcl11A, SOX6, KLF1 And KLF2 Expression Induces Fetal Hemoglobin Synthesis In β-thalassemia Red Blood Cells

Backgroundβ-thalassemia major is a kind of serious hereditary hemolytic anemia disease,which results from severely reduced or absent expression of theβ-chain of adult hemoglobin(a2p2;HbA)because of a variety of deletions and mutations in the[3-globin gene or its upstream regulatory elements.In β-thalassemia,insufficient production of the(3-globin results in an excess of unpaired a-globin chains that can precipitate within erythroid precursors.The precipitation of these free a-globin chains impairs the maturation of these precursors,leading to their death and thereby causing ineffective production of RBCs.Significant anemia results and the consequent expansion of erythroid precursors can lead to secondary problems in bones and other organs.Increased levels of fetal hemoglobin(a2y2;HbF),such as occurs with hereditary persistence of HbF,ameliorate the severity of β-thalassemia,raising the potential for genetic therapy directed at enhancing HbF.Previous studies have shown that BCL11 A,SOX6,KLF1 and KLF2 are transcription factors related to the expression of y-globin,but in(3-thalassemia,the studies about regulation of SOX6,KLF1 and KLF2 expression leading to induction ofγ-globin havent been reported,and in Chinese β-thalassemia,the studies of regulation of BCL11A expression leading to induction of y-globin haven’t been reported.ObjectInduction of y-globin in K562 cell line and human erythrocyte deffrentiated from mononuclear cell of the health donor or theβ-thalassemia patient through down-regulation of BCL11A,SOX6,KLF1 or KLF2 expression by lentivirus transduction。And measure the change of mRNA and protein of y-globin by real time fluorescent quantitative polymerase chain reaction(RT-PCR)and western Blot.Methods1.Mononuclear cells separated by Ficoll-Hypaque density gradient centrifugation are from the health adult donors,who were treated with recombinant human Granulocyte colony stimulating factor(rhG-CSF),or form the(3-thalassemia patient bone marrow or peripheral blood.Mononuclear cells are cultured in the one-step erythrocyte liquid culture system in vitro.The cultured cells morphology under phase-contrast microscope,Geimsa Stain,cell classified counting and the CD235a expression detected by flow cytometry were observed,as well as the mRNA expression of β-globin,y-globin and CD235a.2.Based on microRNA interference principle and liposome transfection technology,K562 cells are transfected by BCL11 A,SOX6,KLF1 or KLF2 candidate plasmids containing shRNA fragment,and verify the knock-down efficiency by RT-PCR and Western Blot to choose the plasmid with the most obvious knock-down efficiency.3.293FT cells are transfected with plasmid containing target shRNA fragment and help plasmid VSVG 1.0 vector and Δ8.9 vector.Harvest lentivirues 72hours after transfection by filtration of media supernatant.1080 cells are incubated with lentivirues and live cells are selected by puromycin,take crystal violet dye to measure lentivirus titer.4.Take lentivirues to infect the K562 cells and human erythrocytes deffrentiated from MNC of the health donor or the β-thalassemia patient.In K562 cell line,verify the knock-down efficiency of BCL11 A,SOX6,KLF1 or KLF2 and the regulating effect of y-globin by RT-PCR and Western Blot.In human erythrocytes,verify the knock-down efficiency of BCL11 A,SOX6,KLF1 and KLF2 and the regulating effect of y-globin by RT-PCR only.Results1.With differentiate going,many jaeinth cell-units which symbolized erythroid cells present in culture system by phase-contrast microscope observation.From Geimsa Stain,pronormoblast,basophilic normoblast,polychromatic normoblast,orthochromatic normoblast and erythrocyte are present.From cell classified counting,the proportion of erythrocyte increases gradually and reach more than 90%on the fifteenth differentiate day.From flow cytometry,CD235a is 87%on the fifteenth differentiate day.The mRNA of β-globin,γ-globin and CD235a increase gradually,while the γ/β mRNA ratio decrease gradually.2.In K562 cell line,mRNA and protein of y-globin is up regulated through down-regulation of BCL11A or SOX6.In human erythrocytes differentiated from MNC of the health donor or the β-thalassemia patient,mRNA of y-globin is up regulated through down-regulation of BCL11A or SOX6 without altering erythroid maturation.3.In K562 cell line,the mRNA and protein of γ-globin is down regulated through down-regulation of KLF1,as well as BCL11A and(3-globin expression.While the mRNA and protein of KLF2 is up regulated.The mRNA and protein of y-globin is down regulated through down-regulation of KLF2.In human erythrocytes from the health donor or the β-thalassemia patient,Y/a mRNA ratio and KLF2 mRNA are up regulated through down-regulation of KLF1,while the erythroid maturation is influenced.The mRNA of y-globin is up regulated through down regulation of KLF2.Conclusion1.Down regulation of BCL11A or SOX6 induces the production of y-globin in K562 cell line and human erythrocyte,and the studies about induction of y-globin through down regulation of SOX6 in human erythrocyte from the β-Thalassemia haven’t been reported.2.The y-globin decreases through down-regulation of KLF1 in K562 cell line,indicating the role of KLF1 on y-globin maybe dual according to different context,different co-coordinate factors and different modified status.The specific mechanism of KLF1 on y-globin need to be explored intensively.Although γ/α mRNA ratio increase in human erythrocyte through down-regulation of KLF1,erythrocyte differentiation efficiency is influenced obviously,so KLF1 maybe not a ideal target factor for regulation of the y-globin.3.The y-globin decreases through down-regulation of KLF2 in K562 cell line,while the the y-globin increases through down-regulation of KLF2 in human erythrocyte deffentiated from the MNC of the β-thalassemia patient.The opposite role of KLF2 on y-globin maybe due to different context,the specific mechanism need to be explored intensively.The study about the role of KLF2 on y-globin gene expression inK562 cell line and human erythrocyte deffentiated in vitro hasn’t been reported.4.KLF2 increases when KLF1 decreases in K562 cell line and human erythrocyte deffentiated from MNC of normal donor and theβ-thalassemia patient,indicating KLF2 compensate for KLF 1.The reports of the direct evidence of KLF2 in KLF1 compensation in K562 cell line and human erythrocyte haven’t been reported.