Regulation Effect Of SiRNA On β-globin In Erythrocytes Of Hemoglobin H Disease

Objective:Haemoglobin H disease（Hb H disease）is caused by a defect in theα-gene,which leads to an unbalance between theα-globin chains andβ-globin chains,therefore reducing the production of excessβ-globin is key to treating Hb H disease.To provide new theoretical support for gene therapy of Hb H disease,the regulatory effect of siRNA（targetingβ-globin chain m RNA）onβ-globin of erythroid cells which culture and directed differentiation in vitro was investigated.Methods:1.An in vitro differentiation culture system for erythroid cells was established.Human CD34⁺haematopoietic stem cells were isolated using CD34 microbeads and the cell purity was determined by flow cytometry,then they were amplified and directionally differentiated into erythrocyte in a two-stage culture system.During cell culture,the cell morphological characteristics were observed under the microscope and the markers of the erythrocyte immunophenotype CD71 and CD253a were assessed by flow cytometry.2.In order to identify the best siRNA sequence,transfection of 100pmol of siRNA₁,siRNA₂,siRNA₃into cells of the three experimental groups was performed by electroporation,using 0 pmol as the blank control group,100pmol siRNA-nge as the negative control group.The expression level of erythrocyteβ-globin gene m RNA was detected by RT-PCR to determine the most effective siRNA with the strongest effect on the down-regulation ofβ-globin gene m RNA.3.To determine the optimal dose of siRNA,0 pmol,50pmol,100 pmol,150 pmol,and 200 pmol optimal siRNA were transfected into erythroid cells to determine the expression level of erythrocyteβ-globin gene m RNA by RT-PCR.The regulation ofβ-globin by optimal siRNA was assessed by western blotting and the toxicity of siRNA to cells was determined by flow cytometry.4.The optimal siRNA is applied to hemoglobin H disease erythroid cells at the optimal concentration,detecting the effect of siRNA on the regulation of erythrocyteβ-globin gene m RNA.Comparing the levels of ROS in normal erythroid cells and Hb H disease erythroid cells,and the changes of ROS levels in these two cells after transfection with siRNA,then detect the apoptosis rate of cells,comprehensive evaluation of the effect of siRNA on Hb H disease erythroid cells.Result:1.A highly pure population of CD34⁺haematopoietic stem cells was sorted by the magnetic beads,in the two-stage culture system,CD34+haematopoietic stem cells gradually differentiated into mature erythrocytes 2.siRNA₂is the best sequence for inhibiting the expression ofβ-globin gene in erythroid cells（P<0.05）.4.All concentrations of siRNA tested（50 pmol,100pmol,150 pmol,and 200 pmol）down-regulated human erythrocyteβ-globin Mrna（P<0.05）,with no obvious effect onα-globin m RNA（P>0.05）.The high-dose group was more effective,with a longer-lasting silencing effect,200pmol of siRNA₂down-regulatedβ-globin of human erythroid cells within 96 h after transfection without significantly increasing the apoptosis rate of cells（P<0.05）.4.The siRNA regulated the m RNA ofβ-globin in erythroid cells of Hb H disease（P<0.05）.5.The ROS in normal erythroid cells was significantly less than that in Hb H disease erythroid cells（P<0.01）.After transfection with siRNA2,the ROS in normal erythroid cells increased（P<0.01）,In contrast,the ROS in Hb H disease erythroid cells decreased（P<0.01）.6.Transfection of siRNA₂can reduce the apoptosis rate of erythroid cells of Hb H disease.there was a significantly decreases the early apoptosis rate and total apoptosis rate 72hours after transfection（P<0.05）.Conclusion:1.This study demonstrates that siRNA targetingβ-globin chain RNA can regulateβ-globin in human erythroid cells cultured in vitro,the gene silencing effect and duration of effect of siRNA are both related to the effect dose.2.Targeting siRNA can down-regulate the expression level ofβ-globin m RNA in Hb H disease erythroid cells and can reduce intracellular ROS production and apoptosis rate,which will help to reduce Hb H disease erythrocyte hemolysis and improve clinical anemia symptoms.It may become a new way of clinical treatment of Hb H disease.