The Effect Of RNA Interfering Lkaros On The Expression Of γ Globin Of K562 Cells

Backgroundβ-thalassemia that caused by decreased synthetic ratios of non-αand a globin chains is the most prevalent group of human monogenic diseases, and Guangdong, Guangxi, Hainan and Taiwan provinces have a high incidence. In clinical routine treatments,β-thalassemia is often used with hematopoietic stem cells transplantation, blood transfusion with iron chelation therapy, or splenectomised methods for therpy. Although curative allogeneic stem cell transplantation and palliative therapies have been developed for these disorders, the majority of patients still suffer significant morbidity and early mortality. It becomes one of the first diseases to be tried for gene therapy with in-depth study of its pathogenesis during these years. It has made great progress in the thalassemia gene therapy during the past three decades.It has been reported that the increase ofγ-globin can ameliorate the clinical symptoms ofβ-thalassemia patients. Due to the excessiveα-globin chains be combining with the increasedγ-globin chains, there is a raise of hemoglobin content. Interactions between transcription factor Ikaros and GATA-1 duringγ→3 globin switching can lead to the silence ofγ-globin expression. So we propose that interfering Ikaros can enhance the expression ofγ-globin. RNA interference (RNAi) is a recently developed technology in genetic field. The aim of our reasrech is to design Ikaros siRNA and to investigate what will happen to the expression ofγ-globin after interferencing Ikaros gene. It maybe provide a potential therapeutic for gene therapy toβ-thalassemia.Objective To interfer the Ikaros gene, and to induce the expression ofγ-globin.Methods1. Celllines and intervening factorsK562 cells are selected for the study. Lipofectamine 2000 is used to transfect siRNA to K562 cells. Sodium butyrate has been known that it can induce erythroid differentiation of K562 cells and increase the expression ofγ-globin.2. Screening the optimum transfection conditions for K562 cellsSiRNA NC labeled by FAM is transfected in K562 cells with lipofectamine 2000 under different conditions. The transfection efficiency is detected by flow cytometry.3. Screening the optimum siRNA for Ikaros.The siRNA sequences targeted of Ikaros gene (gene number:NM₀06060.3) are designed using the software developed by Invitrogen, Inc. Three candidate sequences in Ikaros gene, classified as siRNA-Ikaros#1, siRNA-Ikaros#2 and siRNA-Ikaros#3, are selected for RNAi. A scrambled sequence (siRNA NC) served as a negative control. These sequences show no homology with other known human genes. Equal mount of three candidate Ikaros RNAis are transfected into the K562 cells using lipofectamine 2000 respectively. After culturing 48 hours, K562 cells are collectted and total RNA is isolated using the Trizol reagent. RT-PCR products are resolved on 1.5% agarose gel and visualized by ethidium bromide staining.4. Investigating the effect of siRNA-Ikaros and sodium butyrate on the expression ofγ-globin of K562 cellsSiRNA-Ikaros group, sodium butyrate group, siRNA-Ikaros wiht sodium butyrate group, siRNA NC group and the blank group are set and tested. RT-PCR and Western Blot are used to detect the expression of Ikaros mRNA and protein levels. QT-PCR and Western Blot are used to detect the expression ofγ-globin mRNA and protein levels in different groups. Hemoglobin content of the K562 cells are detected by benzidine staining in each group and used to evaluate the degree of differentiation.Results1. Under the optimun conditions, the transfection rate can reach to 58.7%.2. All of the 3 candidate siRNAs can effectively interference Ikaros. Theinhibited efficiency of the expression of Ikaros in the siRNA-Ikaros#3 group can reach to 86.7%. So siRNA-Ikaros#3 is the optimum siRNA for Ikaros.3. SiRNA-Ikaros#3 can enhance the expression ofγ-globin and the hemoglobin content of K562 cells. Sodium butyrate also can increase the expression ofγ-globin gene, but there are no senergy between the siRNA-Ikaros#3 and sodium butyrate.ConclusionsSiRNA-Ikaros can interfer the expression of Ikaros and enhance the expression ofγ-globin. However, it has no synergistic effect with sodium butyrate. SiRNA-Ikaros maybe an new curial method to the severe beta thalassmia.