AML1/ETO SiRNA Enhances The Sensitivity Of Kasumi-1 Cells To Histone Acetylation Enzyme Inhibitors

Background leukemia with AML1/ETO fusion gene positive is a subtype of acute myeloid leukemia, and it is characterized by chromosome translocation of t(8; 21)(q22; q22) which generates the fusion gene of AML1/ETO leading to leukemia through several channels. AML1/ETO fusion gene could activate JNK signal transduction pathway so as to up-regulate the expression of c-jun and inhibit apoptosis of hematopoietic cells, down-regulate the expression of neutrophils differentiation factor C/EBP a which plays an important role in differentiation of neutrophils and function of blood cells, inhibit the interaction of ETO-2 and N-CoR competitively to hinder the differentiation of neutrophils, inhibit the function of AML1 and its target genes which are hematopoietic related, down-regulate AES to self-renew. In addition, AML1/ETO fusion gene could combine to nuclear receptor inhibiting complex N-CoR/Sin3/HDAC as transcription repressors and activate histone acetylation enzymes (HDACs) which can make histone deacetylation and restrain AML1 transcription, so as to suppress the effect of AML1 gene competitively and interfere the normal hematopoiesis to lead to leukemia. Histone acetylation enzyme inhibitors(HDACIs) valproate (VPA) is a type of antiepileptic drugs which is extensive used in clinical, it also plays effet of antiproliferative by inducing differentiation and apoptosis, arresting cell cycle, and this effect had been confirmed in several tumors. The new study shows that VPA can remit leukemogenesis mediated by AML1/ETO fusion gene through breaking abnormal function of HDAC. RNAi is a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA), which leads to gene silencing in a sequence-specific manner. In this study, we investigate the changes of biologic activity and the sensitivity of Kasumi-1 cells to VPA after silence of AML1/ETO gene and provide theoretically and experimental bases for further clinical use.Objective Construction and identification of RNA interference expression vectors targeting AML1/ETO and to investigate the changes of biologic activity and the sensitivity of Kasumi-1 cells to sodium valproate (VPA), a histone acetylation enzyme inhibitors (HDACI), after silence of AML1/ETO gene.Methods The oligonucleotides of AML1/ETO gene were designed referring to related date of GenBank, double-stranded DNA was derived through annealing, and inserted into pGCsilencerU6/Neo/GFP vector. The resultant recombinant plasmid pGCsiRNA-AML1/ETO was transfected into Kasumi-1 cells by liposomes. Expression of AML1/ETO and Bcl-2 mRNA was assayed by real time PCR(RT-PCR). The effet of VPA on proliferation to Kasumi-1 cells was assessed by MTT. The cell cycle was assessed by flow cytometry. The morphology of apoptosis after Hochest33258 staining was observed under a fluorescence microscope.Results After transfection with pGCsiRNA-AML1/ETO, expression of AML1/ETO mRNA in Kasumi-1 cells reduced by 53.7% compared with the controls(p<0.05), while the expression of Bcl-2 mRNA reduced by 54.5%(p<0.05). The inhibitory effet of VPA on proliferation of Kasumi-1 cells was concentration- and time-dependent, and the inhibitory rates in the transfected group were higher than those in the control group 24h and 48h after transfection, with different VPA concentrations. Rates of the cells at G0/G1 phase in the control group and the transfected group were (42.07±5.23)% and (62.6±5.87)%, respectively (p<0.01). After the treatment with VPA (2mmol/L) for 48h, the rates increased to (69.2±7.02)% and (78.92±6.23) %, respectively (p<0.01). After transfection, cells presented karyopyknosis, nuclear margination and apoptotic bodies.Conclusion The siRNA specifically targeting the AML1/ETO gene can remarkably down-regulate the expression of AML1/ETO gene which induces apoptosis, inhibits proliferation, arrests cell cycle, and enhances the sensitivity of Kasumi-1 cells to VPA.