title: Discrimination of target by siRNA: designing of AML1-MTG8 fusion mRNA-specific siRNA sequences.
abstract: AML1-MTG8 is a chimeric transcription factor produced by t(8;21) chromosome translocation and causes AML. AML1-MTG8 acts as a dominant negative effector on normal AML1 protein, a key transcriptional regulator of hematopoietic differentiation, but its precise mechanism is not known. To analyze the function of AML1-MTG8 in leukemic cells and to explore the possibility of AML1-MTG8-targeted therapy, we designed nine small interfering RNAs (siRNAs) targeting a 25-nucleotide region spanning the fusion point of AML1 and MTG8. Two different siRNAs (AM2 and AM4) significantly reduced AML1-MTG8 expression from a transfected reporter plasmid at both the mRNA and protein levels. Both siRNAs did not reduce AML1b expression, but AM2 siRNA showed slightly reducing activity against MTG8b mRNA that is 86% homologous to the corresponded region of AML1-MTG8 mRNA. Moreover, using a cationic lipid reagent, the siRNAs were efficiently introduced into leukemia cell lines with t(8;21), SKNO-1 (30-40%) and Kasumi-1 (60-70%) cells, and reduced specifically the endogenous AML1-MTG8 expression. The siRNAs reduced neither the wild type AML1 in Kasumi-1 cells nor wild type MTG8b in human erythroblastic leukemia (HEL) cells. These results indicated that the two siRNAs are highly specific for the fusion mRNA. The knockdown of AML1-MTG8 in Kasumi-1 cells resulted in the activation of p14(ARF) promoter activity and increased the expression of integrin alphaIIb, whose expression is related to megakaryocytic differentiation. However, the knockdown of AML1-MTG8 in Kasumi-1 cells did not inhibit the cell growth, suggesting that the siRNA-mediated knockdown of AML1-MTG8 is useful for the functional analysis of the gene, but it alone might not be sufficient for gene therapy of the leukemia.
authors.affiliation: Division of Cancer Treatment, Research Institute for Clinical Oncology, Saitama Cancer Center, 818 Komuro, Ina, Saitama 362-0806, Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Kasashima
authors.forename: Eri
authors.initials: E
authors.lastname: Sakota
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kozu
mesh_terms: D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002875:Chromosomes / Q000235:genetics; D002898:Chromosomes, Human, Pair 8 / Q000235:genetics; D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins / Q000235:genetics*; D015973:Gene Expression Regulation, Leukemic / Q000235:genetics*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D000075142:RUNX1 Translocation Partner 1 Protein; D014157:Transcription Factors / Q000235:genetics*; D014178:Translocation, Genetic / Q000235:genetics
chemical_list: D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins; D015514:Oncogene Proteins, Fusion; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D000075142:RUNX1 Translocation Partner 1 Protein; C493728:RUNX1 protein, human; C082222:RUNX1T1 protein, human; D014157:Transcription Factors