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BMC Proc. 2012; 6(Suppl 6): P29.
Published online Oct 1, 2012. doi:  10.1186/1753-6561-6-S6-P29
PMCID: PMC3467469
siRNAs targeting the ERK2 signaling pathway and AML1/MTG8 fusion gene attenuate the differentiation, proliferation and growth arrest in t(8;21) leukemia
Salem Bashanfer,1 Faisal Mutee Al-Hassan,1 Rosline Hassan,2 Olaf Heidenreich,3 Mohamed Saifulaman Mohamed Said,corresponding author4 and Narazah Mohd Yusoff1
1Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, Bandar Putra Bertam, 13200, Kepala Batas, Penang, Malaysia
2School of Medical Sciences, Universiti Sains Malaysia, 16150, Kota Bharu, Kelantan, Malaysia
3University of Newcastle, Northern Institute for Cancer Research, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
4Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM) 40450, Shah Alam, Selangor, Malaysia
corresponding authorCorresponding author.
Supplement
Beyond the Genome 2012
Conference
Beyond the Genome 2012
27-29 September 2012
Boston, MA, USA
Background
The t(8;21) translocation is one of the most frequent chromosome abnormalities associated with acute myeloid leukemia (AML). This translocation generates the (AML1/MTG8) fusion protein causing blockage of the differentiation process. Moreover, constitutive activation of the mitogen-activated protein kinase (MAPK) pathway as a consequence of this translocation results in an increase in the proliferation rate of leukemic cells.
Materials and methods
To ensure subsequent experiments are clinically relevant, we carried out microarray on clinical samples from patients who manifested the t(8;21) translocation together with the corresponding cell lines (Kasumi-1 and SKNO-1). We carried out microarray, qRT-PCR, gene knockdown by siRNA, flowcytometry and various cell assays for apoptosis and cellular proliferation testing.
Analyses revealed a number of overlapping differentially expressed genes in the AML clinical samples and their corresponding cell lines. These genes were uploaded to the KEGG database through the DAVID software (v6.7) to carry out pathway analyses of these leukemic cells. Furthermore, gene expression profiles of both clinical samples and their relevant cell lines showed increased expression of AML1/MTG8 and ERK2 (MAPK1).
Using 100 nM and 200 nM of ERK2-siRNA and siRNA-AML1/MTG8, respectively, siRNAs were transfected by electroporation to knockdown ERK2 and the fusion gene AML1/MTG8 individually and in combination in the t(8;21) cell lines. Gene knockdowns were validated by qRT-PCR, and demonstrated successful mRNA suppression by approximately 80% to 90%. In addition, a slight increase of ERK2 mRNA expression was observed upon AML1/MTG8 suppression.
FACS experiments showed reduced expression of CD34 cell surface marker when AML1/MTG8 was knocked down, which indicated onset of the differentiation process whereas the ERK2 suppression displayed higher CD34 expression when compared with mock and siRNA controls, indicating an antidifferentiation effect. Cell cycle analyses demonstrated increased growth arrest where G0/G1 phase increased by 10% to 15% individually and 25% to 30% in a combination, which correlated with the MTS assay results. An apoptosis assay revealed anti-apoptotic effect of ERK2 and AML1/MTG8 suppression, which was increased when silenced together in a combination silencing.
A subsequent microarray experiment on cell lines with these silenced genes was consistent with previous results and disclosed several genes that might be involved in controlling these processes.
Conclusions
These results suggest possible roles of ERK2 activation in differentiation induction, while ERK2 inhibition arrested cell growth and reduced proliferating cells in t(8;21) leukemia when combined with AML1/MTG8 knockdown.
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