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.