Suppression Of Aml1/Eto Via Sirna Mediated Gene Knockdown And Its Effects On Foxo3 And C-Myc Expression In Aml T (8,21) Cells
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Date
2017-06
Authors
Ong, Si Min
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Acute Myeloid Leukemia (AML) with t (8,21) translocation is one of the most frequent karyotypic abnormalities observed in AML that results in formation of fusion onco-protein, AML1/ETO. siRNA is a commonly used RNA interference (RNAi) tool to induce short-term silencing of protein coding genes. There are studies shows that silencing of AML1/ETO results in up- or down-regulation of various gene responsible for apoptosis, proliferation, and self-renewal of cells. FOXO3 act as a tumor suppressor gene by triggering apoptosis with its upregulation while c-MYC is a proto-oncogene which is found to be upregulated in many types of cancers. Nonetheless, the correlation between these three genes is not known. Therefore, the objective of this study was to study the suppression of AML1/ETO gene via siRNA mediated knockdown and its effect on FOXO3 and c-MYC gene expression in AML t (8,21) positive Kasumi-1 cell. It was hypothesized that siRNA mediated knockdown is efficient in suppressing AML1/ETO gene and there is a correlation between AML1/ETO, FOXO3 and c-MYC gene. This hypothesis was experimentally tested using a siRNA
mediated gene knockdown by electroporation of AML1/ETO followed by the study of gene expression level of FOXO3 and c-MYC using qPCR. The result of siRNA mediated AML1/ETO gene knockdown experiments using siAGF1 reveal that there is a knockdown of 52% at 24 hours (p=0.005), 65% (p=0.002) at 48 hours and 85% (p=0.018) at 72 hours. Whereas the result of gene expression level of FOXO3 reduced by 3 % at 24 hours (p=0.860), 48 hours showed a 50% of increase (p=0.153) and 72 hours showed a 46% (p=0.154) of decrease in expression. Expression of c-MYC increased by 11% at 24 hours (p=0.821), 48 hours showed 83% of increased (p=0.232) while 72 hours showed 25% of decreased in expression (p=0.209). The results of gene expression after gene knockdown does not show conclusive or significant correlation between AML1/ETO, FOXO3 and c-MYC which a prolong knockdown is suggested to further verify the finding.
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Keywords
The suppression of AML1/ETO gene , via siRNA mediated knockdown