Abstract
Activating mutations in the receptor tyrosine kinase FLT3 are one of the most frequent somatic mutations in acute myeloid leukemia (AML). Internal tandem duplications of the juxtamembrane region of FLT3 (FLT3/ITD) constitutively activate survival and proliferation pathways, and are associated with a poor prognosis in AML. We suspected that alteration of small non-coding microRNA (miRNA) expression in these leukemia cells is involved in the transformation process and used miRNA microarrays to determine the miRNA signature from total RNA harvested from FLT3/ITD expressing FDC-P1 cells (FD-FLT3/ITD). This revealed that a limited set of miRNAs appeared to be affected by expression of FLT3/ITD compared to the control group consisting of FDC-P1 parental cells transfected with an empty vector (FD-EV). Among differentially expressed miRNAs, we selected miR-16, miR-21 and miR-223 to validate the microarray data by quantitative real-time RT-PCR showing a high degree of correlation. We further analyzed miR-16 expression with FLT3 inhibitors in FLT3/ITD expressing cells. MiR-16 was found to be one of most significantly down-regulated miRNAs in FLT3/ITD expressing cells and was up-regulated upon FLT3 inhibition. The data suggests that miR-16 is acting as a tumour suppressor gene in FLT3/ITD-mediated leukemic transformation. Whilst miR-16 has been reported to target multiple mRNAs, computer models from public bioinformatic resources predicted a potential regulatory mechanism between miR-16 and Pim-1 mRNA. In support of this interaction, miR-16 was shown to suppress Pim-1 reporter gene expression. Further, our data demonstrated that over-expression of miR-16 mimics suppressed Pim-1 expression in FD-FLT3/ITD cells suggesting that increased miR-16 expression contributes to depletion of Pim-1 after FLT3 inhibition and that miR-16 repression may be associated with up-regulated Pim-1 in FLT3/ITD expressing cells.
Highlights
Fms-like tyrosine kinase 3 (FLT3) is expressed and activated in many human leukemias, including a significant percentage of acute myeloid leukemia (AML), and infant/childhood acute lymphoblastic leukemia (ALL) [1,2,3]
Our results indicated that a limited set of miRNAs are differentially expressed in FLT3/Internal tandem duplication (ITD) expressing FDC-P1 cells (FD-FLT3/ITD) when compared to empty vector expressing FDC-P1 cells (FD-EV)
The miRNA signature associated with this transformation was subsequently investigated, revealing the differential expression of several miRNAs in FD-FLT3/ITD cells compared to the control cells (Fig. 2)
Summary
Fms-like tyrosine kinase 3 (FLT3) is expressed and activated in many human leukemias, including a significant percentage of acute myeloid leukemia (AML), and infant/childhood acute lymphoblastic leukemia (ALL) [1,2,3]. Activating mutations of FLT3 are found in approximately one third of AML cases and portend a poor prognosis [4]. MiRNA are a highly-conserved family of small non-proteincoding RNA molecules, approximately 22 nucleotides in length, which can negatively regulate their target gene expression posttranscriptionally [13,14]. This occurs through partial base-pairing at miRNA recognition elements (MREs) within the 39-untranslated region (UTR) of target mRNAs, resulting in mRNA destabilization and translational inhibition [15,16]. MiRNAs are expressed in specific haematological cell types and play important regulatory roles in early haematopoietic differentiation, erythropoiesis, granulocytosis, megakaryocytosis and lymphoid development [13,22]
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