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Abstract
A previous study published by us identified twelve differentially expressed microRNAs between AML FAB M1 and M5, from these five candidate microRNAs (microRNA-130a, -135b, -146a, -181a and -181b) were selected for further study based on their predicted targeting for inhibition of nine transcription factor mRNAs involved in myeloid differentiation and development (Lutherborrow et al., 2011). In this study through the use of a luciferase reporter assay six of the predicted gene targets (FOS, HOXA10, MAFB, IRF8, KLF4 and MCL-1) identified were as bona fide targets of four of the five candidate microRNAs. The next aim was to assess the effect of over-expressing the microRNAs in AML cell line models (HL-60 and NB4); optimisation of three transfection methods was undertaken. The lipid based transfection reagent lipofectamine 2000 delivered oligonucleotides into HL-60 and NB4 cells at transfection efficiencies of 10% and 40% respectively, but was unable to successfully introduce plasmid to levels required for functional studies. Electroporation was found to be highly variable and toxic resulting in high levels of cell death when introducing oligonucleotides or plasmids. Nucleofection, a more specific method of electroporation, was optimised and was able to successfully transfect a GFP plasmid into the HL-60 cell line at efficiencies of 21-46%, but was unable to transfect oligonucleotides. In preparation for future studies based on the transfection optimisation results, inducible microRNA expression vectors for the candidate microRNAs were successfully cloned.
The third part of my work was to examine changes in the candidate microRNA expression levels during normal primary macrophage differentiation to determine if published changes seen in cell line experiments occur in primary cells. Interestingly microRNA-146a was identified as being significantly increased during monocyte to macrophage differentiation in contrast to the published results. Since NF-κβ signalling is known to be important in malignancies, an experiment was performed examining the effect of the candidate microRNAs on NF-κβ signalling using a NF-κβ specific luciferase reporter. Of the candidate microRNAs, only microRNA-146a over-expression resulted in a statistically significantly decreased of luciferase activity (by 60%) and therefore NF-κβ signalling.
In summary this work has identified six microRNA:mRNA interactions involved in the myeloid development and it can be hypothesised that these interactions could be involved in the formation of the myeloid differentiation blocks seen in AML. Transfection optimisation has elucidated that stable plasmid over-expression is the best method for microRNA alteration in AML cell lines. Additionally this study has provided a foundation for future projects to further examine the roles of these microRNAs in the pathogenesis of AML.