MicroRNAs (miRNAs) are single stranded non-coding RNAs of ∼ 22 nucleotides involved in gene regulation. Several examples of an association between disrupted expression of miRNAs and cancer have been shown. Using a real-time quantitative PCR, designed to amplify only from the mature miRNA (TaqMan® -MicroRNA Looped-PCR assay, Applied Biosystems), we measured the expression levels of 157 miRNAs in 100 acute myeloid leukaemia (AML) patients representing the spectrum of known karyotypes common in AML, 2 leukaemic cell lines (KG1 and NB4), and the bone marrow from 2 healthy donors. ANOVA analysis using a 5% false discovery rate threshold was performed to identify differentially expressed miRNAs between leukaemia samples and normal bone marrow. MiRNAs specific to karyotype groupings were also identified in the same way. A method was developed to demonstrate the spatial localisation, in situ, of specific miRNA identified in the quantification and confirm the expression of miRNA with relation to karyotype. Commercial locked nucleic acid (LNA) oligonucleotides (Exiqon) were obtained for two miRNAs (miRNA-127 and miRNA-154), as well as for positive and negative control probes. LNAs were labelled with digoxigenin and probes applied to both cytospins and/or trephines of a sample set representative of the different AML subtypes. Detection of hybridisation signals was either by colorimetric or fluorescent reaction and visualised by confocal microscopy. Unsupervised cluster analysis revealed an association of miRNA expression with the karyotype of the samples. Promyelocytic leukaemias (APML) bearing the t(15;17) translocation, show a distinct pattern characterised by the high expression of a subset of 10 miRNAs located in the human 14q32 imprinted domain, including miR-127 and miR-154. ANOVA data analysis revealed the de-regulation of 33 miRNAs across the leukaemic set in respect to bone marrow from healthy donors. Seventeen miRNAs were up-regulated and 16 down-regulated. MiRNAs miR-155, miR-181a, miR-181b, miR-181c, miR-142-5p, miR-221, and miR-222, were among those commonly highly expressed. Down-regulated were miR-26a, miR-34c, and miR-199a. MiRNAs miR-10a and miR-125b showed the highest variability throughout the samples, being associated with specific subgroups. In situ hybridisation analysis of miR-127 and miR-154 confirmed the results obtained by real-time PCR of their expression associated with APML. This study, conducted on about a third of the miRNAs reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub-classify cancer. The expression analysis of larger number of miRNAs coupled with the in situ hybridisation of leukaemic cells will allow the investigation of miRNA expression on stored samples during the disease course and provide valuable insights into the leukaemogenic process.
Disclosure: No relevant conflicts of interest to declare.