miRNA expression profiling in rhabdomyosarcoma cell lines

Rhabdomyosarcomas (RMS) are rare but very aggressive tumours of childhood that arise as a consequence of regulatory disruption of the growth and differentiation pathways of myogenic precursor cells. Based on morphology, two major RMS subtypes can be identified: embryonal RMS (ERMS) and alveolar (ARMS). To better understand the global function of miRNA in RMS, we analyzed the expression profile of 8 different RMS cell lines (3 PAX3/FOXO1 positive and 5 PAX3/FOXO1 negative RMS) using the mirVana miRNA Probe Set V1 (Ambion). The miRNA microarray platform was able to distinguish PAX3/FOXO1 positive (RH4, RH30, RH28) and negative RMS (RD, CCA, SMS-CTR, RH36, RH18) cell lines through the expression pattern of about 90 miRNAs. Since the translocation positive RMS patients fared worse than the negative counterpart our results demonstrated the potential of miRNA expression profiling to classify different RMS subtypes, in agreement to previous gene expression studies, and set the basis for a further functional characterization of selected miRNAs implicated in RMS pathogenesis and in the different clinical behaviour and aggressiveness of the two RMS subtypes. We decided to study miRNAs with the greatest difference in expression between PAX3/FOXO1 positive and negative RMS:miR-23a, miR-27a and miR-199a. microRNA expression profiling was carried out using the mirVana Probe Set V1 (Ambion) that is a collection of about 400 amine-modified DNA oligonucleotides representing a panel of the human, mouse and rat microRNAome in the miRNA Registry (miRBase - Release 9). We analyzed the expression profiles of 8 different rhabdomyosarcoma cell lines: three PAX3/FOXO1 positive (RH4, RH30, RH28) and five negative (RD, CCA, SMS-CTR, RH36, RH18). The miRNA population from each cell line was compared to a reference sample consisting of a pool of the 8 total RNA samples mixed in equal amounts. Two replicates of each experiment were performed using different microarray slides, in which sample and reference RNAs, labeled either with Cy3 or Cy5 fluorochromes, were crossed in both combinations (dye-swapping procedure).