Abstract

Dr. Tiziana Bonaldi, Group Leader, Department of Experimental Oncology of the European Institute of Oncology, Italy .



Background MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level. Computational studies suggest that each miRNA have hundreds targets. Correlation between miRNAs and cancer has been frequently reported. MiR-17-92, for instance, is amplified in several lymphomas and B-CLL cells, acting synergistically with c-myc. A comprehensive screening for miR17-92 targets would elucidate his role in oncogenesis. Since miRNAs act post-transcriptionally, the screening should be carried out at the protein level. We describe quantitative proteomics to profile changes upon miR17-92 over-expression.
Methods: SILAC is a metabolic labelling strategy allowing the accurate comparative measurement of protein levels from different functional states. We SILAC- labelled B-lymphoma cells over-expressing miR17-92. Retroviral-infected and control cells were grown in "light" and "heavy" medium, respectively. After full incorporation, cells were harvested and mixed in equal amounts. Extracted proteins we analyzed by Mass Spectrometry and protein quantification was carried out with MaxQuant software. Three biological replicates were analyzed; a parallel transcriptome analysis (Affymetrix) was carried out. Results: We obtained a high-confidence quantitative proteome of about 4700 proteins, and a corresponding transcriptome of about 10000 transcripts. Statistical analysis indicates that the cellular response to mir17-92 induction is predominantly post-transcriptional, with about 550 significant outliers in the proteome, versus 150 responding transcripts. Remarkably, enrichment analysis of the down-regulated proteins and transcripts comparing miRNA predicted targets with non-targets shows evidence of enrichment for miRNA 19a/b, in agreement with previous reports The intersection of significant protein outliers with a comprehensive database of cancer-related genes produced a list of candidates that may explain Oncomir1 role in lymphomagenesis. Finally, the functional analysis carried out on the total Proteome, in combination with the enrichment analysis on the responders indicates that miR17-92 modulates in both direction several pathways downstream of c-Myc; this is highly suggestive of the existence of multiple circuitries of positive and negative feedback loops, where mir17-92 potentiates or dampens Myc-mediated cellular processes, in different stage of differentiation or tumour progression. Independent validation of the cellular response both for single gene products and for whole processes is ongoing.
Discussion: We have established SILAC in B-lymphoma cells for the first time, acquiring the most extensive quantitative proteome in B-lymphocytes to date, an important resource for scientists. The comparative analysis of proteome and transcriptome changes upon a perturbation represents an extremely innovative system-biology approach to characterize comprehensively functional states, with a focus on post-transcriptional events. .

Host: Dr. Ernesto Guccione