L'INFLUENZA DEL MIR-124 SUL DIFFERENZIAMENTO NEURONALE: UN LINK PROTEOMICO

MicroRNAs are endogenous 23-nucleotides RNAs that can pair to particular sites in messenger RNAs of protein-coding genes to down regulate the expression of these genes thus altering cell physiology. Many studies have been done towards the identification of the miRNA targets at a transcriptomic level, and many algorithms have been developed to predict and identify the putative targets. But none of the approaches currently used to study miRNAs directly measures their influence on protein output, which is the most relevant readout of their regulatory effects. The importance of knowing miRNA targets relies on the fact that these molecules are involved in the temporal and spatial regulation of organism development and in many aspects of the cell physiology, and that a mutation in their expression can lead to different pathologies, such as neurodegenerative diseases and tumours. miR-124 is one of the most abundantly expressed miRNAs in the nervous system, being widely expressed in neurons in the brain, retina and spinal cord. Its expression can be detected in differentiating neurons and persists in mature neurons, suggesting that miR-124 plays important roles during neuronal development and normal physiology. Some of its targets have already been identified by classical methods, but a more comprehensive approach is required to go deeply in its mechanism of action. To identify the possible protein targets of this miRNA, we have adopted a SILAC-based approach, which enabled us to identify the quantitative changes in the protein abundances in Neuro2A cells, the neuroblastoma cells that we have used as model system for this study. These cells are progenitor-like cells, able to differentiate to neuronal-like cells following serum starvation. We have transduced them with a lentiviral vector harbouring a construct able to knockdown the expression of the miRNA of interest, completely labeled the proteome with the heavy forms of lysine and arginine, and then induced the differentiation and the expression of miR-124 by serum starvation for 24 hours. The increment of mi-R124 levels has been verified and the functionality of the vector has been checked by FACS analysis, confirming both the over-expression of the miR-124 after the induction by serum depletion and its knock-down in the presence of the lentivirale vector. The whole proteomes from control and starved cells have been firstly sub-fractionated in order to simplify the protein patterns and to increase the number of identifications. The fractions have been separated by SDS-PAGE, the excised gel bands have been tryptic digested and analysed by high accuracy mass spectrometry (LTQ-OrbiTRAP). The analysis of changes in the identified and quantified proteins will shed light on the mechanisms affected by miRNA-124 that rule neuronal differentiation of progenitors cells.