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ABSTRACT
Replicative senescence is a fate that cultured mammal primary fibroblasts spontaneously undergo. Exposed to continuous mitogenic stimuli, those cells activate the transcription of p19ARF and p16, two proteins encoded by exons located within the same locus INK4A. The transcription factor LRF (leukemia/leucocytosis related factor) controls the expression of p19ARF ; from preliminary studies, it has been seen that the 3' UTR of the LRF messenger possess many interaction sites for various families of microRNA, small fragments of untranslated RNA that regulate the levels of theirs targets, through the inhibition of the translation of their messengers. We focused our attention on the microRNA miR-100 and miR-125b. The aim of this thesis has been to verify whether miR-100 and miR-125b control the expression of LRF mouse embryonic fibroblasts (MEFs).
First of all, human HEK 293T cells had been transfected with a plasmid expressing miR-100 or miR-125b together with another plasmid containing the entire sequence of LRF 3' UTR, cloned at the end of the EGFP (enhanced Green Fluorescent Protein) sequence. Through this heterologous validation test, we show that the two microRNA control the expression of LRF on a post-transcriptional level.
The following step has been to trasfect premature passaged MEF cells with mature miR-100 or miR-125b, in order to characterize the molecular effects of the over-expression of these two microRNA in MEF. Protein LRF has been seen reduced in response to the over-expression of the two microRNA, while the levels of its messenger remain constants; this data confirm that the previously mentioned miRNA reduce LRF, by inhibiting the translation of the messenger, not its messenger expression level. Furthermore, we estimated the levels of p19ARF messenger and protein, alongside with other senescence markers: p21, E2F1 and p16. As a consequence of the reduction of the LRF protein, induced by the over-expression of miR-100 or miR-125b, we observed an approximately three fold increase of p19ARF expression, as well as an increase of the protein itself. The activation of the p19ARF expression correlated with an increase
Abstract
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of the p21 protein, an inhibitor of the cyclins. Moreover, we observed a reduction of the factor E2F1 levels (E2F1 is a master factor in cell cycle progression) while the levels of p16, an other inhibitor of the cyclins, remained constant. Since senescence relies on the activation of the p19ARF-p21 axis, we measured the percentage of senescent cells. However, in spite of the induction of the p19ARF-p21 axis, the trasfected cells with miR-100 or miR-125b do not senescence. On the contrary, we observed a greater increase of cell proliferation in transfected MEF, compared with control cells. This results enlightened partial discrepancy with data obtained with the miR-20, whose over-expression in MEF cells reduced LRF expression and anticipated the onset of senescence.
In conclusion, the lone LRF inhibition due to different miRNA families is not sufficient to address the cells towards a common fate. The key of the complex regulatory activities mediated by microRNA lies within the diversity of their targets, whose modulation induced by different microRNA leads to several cellular responses.
