Succoio, Mariangela (2015) A proteomic approach for the identification of novel direct targets of microRNA-494 and of novel mediators of cellular senescence. [Tesi di dottorato]

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Abstract

Normal cells do not replicate indefinitely, rather after a certain number of divisions, they enter into a state of permanent arrest of proliferation defined cellular senescence. Cellular senescence is accompanied by a reprogramming of gene expression profile, resulting in changes of the regulatory mechanisms that act at both transcriptional and post-trascriptional as well as at epigenetic level. MicroRNAs (miRs) are small non-coding RNAs that play a fundamental role in the negative regulation of gene expression at post-transcriptional level. They recognize sequences in the 3'UTR and block the translation or determine the degradation of messenger RNAs. In the laboratory had previously been identified 24 miRs (SAmiRs) up- or down-regulated in cellular senescence of primary fibroblasts IMR90. Functional studies on SAmiRs up-regulated demonstrated that 5 of them are able to induce premature senescence phenotype when over-expressed in young cells, in particular, the miR-494 elicited the most robust senescent phenotype. Our studies have pursued two main objectives: i) to study the regulatory pathways in which is involved the miR-494 through the identification of its molecular targets; ii) to identify novel common mediators/effectors of cellular senescence induced by telomere shortening or caused by oxidative stress. For both aims, we choose a high-throughput proteomic approach using the technology of 2D-DIGE (Two Dimensional-Difference Gel Electrophoresis) coupled to Mass Spectrometry (LC-MS / MS). In the first objective, to identify possible targets of miR-494, we monitored with 2D-DIGE changes at the protein level consequent to its overexpression in cells IMR90 young, considering the inverse relationship between levels of miRs and protein levels of their targets. The 2D-DIGE analysis allowed us to consider about 1500 protein spots present in both of the two conditions. Among these, 102 protein spots (38 up- and 64 down-regulated) were differentially represented in the cells transfected with miR-494, compared with control cells transfected with miR-scrable. Mass Spectrometry analysis identified 172 different protein species, of which 121 present in the spots down-regulated. In silico analysis, allowed the identification among these of 26 putative targets of miR-494, 9 of which have been selected by us for functional analysis. By using luciferase assays, we showed that four of them (hnRNPA3, PDIA3, RAD23B, SYNCRIP / hnRNPQ) contain functional binding sites for miR-494. We also showed that the expression of hnRNPA3, PDIA3, RAD23B, SYNCRIP decreased or increased in conditions of over-expression or inhibition of miR-494, respectively. The reduced expression of these proteins has also been found in other models of cellular senescence. We also showed that silencing hnRNPA3 or RAD23B in young IMR90 cells induces a senescent phenotype similar to that induced by the over-expression of miR-494, and mediated by the activation of the p53 pathway. Finally, other results have shown that the senescent phenotype induced by miR-494 is prevented if in the cells was introduced hnRNPA3 or RAD23B genes. The other aspect of the project involved the comparative analysis of the protein profile occurring both in replicative senescence and premature senescence induced by oxidative stress. We showed that 20 protein spots common to both conditions are differentially represented (3 up- and 17 down-regulated) and in these the Mass Spectrometry identified 25 different protein components. To check whether the down-regulation of these genes is based on regulatory mechanisms that act at the transcriptional level, the levels of mRNAs of 25 proteins were analyzed in the above reported models of senescence. Among them, 10 genes that had at least 2 times decrease of mRNAs in both conditions, were further analyzed. Then, to study the functional significance of the down-regulation of these proteins, we individually silenced the expression of these 10 genes using specific siRNA in young IMR90 cells. The results show that silencing of LEPRE1, LIMA1 / EPLIN, MAGOHA and MAGOHB genes, induces a senescent phenotype with the presence of specific markers and mediated, in the case of MAGOA and MAGOHB, by the activation of the p53 pathway. We then looked at both protein and mRNA levels in: i) IMR90 senescent cells after DNA damage; ii) WI38 senescent cells caused either by telomere shortening or oxidative stress; iii) fibroblasts from donors of different ages between 21 and 94 years. The results showed that in senescent cells, both in vitro and in vivo, the levels of the proteins and those of the respective mRNAs are decreased. Finally, since it is known that selective histone modifications, in particular the methylation of histone H3 lysine, play a crucial role in the mechanisms of gene expression regulation at epigenetic level, we verified the methylation status of histone H3 in the promoter genes of LEPRE1, LIMA1/EPLIN, MAGOHA and MAGOHB during senescence. For this purpose, we performed chromatin immunoprecipitation experiments using antibodies specific for the repressive H3K27me3 marker or for the active H3K4me3 marker. The results demonstrate that the reduced expression of these genes in senescence is associated with increase in the amount of repressive H3K27me3 and in almost all cases to decrease of active H3K4me3. In conclusion, the data obtained with the approach of differential proteomics have identified novel genes (hnRNPA3 and RAD23B LEPRE1, LIMA1/EPLIN, MAGOHA, and MAGOHB) that have a causative role in the appearance of the senescent phenotype. These results on one hand contribute to gain a deeper knowledge of the molecular mechanisms involved in senescence and on the other creates the basis for considering these genes as possible markers of aging and age-related diseases, as well as candidates for developing therapeutic strategies for the manipulation of in vivo cellular senescence.

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