Identification of miR-494 direct targets involved in senescence of human diploid fibroblasts.

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up-regulation of microRNA (miR)-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (P≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational analysis identified 7 potential evolution-conserved miR-494 targets. Functional miR-494 binding sites were confirmed in 3'-untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR-494 up-regulation in senescent cells. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-ß-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.

Exploring Heterosis in Melon (Cucumis melo L.).

Heterosis is the superiority of an F1 hybrid over its parents. Since this phenomenon is still unclear in melon, a half diallel experiment based on eight genetically distant breeding lines was conducted in six environments of Central Italy, assessing commercially important traits: yield, total soluble solids (TSS), and days to ripening (DTR). To estimate the additive (general combining ability; GCA) and the non-additive gene effects (specific combining ability; SCA), yield was analyzed by Griffing's methods two and four, and the results were compared to the GGE (Genotype plus Genotype by Environment interaction) biplot methodology; TSS and earliness were evaluated only by Griffing's method four. Overall, GCAs were significantly more relevant than SCAs for all examined traits. Least square means (LsM), mid-parent heterosis (MPH), best-parent heterosis (BPH), as well as Euclidean and Mahalanobis' distances were calculated and compared with the genetic distance (GD). As a few correlations were found statistically significant (only for TSS), it was difficult to predict the value of a hybrid combination only by knowing the genetic distance of its parents. Despite this, heterosis was observed, indicating either the presence of epistatic effects (additive × additive interactions) and/or an underestimate of SCAs embedded within Griffing's method. The significant Env × Entries source of variation suggests development of hybrids in specific environments. The results are discussed with a breeding perspective.

Comparative analysis of gene expression data reveals novel targets of senescence-associated microRNAs.

In the last decades, cellular senescence is viewed as a complex mechanism involved in different processes, ranging from tumor suppression to induction of age-related degenerative alterations. Senescence-inducing stimuli are myriad and, recently, we and others have demonstrated the role exerted by microRNAs in the induction and maintenance of senescence, by the identification of a subset of Senescence-Associated microRNAs (SAmiRs) up-regulated during replicative or stress-induced senescence and able to induce a premature senescent phenotype when over-expressed in human primary cells. With the intent to find novel direct targets of two specific SAmiRs, SAmiR-494 and -486-5p, and cellular pathways which they are involved in, we performed a comparative analysis of gene expression profiles available in literature to select genes down-regulated upon replicative senescence of human primary fibroblasts. Among them, we searched for SAmiR's candidate targets by analyzing with different target prediction algorithms their 3'UTR for the presence of SAmiR-binding sites. The expression profiles of selected candidates have been validated on replicative and stress-induced senescence and the targeting of the 3'UTRs was assessed by luciferase assay. Results allowed us to identify Cell Division Cycle Associated 2 (CDCA2) and Inhibitor of DNA binding/differentiation type 4 (ID4) as novel targets of SAmiR-494 and SAmiR-486-5p, respectively. Furthermore, we demonstrated that the over-expression of CDCA2 in human primary fibroblasts was able to partially counteract etoposide-induced senescence by mitigating the activation of DNA Damage Response.

Brg1 chromatin remodeling factor is involved in cell growth arrest, apoptosis and senescence of rat mesenchymal stem cells.

Self-renewal, proliferation and differentiation properties of stem cells are controlled by key transcription factors. However, their activity is modulated by chromatin remodeling factors that operate at the highest hierarchical level. Studies on these factors can be especially important to dissect molecular pathways governing the biology of stem cells. SWI/SNF complexes are adenosine triphosphate (ATP)-dependent chromatin remodeling enzymes that have been shown to be required for cell cycle control, apoptosis and cell differentiation in several biological systems. The aim of our research was to investigate the role of these complexes in the biology of mesenchymal stem cells (MSCs). To this end, in MSCs we caused a forced expression of the ATPase subunit of SWI/SNF (Brg1 - also known as Smarca4) by adenoviral transduction. Forced Brg1 expression induced a significant cell cycle arrest of MSCs in culture. This was associated with a huge increase in apoptosis that reached a peak 3 days after transduction. In addition, we observed signs of senescence in cells having ectopic Brg1 expression. At the molecular level these phenomena were associated with activation of Rb- and p53-related pathways. Inhibition of either p53 or Rb with E1A mutated proteins allowed us to hypothesize that both Rb and p53 are indispensable for Brg1-induced senescence, whereas only p53 seems to play a role in triggering programmed cell death. We also looked at the effects of forced Brg1 expression on canonical MSC differentiation in adipocytes, chondrocytes and osteocytes. Brg1 did not induce cell differentiation per se; however, this protein could contribute, at least in part, to the adipocyte differentiation process. In conclusion, our results suggest that whereas some ATP-dependent chromatin remodeling factors, such as ISWI complexes, promote stem cell self-renewal and conservation of an uncommitted state, others cause an escape from 'stemness' and induction of differentiation along with senescence and cell death phenomena.

An effective method for adenoviral-mediated delivery of small interfering RNA into mesenchymal stem cells.

Mesenchymal stem cells (MSCs) promise as a main actor of cell-based therapeutic strategies, due to their intrinsic ability to differentiate along different mesenchymal cell lineages, able to repair the diseased or injured tissue in which they are localized. The application of MSCs in therapies requires an in depth knowledge of their biology and of the molecular mechanisms leading to MSC multilineage differentiation. The knockdown of target genes through small interfering RNA (siRNA) carried by adenoviruses (Ad) represents a valid tool for the study of the role of specific molecules in cell biology. Unfortunately, MSCs are poorly transfected by conventional Ad serotype 5 (Ad5) vectors. We set up a method to obtain a very efficient transduction of rat MSCs with low doses of unmodified Ad5, carrying the siRNA targeted against the mRNA coding for Rb2/p130 (Ad-siRNA-Rb2), which plays a fundamental role in cell differentiation. This method allowed a 95% transduction rate of Ad-siRNA in MSC, along with a siRNA-mediated 85% decrease of Rb2/p130 mRNA and a 70% decrease of Rb2/p130 protein 48 h after transduction with 50 multiplicities of infection (MOIs) of Ad5. The effect on Rb2/p130 protein persisted 15 days after transduction. Finally, Ad-siRNA did not compromise the viability of transduced MSCs neither induced any cell cycle modification. The effective Ad-siRNA-Rb2 we constructed, together with the efficient method of delivery in MSCs we set up, will allow an in depth analysis of the role of Rb2/p130 in MSC biology and multilineage differentiation.

RB and RB2/P130 genes cooperate with extrinsic signals to promote differentiation of rat neural stem cells.

Mechanisms governing commitment and differentiation of the cells of the nervous system begin to be elucidated: how extrinsic and intrinsic components are related remains poorly understood. To investigate this issue, we overexpressed genes of the retinoblastoma (Rb) family RB and RB2/p130, which play an important role during nerve cell maturation, in rat neural stem cells (NSCs). Immunostaining of neurons, astrocytes and oligodendrocytes in cultures overexpressing pRb or pRb2/p130 revealed that these genes affect lineage specification of differentiating NSCs. We observed modifications in percentage of differentiated cells indicating a shift towards the phenotype induced by culture conditions. Results were confirmed by detection of the expression levels of differentiation markers by RT-PCR. Analysis of BrdU incorporation and detection of an early marker of apoptosis suggest that the effect of pRb and pRb2/p130 overexpression is not dependent on the inhibition of cell proliferation, nor does it rely on the regulation of cell survival. Our findings suggest that Rb family genes are involved in fate determination of the cells of the nervous system. However, their role seems subsidiary to that of the extrinsic signals that promote lineage specification and appear to be mediated by a direct effect on the acquisition of a specific phenotype.

Molecular pathways involved in neural in vitro differentiation of marrow stromal stem cells.

In recent years several reports have claimed to demonstrate trans-differentiation, namely that stem cells have been derived from a given tissue and have differentiated into phenotypes characteristic of different tissues following transplantation or in vitro treatment. For example, the mesenchymal stem cells, also referred to as marrow stromal stem cells (MSCs), present in bone marrow, have been induced to differentiate into neurons. We decided to investigate this phenomenon more in depth by a molecular and morphological follow-up. We analyzed the biochemical pathways that are currently induced to trigger neuron-like commitment and maturation of MSCs. Our studies suggest that: (i) the increase in cAMP, induced to differentiate MSCs, activates the classical PKA pathway and not through the exchange protein directly activated by cAMP (EPAC), a guanine nucleotide exchange factor for the small GTPase Rap1 and Rap2; (ii) MEK-ERK signaling could contribute to neural commitment and differentiation; (iii) CaM KII activity seems dispensable for neuron differentiation. On the contrary, its inhibition could contribute to rescuing differentiating cells from death. Our research also indicates that the currently used in vitro differentiation protocols, while they allow the early steps of neural differentiation to take place, are not able to further sustain this process.

Role of RB and RB2/P130 genes in marrow stromal stem cells plasticity.

Marrow stromal cells (MSCs) are stem-like cells having a striking somatic plasticity. In fact, besides differentiating into mesenchymal lineages (bone, cartilage, and fat), they are capable of differentiating into neurons and astrocytes in vitro and in vivo. The RB and RB2/P130 genes, belonging to the retinoblastoma gene family, play a key role in neurogenesis, and for this reason, we investigated their role in neural commitment and differentiation of MSCs. In MSCs that were either uncommitted or committed toward neural differentiation, we ectopically expressed RB and RB2/P130 genes and analyzed their role in regulating the cell cycle, apoptosis and differentiation. In uncommitted MSCs, the activity of RB and RB2/P130 appeared limited to negatively regulating cell cycle progression, having no role in apoptosis and differentiation (toward either mesenchymal or neural lineages). On the other hand, in MSCs committed toward the neural phenotype, both RB and RB2/P130 reduced cell proliferation rate and affected the apoptotic process. RB protected differentiating cells from programmed cell death. On the contrary, RB2/P130 increased the percentage of cells in apoptosis. All of these activities were accomplished mainly in an HDAC-independent way. The retinoblastoma genes also influenced differentiation in neural committed MSCs. RB2/P130 contributes mainly to the induction of generic neural properties, while RB triggers cholinergic differentiation. These differentiating activities are HDAC-dependent. Our research shows that there is a critical temporal requirement for the RB genes during neuronal differentiation of MSCs: they are not required for cell commitment but play a role in the maturation process. For the above reasons, RB and RB2/P130 may have a role in neural differentiation but not in neural determination.