MicroRNAs (miRNAs) are short 20–22 nucleotide RNA molecules that act as negative regulators of gene expression via translational repression: they have been shown to play a role in development, proliferation, stress response, and apoptosis. The transcriptional regulator LRF (Leukemia/lymphoma Related Factor) has been shown to prevent p19ARF transcription and consequently to inhibit senescence in mouse embryonic fibroblasts (MEF). Here we report, for the first time, that LRF is post-transcriptionally regulated by miR-20a. Using a gene reporter assay, direct interaction of miR-20a with the LRF 3′UTR is demonstrated. To validate the interaction miR-20a/3′UTR LRF miR-20a was over-expressed, either by transient transfection or retroviral infection, in wild type mouse embryo fibroblasts and in LRF-null MEF derived from LRF knock-out mice. We observed LRF decrease, p19ARF increase, inhibition of cell proliferation and induction of senescence. The comparison of miR-20a activity in wt and LRF-null MEF indicates that LRF is the main mediator of the miR-20a-induced senescence and that other targets are cooperating. As LRF down-regulation/p19ARF induction is always accompanied by E2F1 down-regulation and increase of p16, we propose that all these events act in synergy to accomplish miR-20a-induced senescence in MEF. Senescence has been recently revaluated as a tumor suppressor mechanism, alternative to apoptosis; from this point of view the discovery of new physiological “senescence inducer” appears to be promising as this molecule could be used as anticancer drug.

Leukemia/lymphoma-related factor (LRF) is a transcriptional repressor, which by recruiting histone deacetylases specifically represses p19/ARF expression, thus behaving as an oncogene. Conversely, in mouse embryonic fibroblasts (MEF), LRF inhibition causes aberrant p19ARF up-regulation resulting in proliferative defects and premature senescence. We have recently shown that LRF is controlled by microRNAs. Here we show that LRF acts on MEF proliferation and senescence/apoptosis by repressing miR-28 and miR-505, revealing a regulatory circuit where microRNAs (miRNAs) work both upstream and downstream of LRF. By analyzing miRNA expression profiles of MEF transfected with LRF-specific short interfering RNAs, we found that miR-28 and miR-505 are modulated by LRF. Both miRNAs are predicted to target alternative splicing factor/splicing factor 2 (ASF/SF2), a serine/arginine protein essential for cell viability. In vertebrates, loss or inactivation of ASF/SF2 may result in genomic instability and induce G 2 cell cycle arrest and apoptosis. We showed that miR-28 and miR-505 modulate ASF/SF2 by directly binding ASF/ SF2 3-UTR. Decrease in LRF causes a decrease in ASF/SF2, which depends on up-regulation of miR-28 and miR-505. Alteration of each of the members of the LRF/miR-28/miR-505/ASF/ SF2 axis affects MEF proliferation and the number of senescent and apoptotic cells. Consistently, the axis is coordinately modulated as cell senescence increases with passages in MEF culture. In conclusion, we show that LRF-dependent miRNAs miR-28 and miR-505 control MEF proliferation and survival by targeting ASF/SF2 and suggest a central role of LRF-related miRNAs, in addition to the role of LRF-dependent p53 control, in cellular homeostasis. Leukemia/lymphoma-related factor (LRF)2 is a BTB/POZ domain Krüppel-like zinc finger transcription factor belonging to the POK protein family. POK proteins exert transcriptional repressive activity by recruiting histone deacetylases and subsequent remodeling of chromatin (1, 2). LRF can act as both transcriptional repressor and activator depending on the promoter context (3-6). LRF is known to have pleiotropic functions during development and differentiation, and it has recently been shown to act as an oncogene (7). Transgenic mouse cell lines overexpressing LRF developed aggressive tumors, and aberrant LRF overexpression was observed in a high percentage of human lymphomas. Conversely, LRF loss caused proliferative defects, premature senescence, and unresponsiveness to oncogenic stimuli. The main mode of action of LRF seems to be through the direct binding to p19ARF, a major tumor suppressor. Alterations of LRF therefore affect the p19ARF/MDM2/p53 pathway (7,8). However, LRF is also overexpressed in solid tumors, such as colon and bladder cancer, which have frequently lost the p14ARF/p53 pathway (8), and a positive relationship between LRF and p14ARF has been observed in several lung cancers (9). These data suggest possible additional target genes through which LRF might exert its activity.We have...

Thyroid hormone (T3) dyshomeostasis in the cardiac ischemia-reperfusion (IR) setting negatively impacts on mitochondria function and extracellular matrix remodeling. The modulation of cardiac miRNAs may represent the underlying molecular mechanisms, but a systems biology perspective investigating this critical issue in depth is still lacking. A rat model of myocardial IR, with or without an early short-term T3-replacement, was used to predict putative T3-dependent miRNA-gene interactions targeted to mitochondria quality control and wound healing repair. As evidenced by mRNA and miRNA expression profiling, the T3 supplementation reverted the expression of 87 genes and 11 miRNAs that were dysregulated in the untreated group. In silico crossing and functional analysis of the T3-associated differentially expressed transcripts, identified a signature of interconnected miRNA-gene regulatory circuits that confer resistance to noxious cascades of acute stress. In this network the T3-down-regulated Tp53, Jun and Sp1 transcription factors emerge as critical nodes linking intrinsic cell death and oxidative stress pathways to adverse remodeling cascades. The data presented here provide a novel insight into the molecular basis of T3 cardioprotection in the early post-IR phase and highlight the contribution of a previously unappreciated complex T3-regulatory network that may be helpful in translating T3 replacement into clinical practice.

-290 acts as a physiological effector of senescence in mouse embryo fibroblasts. Physiol Genomics 39: 210 -218, 2009. First published September 1, 2009 doi:10.1152/physiolgenomics.00085.2009.-The culture-induced senescence of mouse embryo fibroblasts (MEF) correlates with reduction of cell proliferation. In this work we found that the accumulation of cells with 4C DNA content and the transcriptional change of several microRNAs (miRNAs or miRs) are relevant events in culture senescence. By comparing the miRNA expression profiles of physiologically senescent MEF and that of senescent MEF induced by the downregulation of leukemia-related factor, we identified miR-290 as a common upregulated miRNA. When miR-290 was transfected in presenescent MEF, SA-␤-gal ϩ cells and p16, two markers of culture senescence, increased compared with control, indicating that miR-290 is causally involved in senescence. Interestingly, nocodazole (NCZ), which induces G2/M block, increased the percentage of senescent cells as well as the expression of miR-290 and of the tumor suppressor p16, thus mimicking culture senescence. As miR-290 was overexpressed in NCZ-treated cells and it was able to induce senescence in proliferating MEF, we investigated whether miR-290 and NCZ could share common mechanisms of culture senescence. Whereas the induction of SA-␤-gal ϩ by miR-290 was not strengthened by coupling its transfection with NCZ treatment, the transfection of the antagomir 290 (d-290) plus NCZ treatment, while blocking cells at G2/M, suppressed SA-␤-gal ϩ and p16 induction. On the basis of these findings we conclude that miR-290 might act as a physiological effector of NCZ induced as well as culture senescence via p16 regulation expanding the role of this miRNA from embryonic stem to differentiated cells. microRNAs; nocodazole; cell cycle CELLULAR SENESCENCE REPRESENTS a stress response whereby cells undergo irreversible cell cycle arrest (18). In mouse embryo fibroblasts (MEF) senescence can be induced by cellular stresses such as the persistent mitogenic stimulation during propagation in culture (10), the overexpression (32) or downregulation (20) of single oncogenes, or DNA-damaging drugs (37). Most senescence-dependent cellular dysfunctions are the result of a complex interaction between gene and environment over a cell life span. Genes identified as associated with cellular senescence can be grouped into families regulating redox/homeostasis (7, 34), DNA repair (19), apoptosis (10), and growth factor signaling (6). Deciphering how these genes interact to control senescence might help to elucidate the signaling networks at the basis of senescence.MicroRNAs (miRNAs or miRs), a class of endogenous 22-25 nt single-stranded RNA molecules that bind to the 3Ј-untranslated region of multiple messenger RNAs and inhibit translation (4, 5), represent a network of factors that, together with transcription factors, exerts a spatial and temporal control of gene expression with the final effect depending on the sum of the affected genes and on the g...