MiR-23-TrxR1 as a novel molecular axis in skeletal muscle differentiation

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing protein involved in cellular redox homeostasis which is downregulated in skeletal muscle differentiation. Here we show that TrxR1 decrease occurring during myogenesis is functionally involved in the coordination of this cellular process. Indeed, TrxR1 depletion reduces myoblasts growth by inducing an early myogenesis -related gene expression pattern which includes myogenin and Myf5 up-regulation and Cyclin D1 decrease. On the contrary, the overexpression of TrxR1 during differentiation delays myogenic process, by negatively affecting the expression of Myogenin and MyHC. Moreover, we found that miR-23a and miR-23b - whose expression was increased in the early stage of C2C12 differentiation - are involved in the regulation of TrxR1 expression through their direct binding to the 3′ UTR of TrxR1 mRNA. Interestingly, the forced inhibition of miR-23a and miR-23b during C2C12 differentiation partially rescues TrxR1 levels and delays the expression of myogenic markers, suggesting the involvement of miR-23 in myogenesis via TrxR1 repression. Taken together, our results depict for the first time a novel molecular axis, which functionally acts in skeletal muscle differentiation through the modulation of TrxR1 by miR-23

miR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27(Kip1). We recognize two target sites for the microRNAs in the 3' untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G(1) to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27(Kip1) in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) down-regulation

An animal and cellular study on αB-crystallin activation in cardiac muscle by acute exercise

Alpha-B-Crystallin (CRYAB), a Small Heat Shock Protein sensitive to oxidative stress, is expressed in many tissues and implicated in various biological processes. In cardiac muscle, CRYAB exerts a cardio protective role in ischemia-induced damage preventing apoptosis and necrosis. In the present study we used forty young (7-weeks old) healthy male mice (BALB/c AnNHsd), which after 1 week of acclimatization to the new housing environment, runned 2 days per 10 minutes. The TR mice ran for 60 min at a speed of 5.5 m/min. Mice were sacrificed immediately after, 15 and 120 minutes after the end of the acute bout of endurance exercise (TR-0’, TR-15’ and TR-120’, respectively). We prepared samples from the heart and from the group of posterior muscles study αB-crystallin’ response at different time of recovery from an acute aerobic exercise (1 hour), correlating its modulation with oxidative stress level. We found that a single bout exercise lead to a specific short-term increase of phospho-αB-crystallin level (pCRYAB), without changes of its total expression. Further, the level of 4-hydroxynonenal, a marker of lipidic peroxidation, has shown a similar trend of pCRYAB enhancement. This may indicate that CRYAB in cardiac muscle is activated and it has a putative role in oxidative stress during exercise. These results are supported by our previous data obtained in mouse skeletal tissues (i.e. gastrocnemius, soleus) and in H₂O₂-treated C2C12 myotubes. In particular we observed not only a fiber-dependent response of pCRYAB, but also its translocation into myofibrillar compartment. Experiments are in progress to further investigate on CRYAB role during exercise and its interactions with cytoskeletal structures

Oxaliplatin, 5-fluorouracil/leucovorin and epirubicin as first-line treatment in advanced gastric carcinoma: a phase II study

The association between oxaliplatin and 5-fluorouracil (5-FU) has been extensively reported to improve prognosis of gastric cancer patients. The present study is aimed at evaluating response rate and the toxicity profile of the association with oxaliplatin, 5-FU/lecovorin and epirubicin in gastric cancer patients with locally advanced or metastatic disease. Thirty-six patients have been enrolled and 35 evaluated. The treatment schedule was oxaliplatin (100 mg m−2), 5-FU (400 mg m−2), leucovorin (40 mg m−2) and epirubicin (60 mg m−2) intravenously. administered every 3 weeks for 6 months, for a total of 185 therapy cycles . Response rate and toxicity were assessed according to the international WHO criteria. Every patient received a mean of 5.3 therapy cycles in a day-hospital setting. Sixteen of 35 patients (46%) showed an objective response, two complete response and 14 partial response. Median time to progression was 33 weeks with an overall median survival of 49 weeks. During the study, anaemia grade 3 and neutropenia grade 3 were observed in 9 and 11% of patients respectively. A grade 3 periferic sensorial neuropathy was observed in 6% of patients. No life threatening or cardiac toxicity was recorded. The regimen used showed anticancer activity against gastric carcinoma, a tolerable toxicity profile and excellent patient compliance

Ruolo di miR-221 e miR-222 in modelli cellulari di adenocarcinoma prostatico

I microRNA sono una abbondante classe di piccoli RNA non codificanti che funzionano come inibitori post-trascrizionali appaiandosi a RNA messaggeri specifici e inducendone la degradazione o impedendone la traduzione nel corrispondente prodotto proteico. Negli ultimi anni un numero crescente di evidenze sperimentali ha correlato la modulazione specifica di diversi microRNA a varie forme tumorali umane, suggerendo che queste molecole possano rappresentare una nuova classe di oncogeni ed oncosppressori. In questo lavoro abbiamo osservato che i microRNA 221 e 222 mostrano un’espressione differenziale nei modelli cellulari di carcinoma prostatico umano da noi analizzati; sono infatti abbondantemente espressi nella linea altamente aggressiva PC3, mentre al contrario sembrano essere assenti o scarsamente espressi in linee molto meno tumorigeniche come le LNCaP e le 22Rv1. Tramite predizione bioinformatica abbiamo identificato come bersaglio molecolare di questa coppia di microRNA un noto regolatore del ciclo cellulare, l’oncosoppressore p27Kip1, e successivamente abbiamo dimostrato una correlazione inversa fra l’espressione di miR-221 e 222 ed i livelli proteici di p27. L’espressione ectopica di uno o entrambi i microRNA in cellule LNCaP induce una sensibile riduzione (50%) della quantità proteica di p27, mentre la loro inibizione, induce l’effetto opposto nelle PC3 incrementando abbondantemente i livelli quantitativi della proteina. Con ulteriori analisi abbiamo confermato che questa relazione è mediata in maniera specifica dall’ interazione diretta fra i microRNA e due siti specifici nella regione 3’UTR dell’ mRNA di p27. Abbiamo inoltre fornito evidenze funzionali riguardo al ruolo di questi due microRNA nello sviluppo tumorale, dimostrando che cellule LNCaP overesprimenti i miR-221 e 222 mostrano un significativo aumento della popolazione nella fase S del ciclo cellulare (+ 12-14% rispetto ai campioni di controllo), in accordo col ruolo di p27 come regolatore specifico della transizione G1-S. Questa caratteristica è accompagnata da un sensibile incremento del tasso di crescita, del potenziale clonogenico in terreno di coltura semisolido (28,6 ± 8 colonie per il controllo contro 119,6 ± 9 per miR-221 e 167 ± 20 per miR-222), e della capacità di formare tumori quando iniettate sotto cute in topi SCID CB-17 (107±58,2 di incremento medio di crescita tumorale per miR-221 in confronto a 4±2 per il controllo). In conclusione i nostri risultati mostrano che la sovraespressione di miR-221 e miR-222 contribuisce alla proliferazione e tumorigenicità in vitro ed in vivo di modelli cellulari umani di carcinoma prostatico suggerendo per questi due microRNA un ruolo come possibili prognostici. Lo sviluppo futuro di questo lavoro prevede l’ideazione di nuove strategie terapeutiche volte ad inibire in vivo la crescita e progressione di questo specifico tipo di neoplasia.MicroRNAs are short regulatory RNAs exerting their action by negatively modulating mRNA translation or by inducing degradation of their target mRNAs. Recently, an ever growing amount of evidence has been linking the specific modulation of microRNAs to several cancers, suggesting that they may play a role as a novel class of oncogenes or tumor suppressor genes. In our work we show that miR-221 and miR-222 are highly expressed in PC3, a human strongly aggressive prostate carcinoma cell line, whereas they are downregulated in the poorly tumorigenic prostate cancer cell lines LNCaP and 22Rv1. By bioinformatic prediction we have identified the cell cycle inhibitor p27kip1 as a molecular target of this couple of microRNAs, and we have demonstrated an inverse relationship between the miR-221/222 expression and p27 protein levels. Moreover, we have shown that the ectopic overexpression of these miRNAs in LNCaP induces a reduction of p27 levels (50%), whereas their knock-down in PC3 is able to induce the opposite effect, strongly increasing p27 expression. This relationship is mediated by direct interaction between these microRNAs and two specific p27 3’UTR region sites. We have also verified that the forced expression of miR-221 and miR-222 strongly affects the LNCaP growth potential by shifting the cells from the G1 to S phase of cell cycle (+ 12-14% vs. control cells), in agreement with p27 role as a regulator of G1/S transition. This property is accompanied by a powerful enhancement of LNCaP clonogenic potential in soft agar (28,6 ± 8 colonies for control vs.119, 6 ± 9 for miR-221 and 167 ± 20 for miR-222) and tumor formation capacity after injection in CB-17 SCID mice (107±58,2 of fold increase for miR-221 with respect to 4±2 for control). In conclusion, our findings show that miR-221 and miR-222 overexpression may contribute to the growth and progression of prostate carcinoma cell lines, suggesting that their expression pattern could be exploited as a novel prognostic method. Furthermore, the future progress of our work might provide new clues to develop innovative therapies, targeting specific tumor markers of prostate carcinoma, such as the overexpressed microRNAs

Emerging Contribution of PancRNAs in Cancer

“Cancer” includes a heterogeneous group of diseases characterized by abnormal growth beyond natural boundaries. Neoplastic transformation of cells is orchestrated by multiple molecular players, including oncogenic transcription factors, epigenetic modifiers, RNA binding proteins, and coding and noncoding transcripts. The use of computational methods for global and quantitative analysis of RNA processing regulation provides new insights into the genomic and epigenomic features of the cancer transcriptome. In particular, noncoding RNAs are emerging as key molecular players in oncogenesis. Among them, the promoter-associated noncoding RNAs (pancRNAs) are noncoding transcripts acting in cis to regulate their host genes, including tumor suppressors and oncogenes. In this review, we will illustrate the role played by pancRNAs in cancer biology and will discuss the latest findings that connect pancRNAs with cancer risk and progression. The molecular mechanisms involved in the function of pancRNAs may open the path to novel therapeutic opportunities, thus expanding the repertoire of targets to be tested as anticancer agents in the near future

The early response of αB-crystallin to a single bout of aerobic exercise in mouse skeletal muscles depends upon fiber oxidative features

Besides its substantial role in eye lens, αB-crystallin (HSPB5) retains fundamental function in striated muscle during physiological or pathological modifications. In this study, we aimed to analyse the cellular and molecular factors driving the functional response of HSPB5 protein in different muscles from mice subjected to an acute bout of non-damaging endurance exercise or in C2C12 myocytes upon exposure to pro-oxidant environment, chosen as “in vivo” and “in vitro” models of a physiological stressing conditions, respectively. To this end, red (GR) and white gastrocnemius (GW), as sources of slow-oxidative and fast-glycolytic/oxidative fibers, as well as the soleus (SOL), mainly composed of slow-oxidative type fibers, were obtained from BALB/c mice, before (CTRL) and at different times (0′, 15′, 30′ 120′) following 1-h of running. Although the total level of HSPB5 protein was not affected by exercise, we found a significantly increase of phosphorylated HSPB5 (p- HSPB5) only in GR and SOL skeletal muscle with a higher amount of type I and IIA/X myofibers. The fiberspecific activation of HSPB5 was correlated to its interaction with the actin filaments, as well as to an increased level of lipid peroxidation and carbonylated proteins. The role of the pro-oxidant environment in HSPB5 response was investigated in terminally differentiated C2C12 myotubes, where most of HSPB5/pHSPB5 pool was present in the cytosolic compartment in standard culture conditions. As a result of exposure to pro-oxidizing, but not cytotoxic, H2O2 concentration, the p-38MAPK-mediated phosphorylation of HSPB5 resulted functional to promote its interaction with the myofibrillar components, such as β-actin, desmin and filamin 1. This study provides novel information on the molecular pathway underlying the HSPB5 physiological function in skeletal muscle, confirming the contribution of the pro-oxidant environment in HSPB5 activation and interaction with substrate/client myofibrillar proteins, offering new insights for the study of myofibrillar myopathies and cardiomyopathies

Physical activity in the prevention of human diseases: role of epigenetic modifications

Abstract Epigenetic modification refers to heritable changes in gene function that cannot be explained by alterations in the DNA sequence. The current literature clearly demonstrates that the epigenetic response is highly dynamic and influenced by different biological and environmental factors such as aging, nutrient availability and physical exercise. As such, it is well accepted that physical activity and exercise can modulate gene expression through epigenetic alternations although the type and duration of exercise eliciting specific epigenetic effects that can result in health benefits and prevent chronic diseases remains to be determined. This review highlights the most significant findings from epigenetic studies involving physical activity/exercise interventions known to benefit chronic diseases such as metabolic syndrome, diabetes, cancer, cardiovascular and neurodegenerative diseases