Search

Background: The ability to increase their degree of pigmentation is an adaptive response that confers pigmentable melanoma cells higher resistance to BRAF inhibitors (BRAFi) compared to non-pigmentable melanoma cells. Methods: Here, we compared the miRNome and the transcriptome profile of pigmentable 501Mel and SK-Mel-5 melanoma cells vs. non-pigmentable A375 melanoma cells, following treatment with the BRAFi vemurafenib (vem). In depth bioinformatic analyses (clusterProfiler, WGCNA and SWIMmeR) allowed us to identify the miRNAs, mRNAs and biological processes (BPs) that specifically characterize the response of pigmentable melanoma cells to the drug. Such BPs were studied using appropriate assays in vitro and in vivo (xenograft in zebrafish embryos). Results: Upon vem treatment, miR-192-5p, miR-211-5p, miR-374a-5p, miR-486-5p, miR-582-5p, miR-1260a and miR-7977, as well as GPR143, OCA2, RAB27A, RAB32 and TYRP1 mRNAs, are differentially expressed only in pigmentable cells. These miRNAs and mRNAs belong to BPs related to pigmentation, specifically melanosome maturation and trafficking. In fact, an increase in the number of intracellular melanosomes—due to increased maturation and/or trafficking—confers resistance to vem. Conclusion: We demonstrated that the ability of pigmentable cells to increase the number of intracellular melanosomes fully accounts for their higher resistance to vem compared to non-pigmentable cells. In addition, we identified a network of miRNAs and mRNAs that are involved in melanosome maturation and/or trafficking. Finally, we provide the rationale for testing BRAFi in combination with inhibitors of these biological processes, so that pigmentable melanoma cells can be turned into more sensitive non-pigmentable cells.

Microglial cells are a component of the innate immune system in the brain that support cell-to-cell communication via secreted molecules and extracellular vesicles (EVs). EVs can be divided into two major populations: large (LEVs) and small (SEVs) EVs, carrying different mediators, such as proteins, lipids, and miRNAs. The microglia EVs cargo crucially reflects the status of parental cells and can lead to both beneficial and detrimental effects in many physiopathological states. Herein, a workflow for the extraction and characterization of SEVs and LEVs from human C20 and HMC3 microglia cell lines derived, respectively, from adult and embryonic microglia is reported. EVs were gathered from the culture media of the two cell lines by sequential ultracentrifugation steps and their biochemical and biophysical properties were analyzed by Western blot, transmission electron microscopy, and dynamic light scattering. Although the C20- and HMC3-derived EVs shared several common features, C20-derived EVs were slightly lower in number and more polydispersed. Interestingly, C20- but not HMC3-SEVs were able to interfere with the proliferation of U87 glioblastoma cells. This correlated with the different relative levels of eight miRNAs involved in neuroinflammation and tumor progression in the C20- and HMC3-derived EVs, which in turn reflected a different basal activation state of the two cell types. Our data fill a gap in the community of microglia EVs, in which the preparations from human cells have been poorly characterized so far. Furthermore, these results shed light on both the differences and similarities of EVs extracted from different human microglia cell models, underlining the need to better characterize the features and biological effects of EVs for therein useful and correct application.

MiRNAs represent a mechanism that regulates gene expression in many pathological conditions. Exosomes are known to be secreted from all types of cells, and the exosomes-released molecules are crucial messengers that can regulate cellular processes. We investigated the miRNAs content of exosomes released by cancer cells during the invasion . An invasion stimulus has been generated through scratches created on the confluent cells of cancer cell lines: glioblastoma, breast and prostate cancers. Several miRNAs were found to be significantly differentially abundant during the cell invasion , both in common among different cell lines and exclusive. Understanding the language codes among cells involved in invasion can lead to the development of therapies that can inhibit cellular communication, slowing or eventually stopping their activity.

Melatonin (MEL) is a pleiotropic neurohormone of increasing interest as a neuroprotective agent in ocular diseases. Improving the mucoadhesiveness is a proposed strategy to increase the bioavailability of topical formulations. Herein, the design and optimization of MEL-loaded lipid-polymer hybrid nanoparticles (mel-LPHNs) using Design of Experiment (DoE) was performed. LPHNs consisted of PLGA-PEG polymer nanoparticles coated with a cationic lipid-shell. The optimized nanomedicine showed suitable size for ophthalmic administration (189.4 nm; PDI 0.260) with a positive surface charge (+39.8 mV), high encapsulation efficiency (79.8 %), suitable pH and osmolarity values, good mucoadhesive properties and a controlled release profile. Differential Scanning Calorimetry and Fourier-Transform Infrared Spectroscopy confirmed the encapsulation of melatonin in the systems and the interaction between lipids and polymer matrix. Biological evaluation in an in vitro model of diabetic retinopathy demonstrated enhanced neuroprotective and antioxidant activities of mel-LPHNs, compared to melatonin aqueous solution at the same concentration (0.1 and 1 μM). A modified Draize test was performed to assess the ocular tolerability of the formulation showing no signs of irritation. To the best our knowledge, this study reported for the first time the development of mel-LPHNs, a novel and safe hybrid platform suitable for the topical management of retinal diseases.

Circulating miRNAs (c-miRNAs) are promising biomarkers for HF diagnosis and prognosis. There are no studies on HF pediatric patients undergoing VAD-implantation. Aims of this study were: to examine the c-miRNAs profile in HF children; to evaluate the effects of VAD on c-miRNAs levels; to in vitro validate putative c-miRNA targets. c-miRNA profile was determined in serum of HF children by NGS before and one month after VAD-implant. The c-miRNA differentially expressed were analyzed by real time-PCR, before and at 4 hrs,1,3,7,14,30 days after VAD-implant. A miRNA mimic transfection study in HepG2 cells was performed to validate putative miRNA targets selected through miRWalk database. Thirteen c-miRNAs were modified at 30 days after VAD-implant compared to pre-VAD at NSG, and, among them, six c-miRNAs were confirmed by Real-TimePCR. Putative targets of the validated c-miRNAs are involved in the hemostatic process. The in vitro study confirmed a down-regulatory effect of hsa-miR-409-3p towards coagulation factor 7 (F7) and F2. Of note, all patients had thrombotic events requiring pump change. In conclusion, in HF children, the level of six c-miRNAs involved in the regulation of hemostatic events changed after 30 days of VAD-treatment. In particular, the lowering of c-miR-409-3p regulating both F7 and F2 could reflect a pro-thrombotic state after VAD-implant.

Ventricular Assist Device (VAD) therapy is considered as a part of standard care for end-stage Heart Failure (HF) children unresponsive to medical management, but the potential role of miRNAs in response to VAD therapy on molecular pathways underlying LV remodeling and cardiac function in HF is unknown. The aims of this study were to evaluate the effects of VAD on miRNA expression profile in cardiac tissue obtained from HF children, to determine the putative miRNA targets by an in-silico analysis as well as to verify the changes of predicated miRNA target in the same cardiac samples. The regulatory role of selected miRNAs on predicted targets was evaluated by a dedicated in vitro study. miRNA profile was determined in cardiac samples obtained from 13 HF children [median: 29 months, 19 LVEF%, 9 Kg] by NGS before VAD implant (pre-VAD) and at the moment of heart transplant (Post-VAD). Only hsa-miR-199b-5p, hsa-miR-19a-3p, hsa-miR-1246 were differentially expressed at post-VAD when compared to pre-VAD, and validated by real-time PCR. Putative targets of the selected miRNAs were involved in regulation of sarcomere genes, such as cardiac troponin (cTns) complex. The expression levels of fetal ad adult isoforms of cTns resulted significantly higher after VAD in cardiac tissue of HF pediatric patients when compared with HF adults. An in vitro study confirmed a down-regulatory effect of hsa-miR-19a-3p on cTnC expression. The effect of VAD on sarcomere organization through cTn isoform expression may be epigenetically regulated, suggesting for miRNAs a potential role as therapeutic targets to improve heart function in HF pediatric patients.

Background VAD use in heart failure (HF) children have undergone rapid progress in the last three decades through pump technological innovation and improvement of perioperative care. Studies in HF adults showed that VAD put native heart at rest and lead to molecular changes in cardiac muscle, including at microRNA (miRNA) level. However, little is known on changes induced by VAD implant in cardiac miRNA expression and their putative targets in HF children. Purpose The aims of this study were to evaluate: 1) modification of miRNA expression in cardiac muscle from HF children after VAD support; 2) the putative targets of selected miRNAs by in silico analysis; 2) the role of the identify miRNAs on putative targets by in vitro study. Methods Cardiac biopsies were collected from HF children at the moment of VAD implant [n=8; 20 (7.5–64.5) months, 2 males; 19 (15.75–32.25) LVEF%] and at the time of heart transplant after VAD support [n=5; 32 (5–204) months; 4 males; 13.5 (10–18) LVEF%]. Cardiac miRNA expression was evaluated by NGS. The potential miRNA targets were identified by bioinformatics analyses and their cardiac expression by real-time PCR was evaluated. HL-1 cell line was used for testing the regulatory role of selected miRNA on predicted targets by miRNA mimic transfection study. Results At NGS, 465 miRNA were found on average in each sample and the cardiac expression levels of miR19a-3p, miR-1246 and miR-199b-5p decreased in HF children after VAD support compared to pre-implant (Fig. 1A-B). In silico analysis showed that more than 5000 potential gene targets regulated by miR-19a-3p, miR-1246 and miR-199b-5p. Among them, adiponectin receptors (AdipoR1, AdipoR2, T-CAD) were identified as common targets for 3 miRNAs. Real-time PCR data showed that levels of all adiponectin receptors increased significantly whilst the expression of 3 miRNAs decreased after VAD support (Fig. 1C). Moreover, AdipoR2 and T-CAD were inversely related to miRNA levels (Fig. 1D). In vitro studies confirmed the regulatory role of miR-1246 and miR-199b-5p on AdipoR2 (Fig. 1E-F), whilst only miR-199b-5p reduced the expression of T-CAD (Fig. 1G). Finally, AdipoR1 expression levels are not modified compared to control by miRNAs mimic transfection (data not shown). Conclusion In HF children the use of VAD could modify the expression of several miRNAs potentially involved in the regulation of several pathophysiological mechanisms underlying HF. Specifically, the reductions of miR-1246, mir-19a-3p, miR-199b-5p were associated with an increase of the adiponectin receptors AdipoR2 and T-CAD mRNA, suggesting the existence of a miRNAs related fine tuning of the adiponectin system at cardiac tissue level by VAD implant, able to favour the protective effect of adiponectin in HF cardiac muscle. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): FP7-ICT-2009 Project, Grant Agreement 24863 Figure 1

Here we present miR-CATCHv2.0, an implemented experimental method that allows the identification of the microRNA species directly bound to an RNA of interest. After cross-linking of microRNA::RNA::Ago2 complexes using formaldehyde, the RNA is fragmented using sonication and then subjected to affinity purification using two sets of biotinylated tiling probes (ODD and EVEN). Finally, enriched microRNA species are retrieved by means of small RNA sequencing coupled with an ad hoc analytical workflow. In BRAFV600E mutant A375 melanoma cells, miR-CATCHv2.0 allowed us to identify 20 microRNAs that target X1, the most abundant isoform of BRAF mRNA. These microRNAs fall into different functional classes, according to the effect that they exert (decrease/increase in BRAFV600E mRNA and protein levels) and to the mechanism they use to achieve it (destabilization/stabilization of X1 mRNA or decrease/increase in its translation). microRNA-induced variations in BRAFV600E protein levels are most of the times coupled to consistent variations in pMEK levels, in melanoma cell proliferation in vitro and in sensitivity to the BRAF inhibitor vemurafenib in a xenograft model in zebrafish. However, microRNAs exist that uncouple the degree of activation of the ERK pathway from the levels of BRAFV600E protein. Our study proposes miR-CATCHv2.0 as an effective tool for the identification of direct microRNA-target interactions and, by using such a tool, unveils the complexity of the post-transcriptional regulation to which BRAFV600E and the ERK pathway are subjected in melanoma cells.

MicroRNAs (miRNAs) can regulate the expression of potentially every transcript in the cell, and the definition of miRNA-target interactions is crucial to understand their role in all biological processes. However, the identification of the miRNAs that target a specific mRNA remains a challenge. Here, we describe an innovative method called miR-CATCHv2.0 for the high-throughput identification of the miRNA species bound to an RNA of interest. We also describe how this method can overcome the limitations of the current computational and experimental methods available in this field.

Summary Cerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different layers. The mechanisms controlling their expression in distinct cells are only partially known. We investigated the expression and stability of Tbr1, Bcl11b, Fezf2, Satb2, and Cux1 mRNAs in single developing mouse cortical cells. We observe that Satb2 mRNA appears much earlier than its protein and in a set of cells broader than expected, suggesting an initial inhibition of its translation, subsequently released during development. Mechanistically, Satb2 3′UTR modulates protein translation of GFP reporters during mouse corticogenesis. We select miR-541, a eutherian-specific miRNA, and miR-92a/b as the best candidates responsible for SATB2 inhibition, being strongly expressed in early and reduced in late progenitor cells. Their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate RNA interference as a major mechanism in timing cortical cell identities., Graphical abstract, Highlights • mRNAs for key transcription factors are differentially stable during corticogenesis • miRNAs show a dynamic profile of expression in a model of mouse corticogenesis • miR-541 and miR-92a/b bind Satb2 3′UTR and prevent SATB2 translation • Antagonizing mir-541 and miR-92a/b anticipates SATB2 protein production, In this article, Cremisi and colleagues show that post-transcriptional mechanisms are involved in controlling key functional aspects of SATB2-expressing cortical neurons. They show that mir-541, a eutherian-specific miRNA, delays SATB2 protein production in an in vitro model of cortical cell differentiation. These results may explain the heterochronic shift of SATB2 appearance in the eutherian compared with the metatherian cortex.

MicroRNAs (miRNAs) can regulate the expression of potentially every transcript in the cell, and the definition of miRNA-target interactions is crucial to understand their role in all biological processes. However, the identification of the miRNAs that target a specific mRNA remains a challenge. Here, we describe an innovative method called miR-CATCHv2.0 for the high-throughput identification of the miRNA species bound to an RNA of interest. We also describe how this method can overcome the limitations of the current computational and experimental methods available in this field.

The opening of the ATP-sensitive mitochondrial potassium channel (mitok-ATP) is a common goal of cardioprotective strategies in the setting of acute and chronic myocardial disease. The biologically active thyroid hormone (TH), 3-5-3-triiodothyronine (T3), has been indicated as a potential activator of mitoK-ATP but the underlying mechanisms are still elusive. Here we describe a novel role of T3 in the transcriptional regulation of mitoK and mitoSur, the recently identified molecular constituents of the channel. To mimic human ischemic heart damage, we used a rat model of a low T3 state as the outcome of a myocardial ischemia/reperfusion event, and neonatal rat cardiomyocytes (NRCM) challenged with hypoxia or H2O2. Either in the in vivo or in vitro models, T3 administration to recover the physiological concentrations was able to restore the expression level of both the channel subunits, which were found to be downregulated under the stress conditions. Furthermore, the T3-mediated transcriptional activation of mitoK-ATP in the myocardium and NRCM was associated with the repression of the TH-inactivating enzyme, deiodinase 3 (Dio3), and an up-regulation of the T3-responsive miR-133a-3p. Mechanistically, the loss and gain of function experiments and reporter gene assays performed in NRCM, have revealed a new regulatory axis whereby the silencing of Dio3 under the control of miR-133a-3p drives the T3-dependent modulation of cardiac mitoK and mitoSur transcription.

In clinical practice, inflammatory pain is an important, unresolved health problem, despite the utilization of non-steroidal anti-inflammatory drugs (NSAIDs). In the last decade, different studies have proven that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in the development and maintenance of inflammatory pain and hyperalgesia via the post-translation modification of key proteins, such as manganese superoxide dismutase (MnSOD). It is well-known that inducible cyclooxygenase 2 (COX-2) plays a crucial role at the beginning of the inflammatory response by converting arachidonic acid into proinflammatory prostaglandin PGE2 and then producing other proinflammatory chemokines and cytokines. Here, we investigated the impact of oxidative stress on COX-2 and prostaglandin (PG) pathways in paw exudates, and we studied how this mechanism can be reversed by using antioxidants during hyperalgesia in a well-characterized model of inflammatory pain in rats. Our results reveal that during the inflammatory state, induced by intraplantar administration of carrageenan, the increase of PGE2 levels released in the paw exudates were associated with COX-2 nitration. Moreover, we showed that the inhibition of ROS with Mn (III) tetrakis (4-benzoic acid) porphyrin(MnTBAP) antioxidant prevented COX-2 nitration, restored the PGE2 levels, and blocked the development of thermal hyperalgesia.

One of the main problems with the treatment of metastatic prostate cancer is that, despite an initial positive response, the majority of patients develop resistance and progress. In particular, the resistance to docetaxel, the gold standard therapy for metastatic prostate cancer since 2010, represents one of the main factors responsible for the failure of prostate cancer therapy. According to the present knowledge, different processes contribute to the appearance of docetaxel resistance and non-coding RNA seems to play a relevant role in them. In this review, a comprehensive overview of the miRNA network involved in docetaxel resistance is described, highlighting the pathway/s affected by their activity.

The aim of the present work was the assessment of the environmental status of marine biota along the Sicilian ionic coast (Italy). Due to its complex habitat requirements, the fish fauna is a crucial indicator of the ecological integrity of aquatic systems at different scales, from micro-habitat to catchment. Besides environmental contaminants, biomarkers of oxidative stress answer to the Water Framework Directive’s challenges for improved detection of the impacts of chemical compounds on aquatic organisms. Highly consumed seafood species (Solea solea, Mullus barbatus, Sardina pilchardus and Scomber scombrus) were taken at the Gulf of Catania (Sicily). Levels of metals/metalloids (As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, V, Zn) were estimated in muscle tissue as well as oxidative stress parameter such as malondialdehyde (MDA) and protein carbonyl (PC) derivatives in liver. The total metals/metalloids load indicated as Metal Pollution Index (MPI, unitless) were the following: 0.258 in S. pilchardus, 0.203 in S. scombrus, 0.123 in M. barbatus and 0.071 in S. solea. The greatest contribution was given by As (mean range 2.670–9.748 mg/kg wet weight) and Zn (mean range 2.169–4.736 wet weight) levels. Hg, Ni and Zn presented mean levels progressively higher, with increasing trophic position. From a chemical point of view, the analyzed species were healthy for human consumption and the risk to develop chronic systemic and carcinogenic effects was low; nevertheless, it has highlighted an oxidative stress condition.

miR-28-5p is downregulated in some tumor tissues in which it has been demonstrated to have tumor suppressor (TS) activity. Here, we demonstrate that miR-28-5p acts as a TS in prostate cancer (PCa) cells affecting cell proliferation/survival, as well as migration and invasion. Using the miRNA pull out assay and next generation sequencing, we collected the complete repertoire of miR-28-5p targets, obtaining a data set (miR-28-5p targetome) of 191 mRNAs. Filtering the targetome with TargetScan 7, PITA and RNA22, we found that 61% of the transcripts had miR-28-5p binding sites. To assign a functional value to the captured transcripts, we grouped the miR-28-5p targets into gene families with annotated function and showed that six transcripts belong to the transcription factor category. Among them we selected SREBF2, a gene with an important role in PCa. We validated miR-28-5p/SREBF2 interaction, demonstrating that SREBF2 inhibition affects almost all the tumor processes altered by miR-28-5p re-expression, suggesting that SREBF2 is an important mediator of miR-28-5p TS activity. Our findings support the identification of the targetome of cancer-related miRNAs as a tool to discover genes and pathways fundamental for tumor development, and potential new targets for anti-tumor therapy.

Objectives The purpose of this study was the evaluation of the antioxidant activity of natural and semisynthetic polyphenol derivatives from Olea europea L., by assessing malondialdehyde (MDA), an important marker of oxidative stress. Methods Polyphenol as hydroxytyrosol, oleuropein, oleuropein aglycone as mix of four tautomeric forms and their respective acetyl-derivatives were obtained from olive leaves using semisynthetic protocols. These compounds were administered intraperitoneally to Wistar rats treated with paraquat, an herbicide which is able to cause oxidative stress after central administration. Malondialdehyde was derivatized with 2,4-dinitrophenylhydrazine to produce hydrazone that was purified by solid-phase extraction. Using high-performance liquid chromatography coupled with a diode array, free and total MDA was measured on homogenate rat brain as marker of lipid peroxidation. The analytical method was fully validated and showed linearity in the tested concentration range, with detection limit of 5 ng/ml. Recovery ranged from 94.1 to 105.8%. Key findings Both natural and semisynthetic polyphenol derivatives from a natural source as olive leaves were able to reduce MDA detection. The more lipophilic acetyl-derivatives showed an antioxidant activity greater than parent compounds. This potency seems to put in evidence a strict correlation between lipophilicity and bioavailability.

Background Ventricular Assist Device (VAD) has been increasingly used as bridge to transplantation for the treatment both of adult and pediatric patients with end-stage Heart Failure (HF). Several studies reported that VAD support could affect cardiac molecular mechanisms, including miRNA expression, in HF adult patients. However, little is known about the miRNA profile in pediatric HF patients supported by VAD. Purpose Aim of this study was to evaluate the effects of VAD support on expression profile of miRNAs in cardiac tissue from pediatric patients with HF, and to enrich the analysis by an in silico exploration of their potential functions and pathways. Methods Cardiac biopsies from HF children collected at the time of VAD implant [8 HF children; 57±33 (mean±SD) months; 2 males; 14.2±13.5 weight; 29±8 LVEF%] and at the time of heart transplantation after 155±33 days of VAD support [5 children; 90±46 months; 4 males; 30±15.8 weight; 38±3.9 LVEF%] were used for profiling miRNA expression by Next Generation Sequencing (NGS). Bioinformatic analyses were performed to identify the differentially expressed miRNAs in cardiac tissues after VAD support, to elucidate their potential functions (Gene Ontology), and to predict their target genes (miRWalk database). Results We identified two upregulated miRNAs (miR-29b-1-5p, miR-338-3p) and four downregulated miRNAs (miR-199a-5p, miR-199b-5p, miR-19a-3p, miR-1246) after VAD support. Gene enrichment analysis identified heart development/function, apoptosis and metabolism as main process modulated by the selected miRNAs (Fig A). Moreover, twenty genes were selected as putative miRNA targets involved in the pathophysiology of HF (Fig B). Conclusion In summary, the results of the present study suggest that modification of six miRNAs in cardiac tissue from HF children after VAD support may be involved in the regulation of several pathophysiological mechanisms underlying HF, thus providing novel perspectives for future researches.

Here we present miR-CATCHv2.0, an implemented experimental method that allows the identification of the microRNA species directly bound to an RNA of interest. After cross-linking of microRNA::RNA::Ago2 complexes using formaldehyde, the RNA is fragmented using sonication and then subjected to affinity purification using two sets of biotinylated tiling probes (ODD and EVEN). Finally, enriched microRNA species are retrieved by means of small RNA sequencing coupled with an ad hoc analytical workflow. In BRAFV600E mutant A375 melanoma cells, miR-CATCHv2.0 allowed us to identify 20 microRNAs that target X1, the most abundant isoform of BRAF mRNA. These microRNAs fall into different functional classes, according to the effect that they exert (decrease/increase in BRAFV600E mRNA and protein levels) and to the mechanism they use to achieve it (destabilization/stabilization of X1 mRNA or decrease/increase in its translation). microRNA-induced variations in BRAFV600E protein levels are most of the times coupled to consistent variations in pMEK levels, in melanoma cell proliferation in vitro and in sensitivity to the BRAF inhibitor vemurafenib in a xenograft model in zebrafish. However, microRNAs exist that uncouple the degree of activation of the ERK pathway from the levels of BRAFV600E protein. Our study proposes miR-CATCHv2.0 as an effective tool for the identification of direct microRNA-target interactions and, by using such a tool, unveils the complexity of the post-transcriptional regulation to which BRAFV600E and the ERK pathway are subjected in melanoma cells.

Several medicinal foods abound in traditional medicine with antioxidant potentials that could be of importance for the management of several diseases but with little or no scientific justification to substantiate their use. Thus, the objective of this study was the assessment of the antioxidant effect of two leave extracts of Solanum nigrum L. (SN), which is a medicinal plant member of the Solanaceae family, mainly used for soup preparation in different parts of the world. Then methanolic/water (80:20) (SN1) and water (SN2) leaves extracts were prepared. The total polyphenolic content and the concentration of phenolic acids and flavones compounds were determined. In order to verify whether examined extracts were able to restore the oxidative status, modified by glutamate in primary cultures of astrocytes, the study evaluated the glutathione levels, the intracellular oxidative stress, and the cytotoxicity of SN1 and SN2 extracts. Both extracts were able to quench the radical in an in vitro free cellular system and restore the oxidative status in in vitro primary cultures of rat astroglial cells exposed to glutamate. These extracts prevented the increase in glutamate uptake and inhibited glutamate excitotoxicity, which leads to cell damage and shows a notable antioxidant property.

In the last decade noncoding RNAs (ncRNAs) have been extensively studied in several biological processes and human diseases including cancer. microRNAs (miRNAs) are the best-known class of ncRNAs. miRNAs are small ncRNAs of around 20-22 nucleotides (nt) and are crucial posttranscriptional regulators of protein coding genes. Recently, new classes of ncRNAs, longer than miRNAs have been discovered. Those include intergenic noncoding RNAs (lincRNAs) and circular RNAs (circRNAs). These novel types of ncRNAs opened a very exciting field in biology, leading researchers to discover new relationships between miRNAs and long noncoding RNAs (lncRNAs), which act together to control protein coding gene expression. One of these new discoveries led to the formulation of the "competing endogenous RNA (ceRNA) hypothesis." This hypothesis suggests that an lncRNA acts as a sponge for miRNAs reducing their expression and causing the upregulation of miRNA targets. In this chapter we first discuss some recent discoveries in this field showing the mutual regulation of miRNAs, lncRNAs, and protein-coding genes in cancer. We then discuss the general approaches for the study of ceRNAs and present in more detail a recent computational approach to explore the ability of lncRNAs to act as ceRNAs in human breast cancer that has been shown to be, among the others, the most precise and promising.

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.

Congenital myotonic dystrophy type 1 (CDM1) is characterized by severe symptoms that affect patients from birth, with 40% mortality in the neonatal period and impaired skeletal muscle development. In this paper, we examined the relationship between autophagy and abnormal myogenic differentiation of CDM1 myoblasts. We investigated these pathological features at both ultrastructural and molecular levels, utilizing two CDM1 foetal myoblasts, CDM13 and CDM15, with 1800 and 3200 repeats, respectively. The congenital nature of these CDM1 myoblasts was confirmed by the high methylation level at the DMPK locus. Our results indicated that abnormal autophagy was independent of myogenic differentiation, as CDM13 myoblasts differentiated as well as control myoblasts but underwent autophagy like CDM15, displaying impaired differentiation. miRNA expression profiles revealed that CDM15 myoblasts failed to upregulate the complex network of myo-miRNAs under MYOD and MEF2A control, while this network was upregulated in CDM13 myoblasts. Interestingly, the abnormal differentiation of CDM15 myoblasts was associated with cellular stress accompanied by the induction of the interferon type 1 pathway (innate immune response). Indeed, inhibition of the interferon (IFN) type I pathway restores myogenic differentiation of CDM15 myoblasts, suggesting that the inappropriate activation of the innate immune response might contribute to impaired myogenic differentiation and severe muscle symptoms observed in some CDM1 patients. These findings open up the possibility of new therapeutic approaches to treat CDM1.

In the last decade noncoding RNAs (ncRNAs) have been extensively studied in several biological processes and human diseases including cancer. microRNAs (miRNAs) are the best-known class of ncRNAs. miRNAs are small ncRNAs of around 20-22 nucleotides (nt) and are crucial posttranscriptional regulators of protein coding genes. Recently, new classes of ncRNAs, longer than miRNAs have been discovered. Those include intergenic noncoding RNAs (lincRNAs) and circular RNAs (circRNAs). These novel types of ncRNAs opened a very exciting field in biology, leading researchers to discover new relationships between miRNAs and long noncoding RNAs (lncRNAs), which act together to control protein coding gene expression. One of these new discoveries led to the formulation of the "competing endogenous RNA (ceRNA) hypothesis." This hypothesis suggests that an lncRNA acts as a sponge for miRNAs reducing their expression and causing the upregulation of miRNA targets. In this chapter we first discuss some recent discoveries in this field showing the mutual regulation of miRNAs, lncRNAs, and protein-coding genes in cancer. We then discuss the general approaches for the study of ceRNAs and present in more detail a recent computational approach to explore the ability of lncRNAs to act as ceRNAs in human breast cancer that has been shown to be, among the others, the most precise and promising.

In this study, we report that immortal mouse embryonic fibroblasts (I-MEFs) have a baseline level of cells positive for alkaline phosphatase (AP+) staining. Environmental stresses, including long-lasting growth in the absence of expansion and treatment with drugs, enhance the frequency of AP+I-MEFs. By adapting fast red AP staining to the sorting procedure, we separated AP+and AP–I-MEFs and demonstrated that the differentially expressed genes are consistent with a reprogrammed phenotype. In particular, we found that sestrin 1 is upregulated in AP+I-MEFs. We focused on this gene and demonstrated that increased sestrin 1 expression is accompanied by the growth of I-MEFs in the absence of expansion and occurs before the formation of AP+I-MEFs. Together with sestrin 1 upregulation, we found that AP+I-MEFs accumulated in the G1 phase of the cell cycle, suggesting that the two events are causally related. Accordingly, we found that silencing sestrin 1 expression reduced the frequency and G1 accumulation of AP+I-MEFs. Taken together, our data suggested that I-MEFs stressed by environmental changes acquire the AP+phenotype and achieve a quiescent state characterized by a new transcriptional network.

Sphingosine-1-phosphate is a bioactive lipid and a signaling molecule integrated into many physiological systems such as differentiation, proliferation and migration. In mammals S1P acts through binding to a family of five trans-membrane, G-protein coupled receptors (S1PRs) whose complex role has not been completely elucidated. In this study we use zebrafish, in which seven s1prs have been identified, to investigate the role of s1pr1. In mammals S1PR1 is the most highly expressed S1P receptor in the developing heart and regulates vascular development, but in zebrafish the data concerning its role are contradictory. Here we show that overexpression of zebrafish s1pr1 affects both vascular and cardiac development. Moreover we demonstrate that s1pr1 expression is strongly repressed by miR-19a during the early phases of zebrafish development. In line with this observation and with a recent study showing that miR-19a is downregulated in a zebrafish Holt-Oram model, we now demonstrate that s1pr1 is upregulated in heartstring hearts. Next we investigated whether defects induced by s1pr1 upregulation might contribute to the morphological alterations caused by Tbx5 depletion. We show that downregulation of s1pr1 is able to partially rescue cardiac and fin defects induced by Tbx5 depletion. Taken together, these data support a role for s1pr1 in zebrafish cardiovascular development, suggest the involvement of this receptor in the Tbx5 regulatory circuitry, and further support the crucial role of microRNAs in early phase of zebrafish development.

On the grounds that miRNAs present in the blood of prostate cancer (PCa) patients are released in the growth medium by PCa cells, it is conceivable that PCa cells resistant to docetaxel (DCT) (DCTR) will release miRNAs that may be found in PCa patients under DCT therapy if resistant PCa cells appear. We isolated DCTR clones respectively from 22Rv1 and DU-145 PCa cell lines and performed through next-generation sequencing (NGS) the miRNAs profiles of the released miRNAs. The analysis of the NGS data identified 105 and 1 miRNAs which were differentially released in the growth medium of the 22Rv1/DCTR and DU-145/DCTR clones, respectively. Using additional filters, we selected 12 and 1 miRNA more released by all 22Rv1/DCTR and DU-145/DCTR clones, respectively. Moreover, we showed that 6 of them were more represented in the growth medium of the DCTR cells than the ones of DCT-treated cells. We speculated that they have the pre-requisite to be tested as predictive biomarkers of the DCT resistance in PCa patients under DCT therapy. We propose the utilization of clones resistant to a given drug as in vitro model to identify the differentially released miRNAs, which in perspective could be tested as predictive biomarkers of drug resistance in tumor patients under therapy.

Objective: Ventricular assist device (VAD) utilization is increasing in children with heart failure (HF) unresponsive to medical therapy, allowing for bridge to transplantation. The use of circulating biomarkers is crucial for the management of adult patients with HF, but their role in pediatric setting is unknown. In this study we assessed the effects of VAD implant on plasma levels of inflammatory and cardiac biomarkers as well as of circulating micro RNAs (miRNAs) in pediatric patients with HF. Methods: Blood samples were collected from 12 HF pediatric patients [73.91±27.94 (mean±SD) months, 6 males, 19±3.8 LVEF%, Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profiles 1/2] before and at 4 hrs, 1, 3, 7, 14 and 30 days after VAD implant. Indications for support was idiopathic dilated (9 patients), non compaction (2 patients) and hypertrophic cardiomyopathy (1 patient). Inflammatory and cardiac biomarkers (Figure 1A) were measured in frozen plasma through the use of immunoassay tests. The circulating miRNAs were extracted from serum samples and miRNA profile was determined by NGS. and validated by real-time PCR. Results: Inflammatory and cardiac biomarker levels during time-course were reported in Figure 1A. Cytokines increased significantly up to 4 hrs after VAD implant, while cardiac biomarkers up to 1 day. Only NT-proBNP decreased significantly up to 1 month of VAD implant compared to pre-VAD values. After sequencing, a total of 169 miRNAs were detected in serum samples. Among them, 13 miRNAs were simultaneously modulated at 1 month after VAD implant compared to pre-VAD levels, i.e. 9 miRNAs decrease and 4 increase (Figure 1B). Real-time PCR validation confirmed a reduction for miR-409-3p and miR-432-5p. Finally, significant correlations with bio-humoral markers were observed for these miRNAs (Figure 1 C). Conclusions: VAD implant induced modifications in circulating levels of cytokine, cardiac biomarkers and miRNAs. NT-proBNP levels and miRNAs significantly decreased after 1 month compared to the values pre-VAD, suggesting the involvement of these biomarkers in molecular mechanisms triggered by VAD implant in children.

miR-28-5p is an intragenic miRNA which is underexpressed in several tumor types showing a tumor suppressor (TS) activity. Routinely, the known miR-28-5p targets are validated in specific tumor contexts but it is unclear whether these targets are also being regulated in other tumor types. To this end, we adopted the miRNA pull out assay to capture the miR-28-5p targets in DU-145 prostate cancer (PCa) cells. Firstly, we demonstrated that miR-28-5p acts as a TS-miRNA in PCa, affecting cell proliferation, survival, and apoptosis. Secondly, we evaluated the enrichment of the 10 validated miR-28-5p targets in the pull out sample. We showed that E2F6, TEX-261, MAPK1, MPL, N4BP1, and RAP1B but not BAG1, OTUB1, MAD2L1, and p21 were significantly enriched, suggesting that not all the miR-28-5p targets are regulated by this miRNA in PCa. We then verified whether the miR-28-5p-interacting targets were regulated by this miRNA. We selected E2F6, the most enriched target in the pull out sample, and demonstrated that miR-28-5p downregulated E2F6 at the protein level suggesting that our approach was effective. In general terms, these findings support the miRNA pull out assay as a useful method to identify context-specific miRNA targets.

Somatic mutations and dysregulation by microRNAs (miRNAs) may have a pivotal role in the Congenital Heart Defects (CHDs). The purpose of the study was to assess both somatic and germline mutations in the GATA4 and NKX2.5 genes as well as to identify 3'UTR single nucleotide polymorphisms (SNPs) in the miRNA target sites. We enrolled 30 patients (13 males; 13.4±8.3 years) with non-syndromic CHD. GATA4 and NKX2.5 genes were screened in cardiac tissue of sporadic and in blood samples of familial cases. Computational methods were used to detect putative miRNAs in the 3'UTR region and to assess the Minimum Free Energy of hybridization (MFE, kcal/mol). Difference of MFEs (ΔMFE) ≥4 kcal/mol between alleles was considered biologically relevant on miRNA binding. The sum of all ΔMFEs (|ΔMFEtot|=∑|ΔMFE|) was calculated in order to predict the biological importance of SNPs binding more miRNAs. No evidence of novel GATA4 and NKX2.5 mutations was found both in sporadic and familial patients. Bioinformatic analysis revealed 27 putative miRNAs binding to identified SNPs in the 3'UTR of GATA4. ΔMFE ≥4 kcal/mol between alleles was obtained for the +354A>C (miR-4299), +587A>G (miR-604), +1355G>A (miR-548v, miR-139-5p) and +1521C>G (miR-583, miR-3125, miR-3928) SNPs. The +1521C>G SNP showed the highest ΔMFEtot (21.66 kcal/mol). Luciferase reporter assays indicated that miR-583 was dose-dependently effective in regulating +1521 C allele compared with +1521 G allele. Based on the analysis of 100 CHD cases and 204 healthy newborns, the +1521 G allele was also associated with a lower risk of CHD (OR=0.5, 95% CI 0.3-0.9, p=0.03), likely due to the relatively low binding of the miRNA and high levels of protein. These results suggest that common SNPs in the 3'UTR of GATA4 alter miRNA gene regulation contributing to the pathogenesis of CHDs.

The concept of a hype cycle is a well-established business concept, in which novel ideas are said to have an initial wave of hype followed by disillusionment. Only after that, the novel concept takes off and become truly useful entering a so-called plateau of productivity. In biomedical science, the field of microRNAs (miRNAs) certainly had a peak of interest in the end of the last decade. This led by high impact publications (1) and characterization of both novel miRNA-entities as well as their associations to a broad range of diseases. Nonetheless, no clear pharmaceutical successes emerged: miRNA targets are being pursued as therapeutic targets, but none have as of yet successfully made it through clinical trials (2). Likewise the use of miRNA-based treatment strategies targeting regular mRNA is an area of interest (3). In this editorial we focus on a third aspect of miRNAs: the use of miRNAs as prognostic biomarkers in disease, asking the question if miRNAs are now entering this plateau of productivity in which actual benefit will be seen.

Recently, it has been reported that, in several tumor cell lines, short double-stranded RNAs tailored for promoter regions of specific genes are able to activate their transcription. Such molecules (named RNA activators) act opposite to other double-stranded RNA molecules (named RNA inhibitors) in that the overexpression instead of underexpression of a given gene is triggered. In Dohh2 non-Hodgkin lymphoma cells, the transcriptional repressor BCL6, which negatively controls both p53 and p21, is overexpressed, so that the cells can escape the check point governed by p53 and proliferate. The aim of this work was to investigate whether the RNA activator p21 can represent a tool to circumvent the transcriptional control of BCL6 and induce the blockage of cell proliferation in Dohh2 non-Hodgkin lymphoma cells. For that, Dohh2 cells were transfected with either a control RNA activator (ds-NC) or an RNA activator specific for human p21 promoter (ds-p21). At various time points after transfection, the cells were collected and p21 was measured. Dohh2 cells transfected with ds-p21 showed a slight but significant overexpression of p21 at both mRNA and protein levels. Nonetheless, cell proliferation, cell cycle, and apoptosis were not significantly modified. In contrast, the exposure of Dohh2 cells transfected with ds-p21 to fludarabine potentiates the cytotoxicity of the drug, suggesting the RNA activator p21 complements the fludarabine-dependent cell death pathways.

Expression of microRNAs changes markedly in tumours and evidence indicates that they are causatively related to tumourigenesis, behaving as tumour suppressor microRNAs or onco microRNAs; in some cases they can behave as both depending on the type of cancer. Some tumour suppressor microRNAs appear to be an integral part of the p53 and Retinoblastoma (RB) network, the main regulatory pathways controlling senescence, a major tumour suppressor mechanism. The INK4a/ARF locus which codifies for two proteins, p19ARF and p16INK4a, plays a central role in senescence by controlling both p53 and RB. Recent evidence shows that the proto-oncogene leukaemia/lymphoma related factor, a p19ARF specific repressor, is controlled by miRNAs and that miRNAs, in particular miR-20a and miR-290, are causatively involved in mouse embryo fibroblasts (MEF) senescence in culture. Intriguingly, both miR-20a, member of the oncogenic miR-17–92 cluster, and miR-290, belonging to the miR-290–295 cluster, are highly expressed in embryonic stem (ES) cells. The pro-senescence role of miR-20a and miR-290 in MEF is apparently in contrast with their proliferative role in tumour and ES cells. We propose that miRNAs may exert opposing functions depending on the miRNAs repertoire as well as target/s level/s present in different cellular contexts, suggesting the importance of evaluating miRNAs activity in diverse genetic settings before their therapeutic use as tumour suppressors.

Genomic and clinical evidence suggest a major role of microRNAs (miRNAs) in the regulatory mechanisms of gene expression, with a clear impact on development and physiology; miRNAs are a class of endogenous 22-25 nt single-stranded RNA molecules, that negatively regulate gene expression post-transcriptionally, by imperfect base pairing with the 3’ UTR of the corresponding mRNA target. Because of this imperfection, each miRNA can bind multiple targets, and multiple miRNAs can bind the same mRNA target; although digital, the miRNAs control mechanism is characterized by an imprecise action, naturally understandable in the theoretical framework of fuzzy logic. A major practical application of fuzzy logic is represented by the design and the realization of efficient and robust control systems, even when the processes to be controlled show chaotic, deterministic as well unpredictable, behaviours. The vagueness of miRNA action, when considered together with the controlled and chaotic gene expression, is a hint of a cellular fuzzy control system. As a demonstration of the possibility and the effectiveness of miRNA based fuzzy mechanism, a fuzzy cognitive map -a mathematical formalism combining neural network and fuzzy logic- has been developed to study the apoptosis/proliferation control performed by the miRNA-17-92 cluster/E2F1/cMYC circuitry. When experimentally demonstrated, the concept of fuzzy control could modify the way we analyse and model gene expression, with a possible impact on the way we imagine and design therapeutic intervention based on miRNA silencing.

Copyright: Genes and their expression regulation are among the key factors in the comprehension of the genesis and development of complex diseases. In this context, microRNAs (miRNAs) are post-transcriptional regulators that play an important role in gene expression since they are frequently deregulated in pathologies like cardiovascular disease and cancer. In vitro validation of miRNA - targets regulation is often too expensive and time consuming to be carried out for every possible alternative. As a result, a tool able to provide some criteria to prioritize trials is becoming a pressing need. Moreover, before planning in vitro experiments, the scientist needs to evaluate the miRNA-target genes interaction network. In this paper we describe the miRable method whose purpose is to identify new potentially relevant genes and their interaction networks associate to a specific pathology. To achieve this goal miRable follows a system biology approach integrating together general-purpose medical knowledge (literature, Protein-Protein Interaction networks, prediction tools) and pathology specific data (gene expression data). A case study on Prostate Cancer has shown that miRable is able to: 1) find new potential miRNA-targets pairs, 2) highlight novel genes potentially involved in a disease but never or little studied before, 3) reconstruct all possible regulatory subnetworks starting from the literature to expand the knowledge on the regulation of miRNA regulatory mechanisms.