microRNAs (miRNAs) are non-coding RNAs that regulate gene expression post-transcriptionally, and mounting evidence supports the prevalence and functional significance of their interplay with transcription factors (TFs). Here we describe the identification of a regulatory circuit between muscle miRNAs (miR-1, miR-133 and miR-206) and Yin Yang 1 (YY1), an epigenetic repressor of skeletal myogenesis in mouse. Genome-wide identification of potential down-stream targets of YY1 by combining computational prediction with expression profiling data reveals a large number of putative miRNA targets of YY1 during skeletal myoblasts differentiation into myotubes with muscle miRs ranking on top of the list. The subsequent experimental results demonstrate that YY1 indeed represses muscle miRs expression in myoblasts and the repression is mediated through multiple enhancers and recruitment of Polycomb complex to several YY1 binding sites. YY1 regulating miR-1 is functionally important for both C2C12 myogenic differentiation and injury-induced muscle regeneration. Furthermore, we demonstrate that miR-1 in turn targets YY1, thus forming a negative feedback loop. Together, these results identify a novel regulatory circuit required for skeletal myogenesis and reinforce the idea that regulatory circuitries involving miRNAs and TFs are prevalent mechanisms.

BACKGROUND Computational methods for microRNA target prediction are a fundamental step to understand the miRNA role in gene regulation, a key process in molecular biology. In this paper we present miREE, a novel microRNA target prediction tool. miREE is an ensemble of two parts entailing complementary but integrated roles in the prediction. The Ab-Initio module leverages upon a genetic algorithmic approach to generate a set of candidate sites on the basis of their microRNA-mRNA duplex stability properties. Then, a Support Vector Machine (SVM) learning module evaluates the impact of microRNA recognition elements on the target gene. As a result the prediction takes into account information regarding both miRNA-target structural stability and accessibility. RESULTS The proposed method significantly improves the state-of-the-art prediction tools in terms of accuracy with a better balance between specificity and sensitivity, as demonstrated by the experiments conducted on several large datasets across different species. miREE achieves this result by tackling two of the main challenges of current prediction tools:(1) The reduced number of false positives for the Ab-Initio part thanks to the integration of a machine learning module (2) the specificity of the machine learning part, obtained through an innovative technique for rich and representative negative records generation. The validation was conducted on experimental datasets where the miRNA:mRNA interactions had been obtained through (1) direct validation where even the binding site is provided, or through (2) indirect validation, based on gene expression variations obtained from high-throughput experiments where the specific interaction is not validated in detail and consequently the specific binding site is not provided. CONCLUSIONS The coupling of two parts: a sensitive Ab-Initio module and a selective machine learning part capable of recognizing the false positives, leads to an improved balance between sensitivity and specificity. miREE obtains a reasonable trade-off between filtering false positives and identifying targets. miREE tool is available online at http://didattica-online.polito.it/eda/miREE/

MicroRNAs (miRNAs) influence many biological processes, including cancer. They do so by posttranscriptionally repressing target mRNAs to which they have sequence complementarity. Although it has been postulated that miRNAs can regulate other miRNAs, this has never been shown experimentally to our knowledge. Here, we demonstrate that miR-107 negatively regulates the tumor suppressor miRNA let-7 via a direct interaction. miR-107 was found to be highly expressed in malignant tissue from patients with advanced breast cancer, and its expression was inversely correlated with let-7 expression in tumors and in cancer cell lines. Ectopic expression of miR-107 in human cancer cell lines led to destabilization of mature let-7, increased expression of let-7 targets, and increased malignant phenotypes. In contrast, depletion of endogenous miR-107 dramatically increased the stability of mature let-7 and led to downregulation of let-7 targets. Accordingly, miR-107 expression increased the tumorigenic and metastatic potential of a human breast cancer cell line in mice via inhibition of let-7 and upregulation of let-7 targets. By mutating individual sites within miR-107 and let-7, we found that miR-107 directly interacts with let-7 and that the internal loop of the let-7/miR-107 duplex is critical for repression of let-7 expression. Altogether, we have identified an oncogenic role for miR-107 and provide evidence of a transregulational interaction among miRNAs in human cancer development.

Bacterial sRNAs are a class of small regulatory RNAs involved in regulation of expression of a variety of genes. Most sRNAs act in trans via base-pairing with target mRNAs, leading to repression or activation of translation or mRNA degradation. To date, more than 1,000 sRNAs have been identified. However, direct targets have been identified for only approximately 50 of these sRNAs. Computational predictions can provide candidates for target validation, thereby increasing the speed of sRNA target identification. Although several methods have been developed, target prediction for bacterial sRNAs remains challenging. Here, we propose a novel method for sRNA target prediction, termed sTarPicker, which was based on a two-step model for hybridization between an sRNA and an mRNA target. This method first selects stable duplexes after screening all possible duplexes between the sRNA and the potential mRNA target. Next, hybridization between the sRNA and the target is extended to span the entire binding site. Finally, quantitative predictions are produced with an ensemble classifier generated using machine-learning methods. In calculations to determine the hybridization energies of seed regions and binding regions, both thermodynamic stability and site accessibility of the sRNAs and targets were considered. Comparisons with the existing methods showed that sTarPicker performed best in both performance of target prediction and accuracy of the predicted binding sites. sTarPicker can predict bacterial sRNA targets with higher efficiency and determine the exact locations of the interactions with a higher accuracy than competing programs. sTarPicker is available at http://ccb.bmi.ac.cn/starpicker/.

MicroRNAs (miRNAs) are short, non-coding sequences that control gene expression via translational regulation. Through interactions with the 3'-untranslated region of messenger RNA, miRNAs trigger translational repression and play a key role in developmental timing. Furthermore, many miRNA groups have now been shown to regulate various processes in tumorigenesis, including angiogenesis and metastasis. These links highlight the importance of microRNA research in further understanding cancer development. This review article summarizes the current state of microRNA research, with a focus on the roles of microRNAs in various cancer types. Up to date knowledge of the structure and biogenesis pathway of microRNA are also reviewed.

Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, we found that the Rho-GTPase Cdc42 cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart-specific interference with Cdc42 function is sufficient to cause these same defects. We also identified K(+) channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42-Tinman interaction. To determine whether a Cdc42-Nkx2-5 interaction is conserved in the mammalian heart, we examined compound heterozygous mutant mice and found conduction system and cardiac output defects. In exploring the mechanism of Nkx2-5 interaction with Cdc42, we demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. We conclude that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1.

Many computational microRNA target prediction tools are focused on several key features, including complementarity to 5'seed of miRNAs and evolutionary conservation. While these features allow for successful target identification, not all miRNA target sites are conserved and adhere to canonical seed complementarity. Several studies have propagated the use of energy features of mRNA:miRNA duplexes as an alternative feature. However, different independent evaluations reported conflicting results on the reliability of energy-based predictions. Here, we reassess the usefulness of energy features for mammalian target prediction, aiming to relax or eliminate the need for perfect seed matches and conservation requirement. We detect significant differences of energy features at experimentally supported human miRNA target sites and at genome-wide sites of AGO protein interaction. This trend is confirmed on datasets that assay the effect of miRNAs on mRNA and protein expression changes, and a simple linear regression model leads to significant correlation of predicted versus observed expression change. Compared to 6-mer seed matches as baseline, application of our energy-based model leads to ∼3-5-fold enrichment on highly down-regulated targets, and allows for prediction of strictly imperfect targets with enrichment above baseline. In conclusion, our results indicate significant promise for energy-based miRNA target prediction that includes a broader range of targets without having to use conservation or impose stringent seed match rules.

MicroRNAs (miRNAs) regulate gene expression by mediating translational repression or mRNA degradation of their targets, and several miRNAs control developmental decisions through embryogenesis. In the developing heart, miRNA targets comprise key players mediating cardiac lineage determination. However, although several miRNAs have been identified as differentially regulated during cardiac development and disease, their distinct cell-specific localization remains largely undetermined, likely owing to a lack of adequate methods. We therefore report the development of a markedly improved approach combining fluorescence-based miRNA-in situ hybridization (miRNA-ISH) with immunohistochemistry (IHC). We have applied this protocol to differentiating embryoid bodies (EBs) as well as embryonic and adult mouse hearts, to detect miRNAs that were upregulated during EB cardiomyogenesis, as determined by array-based miRNA expression profiling. In this manner, we found specific co-localization of miR-1 to myosin positive cells (cardiomyocytes) of EBs, developing and mature hearts. In contrast, miR-125b and -199a did not localize to cardiomyocytes, as previously suggested for miR-199a, but were rather expressed in connective tissue cells of the heart. More specifically, by co-staining with α-smooth muscle actin (α-SMA) and collagen-I, we found that miR-125b and -199a localize to perivascular α-SMA(-) stromal cells. Our approach thus proved valid for determining cell-specific localization of miRNAs, and the findings we present highlight the importance of determining exact cell-specific localization of miRNAs by sequential miRNA-ISH and IHC in studies aiming at understanding the role of miRNAs and their targets. This approach will hopefully aid in identifying relevant miRNA targets of both the heart and other organs.

MicroRNAs (miRNAs) are a class of non-coding regulatory RNAs of ~22 nucleotides in length. miRNAs regulate gene expression post-transcriptionally, primarily by associating with the 3' untranslated region (UTR) of their regulatory target mRNAs. Recent work has begun to reveal roles for miRNAs in a wide range of biological processes, including cell proliferation, differentiation and apoptosis. Many miRNAs are expressed in cardiac and skeletal muscle, and dysregulated miRNA expression has been correlated with muscle-related disorders. We have previously reported that the expression of muscle-specific miR-1 and miR-133 is induced during skeletal muscle differentiation and miR-1 and miR-133 play central regulatory roles in myoblast proliferation and differentiation in vitro. In this study, we measured the expression of miRNAs in the skeletal muscle of mdx mice, an animal model for human muscular dystrophy. We also generated transgenic mice to overexpress miR-133a in skeletal muscle. We examined the expression of miRNAs in the skeletal muscle of mdx mice. We found that the expression of muscle miRNAs, including miR-1a, miR-133a and miR-206, was up-regulated in the skeletal muscle of mdx mice. In order to further investigate the function of miR-133a in skeletal muscle in vivo, we have created several independent transgenic founder lines. Surprisingly, skeletal muscle development and function appear to be unaffected in miR-133a transgenic mice. Our results indicate that miR-133a is dispensable for the normal development and function of skeletal muscle.

BACKGROUND Left ventricular (LV) reverse remodelling after valve replacement in aortic stenosis (AS) has been classically linked to the hydraulic performance of the replacement device, but myocardial status at the time of surgery has received little attention. OBJECTIVE To establish predictors of LV mass (LVM) regression 1 year after valve replacement in a surgical cohort of patients with AS based on preoperative clinical and echocardiographic parameters and the myocardial gene expression profile at surgery. METHODS Transcript levels of remodelling-related proteins and regulators were determined in LV intraoperative biopsies from 46 patients with AS by RT-PCR. Using multiple linear regression analysis, an equation was developed (adjusted R²=0.73; p<0.0001) that included positive [preoperative LVM, microRNA-133a, serum response factor (SRF, which is known to be a transactivator of miR-133) and age] and negative [body mass index (BMI), Wolf-Hirschhorn syndrome candidate-2 (WHSC2, which is a target for repression by miR-133a), β-myosin heavy chain, myosin light chain-2, diabetes mellitus, and male gender] independent predictors of LVM reduction. RESULTS Aortic valve area gain or the reduction in transvalvular gradient maintained no significant relationships with the dependent variable. Logistic regression analysis identified microRNA-133a as a significant positive predictor of LVM normalisation, whereas β-myosin heavy chain and BMI constituted negative predictors. CONCLUSIONS Hypertrophy regression 1 year after pressure overload release is related to the preoperative myocardial expression of remodelling-related genes, in conjunction with the patient's clinical background. In this scenario, miR-133 emerges as a key element of the reverse remodelling process. Postoperative improvement of valve haemodynamics does not predict the degree of hypertrophy regression or LVM normalisation. These results led us to reconsider the current reverse remodelling paradigm and (1) to include criteria of hypertrophy reversibility in the decision algorithm used to decide timing for the operation; and (2) to modify other prevailing factors (overweight, diabetes, etc) known to maintain LV hypertrophy.

Discovered in Caenorhabditis elegans in 1993, microRNAs (miRNAs) make up a novel class of tiny,~21-24 nucleotide, non-coding RNA species. Since its identification as a key component of a broadly conserved mechanism that regulates gene expression post-transcriptionally, the miRNA pathway has emerged as one of the most extensively investigated pathways of the past decade. Because of their potential to regulate a large number of protein-encoding genes, miRNAs have been implicated in numerous biological processes, including development, stem cell regulation and human diseases. In this Commentary, we focus on miRNAs and their roles in mammalian stem cells. Following an introduction to the miRNA biogenesis pathway with an emphasis on its regulatory features, we then discuss what is currently known about the roles that miRNAs have in the differentiation and maintenance of embryonic and somatic stem cells of diverse origins. In particular, their roles in stem cell differentiation have been well documented. Insights from these studies provide a paradigm for the function of miRNAs in facilitating cellular transitions during differentiation. By contrast, the roles that miRNAs have in the maintenance of stem cells are less well understood. However, with recent advances, their role as a rheostat that fine-tunes stem cell self-renewal has begun to emerge. Finally, we discuss future studies that will hopefully lead to a comprehensive understanding of the miRNA pathway in stem cells.

The heart responds to myriad stresses by well-described transcriptional responses that involve long-term changes in gene expression as well as more immediate, transient adaptations. MicroRNAs quantitatively regulate mRNAs and thus may affect the cardiac transcriptional output and cardiac function. Here we investigate miR-499, a microRNA embedded within a ventricular-specific myosin heavy chain gene, which is expressed in heart and skeletal muscle. We assessed miR-499 expression in human tissue to confirm its potential relevance to human cardiac gene regulation. Using a transgenic mouse model, we found that elevated miR-499 levels caused cellular hypertrophy and cardiac dysfunction in a dose-dependent manner. Global gene expression profiling revealed altered levels of the immediate early stress response genes (Egr1, Egr2 and Fos), ß-myosin heavy chain (Myh7), and skeletal muscle actin (Acta1). We verified the effect of miR-499 on the immediate early response genes by miR-499 gain- and loss-of-function in vitro. Consistent with a role for miR-499 in blunting the response to cardiac stress, asymptomatic miR-499-expressing mice had an impaired response to pressure overload and accentuated cardiac dysfunction. Elevated miR-499 levels affect cardiac gene expression and predispose to cardiac stress-induced dysfunction. miR-499 may titrate the cardiac response to stress in part by regulating the immediate early gene response.

Ezrin, a membrane organizer and linker between plasma membrane and cytoskeleton, is well documented to play an important role in the metastatic capacity of cancer cells especially for osteosarcoma cells. It has provided an ideal target for cancer gene therapy. RNA-cleaving 10-23 DNAzymes, consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, can cleave the target mRNA at purine-pyrimidine dinucleotide effectively. In the present study, we designed and screened the target sites for 10-23 DNAzymes against ezrin mRNA by using multiple computational methods with combination of secondary structural and hybridization thermodynamic parameters. Then, we testified the activities of the DNAzymes directed against these selected target sites in vitro. Our results show that AU1751 is the most effective target site of ezrin mRNA for DNAzymes because of its ideal secondary structure and hybridization thermodynamics. So, there is a significant correlation between the multiple computational methods and the efficacy of the corresponding DNAzymes. These provide a rational, efficient way for DNAzymes selection.

MicroRNAs are small, highly conserved non-coding RNA molecules involved in the regulation of gene expression. MicroRNAs are transcribed by RNA polymerases II and III, generating precursors that undergo a series of cleavage events to form mature microRNA. The conventional biogenesis pathway consists of two cleavage events, one nuclear and one cytoplasmic. However, alternative biogenesis pathways exist that differ in the number of cleavage events and enzymes responsible. How microRNA precursors are sorted to the different pathways is unclear but appears to be determined by the site of origin of the microRNA, its sequence and thermodynamic stability. The regulatory functions of microRNAs are accomplished through the RNA-induced silencing complex (RISC). MicroRNA assembles into RISC, activating the complex to target messenger RNA (mRNA) specified by the microRNA. Various RISC assembly models have been proposed and research continues to explore the mechanism(s) of RISC loading and activation. The degree and nature of the complementarity between the microRNA and target determine the gene silencing mechanism, slicer-dependent mRNA degradation or slicer-independent translation inhibition. Recent evidence indicates that P-bodies are essential for microRNA-mediated gene silencing and that RISC assembly and silencing occurs primarily within P-bodies. The P-body model outlines microRNA sorting and shuttling between specialized P-body compartments that house enzymes required for slicer -dependent and -independent silencing, addressing the reversibility of these silencing mechanisms. Detailed knowledge of the microRNA pathways is essential for understanding their physiological role and the implications associated with dysfunction and dysregulation.

Many molecular pathways involved in heart disease have their roots in evolutionarily ancient developmental programs that depend critically on gene dosage and timing. MicroRNAs (miRNAs) modulate gene dosage posttranscriptionally, and among these, the muscle-specific miR-1 is particularly important for developing and maintaining somatic/skeletal and cardiac muscle. To identify pathways regulated by miR-1, we performed a forward genetic screen in Drosophila using wing-vein patterning as a biological assay. We identified several unexpected genes that genetically interacted with dmiR-1, one of which was kayak, encodes a developmentally regulated transcription factor. Additional studies directed at this genetic relationship revealed a previously unappreciated function of dmiR-1 in regulating the polarity of cardiac progenitor cells. The mammalian ortholog of kayak, c-Fos, was dysregulated in hearts of gain- or loss-of-function miR-1 mutant mice in a stress-dependent manner. These findings illustrate the power of Drosophila-based screens to find points of intersection between miRNAs and conserved pathways in mammals.

Almost 15 years of careful study have established the related basic Helix-Loop-Helix (bHLH) transcription factors Hand1 and Hand2 as critical for heart development across evolution. Hand factors make broad contributions, revealed through animal models, to the development of multiple cellular lineages that ultimately contribute to the heart. They perform critical roles in ventricular cardiomyocyte growth, differentiation, morphogenesis, and conduction. They are also important for the proper development of the cardiac outflow tract, epicardium, and endocardium. Molecularly, they function both through DNA binding and through protein-protein interactions, which are regulated transcriptionally, posttranscriptionally by microRNAs, and posttranslationally through phosphoregulation. Although direct Hand factor transcriptional targets are progressively being identified, confirmed direct targets of Hand factor transcriptional activity in the heart are limited. Identification of these targets will be critical to model the mechanisms by which Hand factor bHLH interactions affect developmental pathways. Improved understanding of Hand factor-mediated transcriptional cascades will be necessary to determine how Hand factor dysregulation translates to human disease phenotypes. This review summarizes the insight that animal models have provided into the regulation and function of these factors during heart development, in addition to the recent findings that suggest roles for HAND1 and HAND2 in human congenital heart disease.

Since 1993, when the first small non-coding RNA was identified, our knowledge about microRNAs has grown exponentially. In this review, we focus on the main progress in this field and discuss the most important findings under a historical perspective. In addition, we examine microRNAs as markers of disease diagnosis and prognosis, and as new therapeutic targets.

Although β(2)-adrenergic receptors (β(2)AR) are expressed on most cell types, mechanisms that establish expression levels and regulate expression by chronic agonist remain unclear. The 3' UTR of ADRB2 has a conserved 8-nucleotide seed region that we hypothesized is targeted by the let-7 family of miRNAs leading to translational repression. In luciferase assays with transfected cells, luc-β(2)WT3'UTR had decreased expression when cotransfected with let-7f, but a mutated luc-β(2)3'UTR lacking the seed was unaffected by let-7f; a mutated let-7f also had no effect on luc-β(2)WT3'UTR expression. ADRB2 mRNA was in greater abundance in immunoprecipitates of Ago2, a core component of the miRNA-induced silencing complex, when cells were transfected with let-7f, but not with a mutated let-7f, indicating a direct interaction with the silencing mechanism. H292 cells transfected with let-7f caused ∼60% decrease in native β(2)AR expression, but transfection with let-7f-specific locked nucleic acid anti-miRNA increased β(2)AR expression by ∼twofold. We considered that an increase in let-7f leading to greater repression of translation contributes to agonist-promoted down-regulation. Paradoxically, in cells and in lungs from mice treated in vivo, an ∼50% decrease in let-7f occurs during long-term agonist exposure, indicating a counterregulatory event. Consistent with this notion, let-7f locked nucleic acid transfection caused depressed agonist-promoted down-regulation. Thus, let-7f miRNA regulates baseline β(2)AR expression and decreases in let-7f evoked by agonist attenuate down-regulation. This positive feedback loop has not previously been described for a G protein-coupled receptor and its miRNA. Methods to decrease let-7f expression in targeted cells may increase therapeutic responses to β-agonist by increasing β(2)AR expression or minimizing tachyphylaxis.

INTRODUCTION: Coronary artery disease (CAD) is still the leading cause of death in industrialized nations. Even though revascularization strategies such as percutaneous coronary intervention (PCI) and coronary artery bypass graft surgery (CABG) as well as drug therapy have significantly reduced mortality, about 30% of patients will develop chronic heart failure over time. Ischemic heart disease and heart failure are characterized by an adverse remodeling of the heart, featuring cardiomyocyte hypertrophy, increased fibrosis and capillary rarification. AREAS COVERED: Beside an assessment of current vector systems, this review focuses on potential target genes affecting angiogenesis/arteriogenesis and contractility. The potential of micro RNA (miRNA) modulation for the de-repression of survival and pro-angiogenic genes is discussed. Since gene therapy of the target region is preferable to avoid systemic contamination, application routes are discussed. Expert opinion: miRNAs are a promising new development for successful gene therapy, especially for acute myocardial infarction since their miRNA antagonists are easy to apply and appear to be selectively absorbed by the ischemic myocardial tissue. Rapid uptake and prolonged presence of known antimirs and antagomirs support this notion. For ischemic heart disease the most promising gene therapeutic approach seems to be the regional intravenous application of suitable AAV vectors and vascular growth factors, providing the full scope of angiogenesis, vessel maturation and collateral growth optionally combined with genes enhancing contractility.

MicroRNAs (miRNAs) are endogenous, short (~22 nucleotide), evolutionarily conserved, non-coding RNAs that regulate gene expression at the post-transcriptional level. Recent evidence suggests that miRNAs are differentially expressed in the failing myocardium and play an important role in progression of heart failure by targeting genes that govern diverse functions in cardiac remodeling process including myocyte hypertrophy, excitation-contraction coupling, increased myocyte loss, and myocardial fibrosis. In addition to their role in adverse cardiac remodeling, miRNAs hold promise as biomarkers of disease progression in heart failure given their presence in circulation and enhanced stability. Further development of miR-based therapeutics may allow for modulation of cardiac and/or systemic levels of specific miRNAs in patients with heart failure . Here, we summarize current knowledge of miRNAs in relation to their role in regulating various aspects of the cardiac remodeling process and discuss their potential use as biomarkers and/or therapeutic targets in heart failure.