Translational Control of FOG-2 Expression in Cardiomyocytes by MicroRNA-130a

MicroRNAs are increasingly being recognized as regulators of embryonic development; however, relatively few microRNAs have been identified to regulate cardiac development. FOG-2 (also known as zfpm2) is a transcriptional co-factor that we have previously shown is critical for cardiac development. In this report, we demonstrate that FOG-2 expression is controlled at the translational level by microRNA-130a. We identified a conserved region in the FOG-2 3′ untranslated region predicted to be a target for miR-130a. To test the functional significance of this site, we generated an expression construct containing the luciferase coding region fused with the 3′ untranslated region of FOG-2 or a mutant version lacking this microRNA binding site. When these constructs were transfected into NIH 3T3 fibroblasts (which are known to express miR-130a), we observed a 3.3-fold increase in translational efficiency when the microRNA target site was disrupted. Moreover, knockdown of miR-130a in fibroblasts resulted in a 3.6-fold increase in translational efficiency. We also demonstrate that cardiomyocytes express miR-130a and can attenuate translation of mRNAs with a FOG-2 3′ untranslated region. Finally, we generated transgenic mice with cardiomyocyte over-expression of miR-130a. In the hearts of these mice, FOG-2 protein levels were reduced by as much as 80%. Histological analysis of transgenic embryos revealed ventricular wall hypoplasia and ventricular septal defects, similar to that seen in FOG-2 deficient hearts. These results demonstrate the importance of miR-130a for the regulation of FOG-2 protein expression and suggest that miR-130a may also play a role in the regulation of cardiac development

MiR-130a inhibition protects rat cardiac myocytes from hypoxia-triggered apoptosis by targeting Smad4

Background: Cardiomyocyte death facilitates the pathological process underlying ischaemic heart diseases, such as myocardial infarction. Emerging evidence suggests that microRNAs play a critical role in the pathological process underlying myocardial infarction by regulating cardiomyocyte apoptosis. However, the relevance of miR-130a in regulating cardiomyocyte apoptosis and the underlying mechanism are still uncertain. Aim: We sought to explore the regulatory effect of miR-130a on hypoxic cardiomyocyte apoptosis. Methods: The expression of miR-130a was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell survival was determined by the MTT assay. The lactate dehydrogenase (LDH) assay was performed to deter­mine the severity of hypoxia-induced cell injury. Apoptosis was assessed via caspase-3 analysis. Protein expression level was determined by Western blotting. The genes targeted by miR-130a were predicted using bioinformatics and were validated via the dual-luciferase reporter assay system. Results: We found that miR-130a expression was greatly increased in hypoxic cardiac myocytes, and that the downregulation of miR-130a effectively shielded cardiac myocytes from hypoxia-triggered apoptosis. In bioinformatic analysis the Smad4 gene was predicted to be the target of miR-130a. This finding was validated through the Western blot assay, dual-luciferase reporter gene assay, and qRT-PCR. MiR-130a inhibition significantly promoted the activation of Smad4 in hypoxic cardiomyocytes. Inter­estingly, knockdown of Smad4 markedly reversed the protective effects induced by miR-130a inhibition. Moreover, we found that the inhibition of miR-130a promoted the activation of transforming growth factor-b1 signalling. Blocking of Smad4 signal­ling significantly abrogated the protective effects of miR-130a inhibition. Conclusions: The findings indicate that inhibition of miR-130a, which targets the Smad4 gene, shields cardiac myocytes from hypoxic apoptosis. This study offers a novel perspective on the molecular basis of hypoxia-induced cardiomyocyte apoptosis and suggests a possible drug target for the treatment of myocardial infarction

Proteomic and epigenomic markers of sepsis-induced delirium (SID)

In elderly population sepsis is one of the leading causes of intensive care unit (ICU) admissions in the United States. Sepsis-induced delirium (SID) is the most frequent cause of delirium in ICU (Martin et al., 2010). Together delirium and SID represent under-recognized public health problems which place an increasing financial burden on the US health care system, currently estimated at 143-152 billion dollars per year (Leslie et al., 2008). The interest in SID was recently reignited as it was demonstrated that, contrary to prior beliefs, cognitive deficits induced by this condition may be irreversible and lead to dementia (Pandharipande et al., 2013; Brummel et al., 2014). Conversely, it is construed that diagnosing SID early or mitigating its full blown manifestations may preempt geriatric cognitive disorders. Biological markers specific for sepsis and SID would facilitate the development of potential therapies, monitor the disease process and at the same time enable elderly individuals to make better informed decisions regarding surgeries which may pose the risk of complications, including sepsis and delirium. This article proposes a battery of peripheral blood markers to be used for diagnostic and prognostic purposes in sepsis and SID. Though each individual marker may not be specific enough, we believe that together as a battery they may achieve the necessary accuracy to answer two important questions: who may be vulnerable to the development of sepsis, and who may develop SID and irreversible cognitive deficits following sepsis?

Estudio de la asociación entre el perfil de expresión de microRNAs con la capacidad de regeneración de las células satélite durante las distrofias musculares Duchenne y Becker

The objective of this work was to determine the association between miRNAs and the differentiation process of satellite cells (SC) during Duchenne and Becker muscular dystrophy. Using expression microarrays, it was found that 15 miRNAs (miR-1-2, miR-184, miR-130a, miR-106b, miR-155, miR-153-1, let-7f-2, miR-130b, let -7e, miR-141, miR-20, miR-133a-1 miR-19a, miR-183, miR-34c) showed higher expression in DMD and BMD patients than in healthy controls. Subsequently, through an in-silico analysis, it was possible to propose that 9 of these miRNAs (miR1-2, let-7f-2 and let-7e, miR-133a, miR-130a, miR-155, miR-153-1, miR-130b, miR-19) are involved in the regulation of PAX3, PAX7, MDFIC, and PAXBP1 proteins, and the activation and differentiation of satellite cells. Additionally, a systematic review was carried out in which five miRNAs stand out (miR-1, -155, let7f, let-7e, -133a) of the nine selected from the in silico analysis, which could regulate the proteins involved in CS activation and differentiation processes and fibrosis and inflammation like PAX7, MYOGENIN, MEF-2C and MYOD, HDAC2, MYF5, SOCS1, IL-6, INFr, MCP-1, TNF-α, COL1A1, COL1A2, COL3A1, COL24A1, COL27A1, ITGA1, ITGA4, SCD1, THBS1, TGF-β, SMAD3, and SMAD5.El objetivo de este trabajo fue determinar la asociación entre miRNAs y el proceso de diferenciación de células satélite (CS) durante la distrofia muscular de Duchenne y Becker. Usando microarreglos de expresión, se encontró que 15 miRNAs (miR1-2, miR-184, miR-130a, miR-106b, miR-155, miR-153-1, let-7f-2, miR-130b, let - 7e, miR-141, miR-20, miR-133a-1 miR-19a, miR-183, miR-34c) se expresaron más en pacientes con DMD y BMD que en controles sanos. Posteriormente, mediante un análisis in silico, fue posible proponer que 9 de estos miRNAs (miR1-2, let-7f-2 y let-7e, miR-133a, miR-130a, miR-155, miR-153-1, miR-130b, miR-19) están involucradas en la regulación de las proteínas PAX3, PAX7, MDFIC y PAXBP1, y en la activación y diferenciación de células satélite. Adicionalmente, se realizó una revisión sistemática en la que se destacan cinco miRNAs (miR-1, -155, let-7f, let7e, -133a) de los nueve seleccionados del análisis in silico, que podrían regular las proteínas implicados en proceso de activación y de diferenciación de CS y en fibrosis e inflamación como PAX7, MIOGENINA, MEF-2C y MYOD, HDAC2, MYF5, SOCS1, IL-6, INFr, MCP-1, TNF-α, COL1A1, COL1A2, COL3A1, COL24A1, COL27A1, ITGA1, ITGA4, SCD1, THBS1, TGF-β, SMAD3, y SMAD5

MicroRNA regulation of endothelial homeostasis and commitment—implications for vascular regeneration strategies using stem cell therapies

Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration

N-Myc-activated microRNAs Inhibit Protein Synthesis of RUNX1 and RUNX3 in Neuroblastoma Cell Lines

　RUNX1 and RUNX3 are master transcription factors in sensory neuron lineage specifications. Protein levels of such developmental regulators are tightly controlled during carcinogenesis, in order to block differentiation and drive proliferation. Here we report that neuroblastoma specific microRNAs inhibit protein syntheses of RUNX1 and RUNX3 through 3’UTR sequences. Computational prediction identified two putative binding sequences for N-Myc-activated microRNAs both in RUNX1 and RUNX3 3’UTRs. Streptavidin RNA aptamer-tagged 3’UTR sequences pulled down miR-17, miR-18a, miR-19a, miR-20a or miR-130a from neuroblastoma cell lysate. 3’UTR target protection from N-Myc-activated microRNAs increased protein synthesis of RUNX1 or RUNX3 and induced differentiation in neuroblastoma cell lines. Together, protein levels of RUNX1 and RUNX3 are post-transcriptionally regulated by N-Myc-activated microRNAs, highlighting the mutual negative feedback between N-Myc oncogene and RUNX3 tumor suppressor in neuroblastoma