Sebastian Vencken

miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production

Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o− tracheal, CFBE41o− and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases. Overexpression of miR-17 in cystic fibrosis airway epithelial cells decreases interleukin-8 protein production http://ow.ly/MZbXB

Non-coding RNA as lung disease biomarkers

Biomarkers are quantifiable indicators of disease. These surrogates should be specific, sensitive, predictive, robust and easily accessible. A major class of RNA described as non-coding RNA fulfils many of these criteria, and recent studies have demonstrated that the two major subclasses of non-coding RNA, long non-coding RNA and, in particular, microRNA are promising potential biomarkers. The ability to detect non-coding RNAs in biofluids has highlighted their usefulness as non-invasive markers of lung disease. Because expression of specific non-coding RNAs is altered in many lung diseases and their levels in the circulation often reflect the changes in expression of their lung-specific counterparts, exploiting these biomolecules as diagnostic tools seems an obvious goal. New technology is driving developments in this area and there has been significant recent progress with respect to lung cancer diagnostics. The non-coding RNA biomarker field represents a clear example of modern-day bench-to-bedside research.

Reduced miR-659-3p Levels Correlate with Progranulin Increase in Hypoxic Conditions: Implications for Frontotemporal Dementia

Progranulin (PGRN) is a secreted protein expressed ubiquitously throughout the body, including the brain, where it localizes in neurons and is activated microglia. Loss-of-function mutations in the GRN gene are an important cause of familial frontotemporal lobar degeneration (FTLD). PGRN has a neurotrophic and anti-inflammatory activity, and it is neuroprotective in several injury conditions, such as oxygen or glucose deprivation, oxidative injury, and hypoxic stress. Indeed, we have previously demonstrated that hypoxia induces the up-regulation of GRN transcripts. Several studies have shown microRNAs (miRNAs) involvement in hypoxia. Moreover, in FTLD patients with a genetic variant of GRN (rs5848), the reinforcement of miR-659-3p binding site has been suggested to be a risk factor. Here, we report that miR-659-3p interacts directly with GRN 3′UTR as shown by luciferase assay in HeLa cells and ELISA and Western Blot analysis in HeLa and Kelly cells. Moreover, we demonstrate the physical binding between GRN mRNA and miR-659-3p employing a miRNA capture-affinity technology in SK-N-BE and Kelly cells. In order to study miRNAs involvement in hypoxia-mediated up-regulation of GRN, we evaluated miR-659-3p levels in SK-N-BE cells after 24 h of hypoxic treatment, finding them inversely correlated to GRN transcripts. Furthermore, we analyzed an animal model of asphyxia, finding that GRN mRNA levels increased at post-natal day (pnd) 1 and pnd 4 in rat cortices subjected to asphyxia in comparison to control rats and miR-659-3p decreased at pnd 4 just when GRN reached the highest levels. Our results demonstrate the interaction between miR-659-3p and GRN transcript and the involvement of miR-659-3p in GRN up-regulation mediated by hypoxic/ischemic insults.

miR-CATCH: MicroRNA Capture Affinity Technology

Several experimental methods exist to explore the microRNA (miRNA) regulome. These methods almost exclusively focus on multiple targets bound to a single, or perhaps a few miRNAs of interest. Here, we describe a microRNA capture affinity technology (miR-CATCH) which uses an affinity capture oligonucleotide to co-purify a single target messenger RNA (mRNA) together with all its endogenously bound miRNAs. This bench-top method is similar to RNA immunoprecipitation (RIP) and provides an experimental alternative to computational miRNA target prediction.

microRNA regulatory circuits in a mouse model of inherited retinal degeneration

miRNA dysregulation is a hallmark of many neurodegenerative disorders, including those involving the retina. Up-regulation of miR-1/133 and miR-142, and down-regulation of miR-183/96/182 has been described in the RHO-P347S mouse retina, a model for a common form of inherited blindness. High-throughput LC-MS/MS was employed to analyse the protein expression of predicted targets for these miRNAs in RHO-P347S mouse retinas; 133 potential target genes were identified. Pathway over-representation analysis suggests G-protein signaling/visual transduction, and synaptic transmission for miR-1, and transmembrane transport, cell-adhesion, signal transduction and apoptosis for miR-183/96/182 as regulated functions in retina. Validation of miRNA-target mRNA interactions for miR-1, miR-96/182 and miR-96 targeting Ctbp2, Rac1 and Slc6a9, respectively, was demonstrated in vitro. In vivo interaction of miR-183/96/182 and Rac1 mRNA in retina was confirmed using miR-CATCH. Additional miRNAs (including miR-103-3p, miR-9-5p) were both predicted to target Rac1 mRNA and enriched by Rac1-miR-CATCH. Other Rac1-miR-CATCH-enriched miRNAs (including miR-125a/b-5p, miR-378a-3p) were not predicted to target Rac1. Furthermore, levels of ~25% of the retinal Rac1 interactors were determined by LC-MS/MS; expression of Rap1gds1 and Cav1 was elevated. Our data suggest significant utilisation of miRNA-based regulation in retina. Possibly more than 30 miRNAs interact with Rac1 in retina, targeting both UTRs and coding regions.

Toll-Like Receptors in Cystic Fibrosis: Impact of Dysfunctional microRNA on Innate Immune Responses in the Cystic Fibrosis Lung.

Toll-like receptors (TLRs) are a class of pattern recognition receptors that are particularly expressed in the sentinel and epithelial cells in the body, including the lung. They are central players in the innate immune system in response to microbial infection, and are the triggers of a complex pathway network that both promotes the inflammatory response and influences the adaptive immune response. These pathways are transiently and finely tuned by cellular factors, including a cells microRNA response program. MicroRNAs are small, non-coding RNAs that specifically regulate gene expression. In this article, we review the disease-specific microRNA regulatory network of cystic fibrosis, a debilitating and ultimately fatal disease and, specifically, its effect on TLR signalling.

Identification of MiR-21-5p as a functional regulator of mesothelin expression using microRNA capture affinity coupled with next generation sequencing

MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3’untranslated region (3’UTR). The identification of bona fide targets is challenging for researchers working on the functional aspect of miRNAs. Recently, we developed a method (miR-CATCH) based on biotinylated DNA antisense oligonucleotides that capture the mRNA of interest and facilitates the characterisation of miRNAs::mRNA interactions in a physiological cellular context. Here, the miR-CATCH technique was applied to the mesothelin (MSLN) gene and coupled with next generation sequencing (NGS), to identify miRNAs that regulate MSLN mRNA and that may be responsible for its increased protein levels found in malignant pleural mesothelioma (MPM). Biotinylated MSLN oligos were employed to isolate miRNA::MSLN mRNA complexes from a normal cell line (Met-5A) which expresses low levels of MSLN. MiRNAs targeting the MSLN mRNA were identified by NGS and miR-21-5p and miR-100-5p were selected for further validation analyses. MiR-21-5p was shown to be able to modulate MSLN expression in miRNA mimic experiments in a panel of malignant and non-malignant cell lines. Further miRNA inhibitor experiments and luciferase assays in Mero-14 cells validated miR-21-5p as a true regulator of MSLN. Moreover, in vitro experiments showed that treatment with miR-21-5p mimic reduced proliferation of MPM cell lines. Altogether, this work shows that the miR-CATCH technique, coupled with NGS and in vitro validation, represents a reliable method to identify native miRNA::mRNA interactions. MiR-21-5p is suggested as novel regulator of MSLN with a possible functional role in cellular growth.

A review of the regulatory framework for nanomedicines in the European Union

Abstract The inclusion of nanotechnology into the development and manufacturing of drugs and medical devices has brought about major gains in efficacy and accuracy. However, with these gains, the novel field of nanomedicine has also introduced many unknowns, the majority of which concern their safety to human health and the environment. Nanomedicine safety and the regulation thereof have been major topics of investigation and discussion by stakeholders in the nanomedicine field. These stakeholders also include the governmental agencies responsible for the regulation of medicines and medical devices. This chapter describes the history of nanomedicine regulation by the institutions of European Union and its member states.

Identification of miR-21 as a regulator of mesothelin expression using microRNA capture affinity coupled with next generation sequencing

Mesothelin (MSLN) protein levels are increased in malignant pleural mesothelioma (MPM). Whether this is due to alterations in microRNAs that regulate MSLN mRNA has not been studied. Here, the miR-CATCH technique (Hassan T, et al. Nucleic Acids Res. 2013; 41:e71) was applied to the MSLN gene and coupled with next generation sequencing (NGS) to identify miRNAs that regulate MSLN mRNA, and that may be responsible for its increased protein levels in MPM. Biotinylated antisense- MSLN mRNA oligonucleotides were employed to isolate miRNA:: MSLN mRNA complexes from a normal mesothelial cell line, Met5A. Library construction was performed using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1) and sequencing was performed on an Illumina MiSeq Platform using the Miseq Reagent Kit v2. Thirteen miRNAs were captured in the MSLN- vs scrambled-captured samples. miR-21 and miR-100 were selected for further validation in the Mero-14 mesothelioma cell line. A miR-21, but not miR-100, mimic inhibited MSLN protein expression in these cells (P MSLN mRNA (starting at residues 1237, 1302, 1512 and 1507). Thus miR-21 is a novel regulator of MSLN expression and may have a role in MSLN expression in MPM.