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      Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage

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          Abstract

          Intraventricular hemorrhage (IVH) represents a high risk of neonatal mortality and later neurodevelopmental impairment in prematurity. IVH is accompanied with inflammation, hemolysis, and extracellular hemoglobin (Hb) oxidation. However, microRNA (miRNA) expression in cerebrospinal fluid (CSF) of preterm infants with IVH has been unknown. Therefore, in the present study, candidate pro-inflammatory cell-free miRNAs were analyzed in CSF samples from 47 preterm infants with grade III or IV IVH vs. clinical controls ( n = 14). miRNAs were quantified by RT-qPCR, normalized to “spike-in” cel-miR-39. Oxidized Hb and total heme levels were determined by spectrophotometry as well as IL-8, VCAM-1, ICAM-1, and E-selectin concentrations by ELISA. To reveal the origin of the investigated miRNAs, controlled hemolysis experiments were performed in vitro; in addition, human choroid plexus epithelial cell (HCPEpiC) cultures were treated with metHb, ferrylHb, heme, or TNF-α to replicate IVH-triggered cellular conditions. Levels of miR-223, miR-155, miR-181b, and miR-126 as well as Hb metabolites along with IL-8 were elevated in CSF after the onset of IVH vs. controls. Significant correlations were observed among the miRNAs, oxidized Hb forms, and the soluble adhesion molecules. During the post-IVH follow-up, attenuated expression of miRNAs and protein biomarkers in CSF was observed upon elimination of Hb metabolites. These miRNAs remained unaffected by a series of artificially induced hemolysis, which excluded red blood cells as their origin, while stimulation of HCPEpiCs with oxidized Hb fractions and heme resulted in increased extracellular miRNA levels in the cell culture supernatant. Overall, the hemorrhage-induced CSF miRNAs reflected inflammatory conditions as potential biomarkers in preterm IVH.

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          Regulation of mRNA translation and stability by microRNAs.

          MicroRNAs (miRNAs) are small noncoding RNAs that extensively regulate gene expression in animals, plants, and protozoa. miRNAs function posttranscriptionally by usually base-pairing to the mRNA 3'-untranslated regions to repress protein synthesis by mechanisms that are not fully understood. In this review, we describe principles of miRNA-mRNA interactions and proteins that interact with miRNAs and function in miRNA-mediated repression. We discuss the multiple, often contradictory, mechanisms that miRNAs have been reported to use, which cause translational repression and mRNA decay. We also address the issue of cellular localization of miRNA-mediated events and a role for RNA-binding proteins in activation or relief of miRNA repression.
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            Incidence and evolution of subependymal and intraventricular hemorrhage: A study of infants with birth weights less than 1,500 gm

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              Haemolysis during Sample Preparation Alters microRNA Content of Plasma

              The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                19 September 2020
                September 2020
                : 21
                : 18
                : 6870
                Affiliations
                [1 ]Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary; fejes.zsolt@ 123456med.unideb.hu (Z.F.); pocsi.marianna@ 123456med.unideb.hu (M.P.); jun_takai@ 123456hotmail.com (J.T.); kappelmayer@ 123456med.unideb.hu (J.K.)
                [2 ]Kálmán Laki Doctoral School of Biomedical and Clinical Sciences, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary
                [3 ]MTA-DE Lendület Vascular Pathophysiology Research Group, Research Centre for Molecular Medicine, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary; jutka.erdei@ 123456gmail.com (J.E.); jeneyv@ 123456belklinika.com (V.J.)
                [4 ]Doctoral School of Molecular Cell and Immune Biology, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary
                [5 ]Department of Pediatrics, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary; nagyand@ 123456med.unideb.hu
                [6 ]Department of Immunology, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary; varga.aliz@ 123456med.unideb.hu (A.V.); etele@ 123456med.unideb.hu (A.B.)
                [7 ]Department of Neurosurgery, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary; bognar.laszlo@ 123456med.unideb.hu (L.B.); lnovak@ 123456med.unideb.hu (L.N.)
                Author notes
                [* ]Correspondence: nagy.bela@ 123456med.unideb.hu ; Tel.: +36-52-340-006
                Author information
                https://orcid.org/0000-0002-1387-2970
                https://orcid.org/0000-0003-1652-4506
                https://orcid.org/0000-0003-4942-7091
                Article
                ijms-21-06870
                10.3390/ijms21186870
                7557369
                32961661
                2e4f2e65-c092-4ef5-a32e-fcf8d6252efa
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 25 August 2020
                : 16 September 2020
                Categories
                Article

                Molecular biology
                cerebrospinal fluid,intraventricular hemorrhage,inflammation,microrna,oxidized hemoglobin,heme

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