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      Regulation of NADPH Oxidase Activity Is Associated with miRNA-25-Mediated NOX4 Expression in Experimental Diabetic Nephropathy

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          Abstract

          Background/Aims: Although numerous studies have explored the mechanisms regulating the enzyme activity of NADPH oxidase in diabetic nephropathy (DN), little information is available for the contribution of microRNAs (miRNAs) to the regulation of NADPH oxidase expression. Therefore, the present study was to test whether miRNAs importantly contribute to the regulation of NOX4 expression, a major catalytic subunit of NADPH oxidase under hyperglycemia. Methods: Diabetic rats were induced by streptozotocin. miRNA microarray, Western blot, real-time RT-PCR and luciferase reporter assays were employed in this study. Results: Among 5 miRNAs, which are predicted to have a binding capacity to rat NOX4, the miRNA-25 level was significantly reduced both in the kidney from diabetic rats and in high glucose-treated mesangial cells, accompanied by the increases in NOX4 expression levels. In an in vitrostudy, we found that NADPH activity was increased by 226.2% in miRNA-25 inhibitor transfected cells and decreased by 51.0% in miRNA-25 precursor transfected cells. miR-25 inhibitor dramatically increased both NOX4 mRNA and protein levels. We then showed that miR-25 negatively regulated NOX4 expression by directly targeting the 3′-UTR by luciferase reporter assays. It was found that transfection of miR-25 precursor significantly decreased the luciferase activity of NOX4 3′-UTR by 39.5%, whereas the mutant sequence restored levels to 79.4%. Finally, our results indicated that the miR-25-mediated NOX4 mRNA level may result from the regulation of mRNA stability. Conclusions: These findings for the first time indicate that miRNA-25 may serve as an endogenous gene silencing factor and contributes to the regulation of NOX4 expression and function in DN.

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          Most cited references 22

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          Genetic variation in microRNA networks: the implications for cancer research.

          Many studies have highlighted the role that microRNAs have in physiological processes and how their deregulation can lead to cancer. More recently, it has been proposed that the presence of single nucleotide polymorphisms in microRNA genes, their processing machinery and target binding sites affects cancer risk, treatment efficacy and patient prognosis. In reviewing this new field of cancer biology, we describe the methodological approaches of these studies and make recommendations for which strategies will be most informative in the future.
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            Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation.

            MicroRNAs (miRNAs) are approximately 22 nucleotide RNAs that negatively regulate the expression of protein-coding genes. In a present model of miRNA function in animals, miRNAs that form imperfect duplexes with their targets inhibit protein expression without affecting mRNA levels. Here, we report that in C. elegans, regulation by the let-7 miRNA results in degradation of its lin-41 target mRNA, despite the fact that its 3'UTR regulatory sequences can only partially base-pair with the miRNA. Furthermore, lin-14 and lin-28 are targets of the lin-4 miRNA, and we show that the mRNA levels for these protein-coding genes significantly decrease in response to lin-4 expression. This study reveals that mRNAs containing partial miRNA complementary sites can be targeted for degradation in vivo, raising the possibility that regulation at the level of mRNA stability may be more common than previously appreciated for the miRNA pathway.
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              MicroRNAs direct rapid deadenylation of mRNA.

              MicroRNAs (miRNAs) are ubiquitous regulators of eukaryotic gene expression. In addition to repressing translation, miRNAs can down-regulate the concentration of mRNAs that contain elements to which they are imperfectly complementary. Using miR-125b and let-7 as representative miRNAs, we show that in mammalian cells this reduction in message abundance is a consequence of accelerated deadenylation, which leads to rapid mRNA decay. The ability of miRNAs to expedite poly(A) removal does not result from decreased translation; nor does translational repression by miRNAs require a poly(A) tail, a 3' histone stem-loop being an effective substitute. These findings suggest that miRNAs use two distinct posttranscriptional mechanisms to down-regulate gene expression.
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                Author and article information

                Journal
                AJN
                Am J Nephrol
                10.1159/issn.0250-8095
                American Journal of Nephrology
                S. Karger AG
                0250-8095
                1421-9670
                2010
                December 2010
                11 November 2010
                : 32
                : 6
                : 581-589
                Affiliations
                aDepartment of Pharmacology, Shandong University School of Medicine, bDepartment of Medicinal Chemistry, Shandong University School of Pharmacy, and Departments of cObstetrics and Gynaecology and dNeurosurgery, Provincial Hospital Affiliated to Shandong University, Jinan, PR China
                Author notes
                *Prof. Fan Yi, PhD, Department of Pharmacology, Shandong University School of Medicine, 44, Wenhua Xi Road, Jinan, Shandong 250012 (PR China), Tel. +86 0531 8838 2616, Fax +86 0531 8838 2616, E-Mail fanyi@sdu.edu.cn
                Article
                322105 Am J Nephrol 2010;32:581–589
                10.1159/000322105
                21071935
                © 2010 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Counts
                Figures: 4, References: 24, Pages: 9
                Categories
                Original Report: Laboratory Investigation

                Cardiovascular Medicine, Nephrology

                Diabetic nephropathy, Mesangial cells, MicroRNA, Oxidative stress

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