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Lookup NU author(s): Dr Sam Tingle, Dr Avinash Sewpaul, Lucy BatesORCiD, Dr Emily ThompsonORCiD, Victoria Shuttleworth, Rodrigo Figueiredo, Dr Ibrahim Ibrahim, Professor Simi Ali, Colin Wilson, Professor Neil SheerinORCiD
This is the authors' accepted manuscript of an article that has been published in its final definitive form by Lippincott Williams & Wilkins, 2020.
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Background: MicroRNAs (miRNA) are short non-coding RNAs which each cause repression of many target genes. Previous work has demonstrated that therapeutic blockade of single microRNAs is possible. miR-24-3p and miR-145-5p are reported to have a detrimental role in ischaemia reperfusion injury (IRI). As the action of miRNAs is inhibitory, we hypothesised that dual blockade of both miRNAs could synergistically upregulate shared target genes.Methods: Quantification of miRNA expression in donated kidneys was performed using PCR panels. IRI was modelled in vitro by placing Human Umbilical Vein Endothelial Cells (HUVECs) into a hypoxic incubator (1% O2) for 24hrs, with reoxygenation for 6hrs. RNA expression was quantified with RT-qPCR and protein expression assessed with Western blot. Antisense oligonucleotides (ASOs) were used to inhibit miRNAs.Results: miR-24-3p and miR-145-5p were highly expressed in human kidneys following extended cold ischemia. In vitro, hypoxia caused significant upregulation of miR-24-3p (p≤0.001) and miR-145-5p (p≤0.001), and significant downregulation in mRNA of shared targets SOD2 (p≤0.001) and HMOX1 (p≤0.001). These changes were mirrored at the protein level. Dual inhibition of both miR-24-3p and miR-145-5p prior to hypoxia-reoxygenation caused significant upregulation of SOD2 and HMOX1 protein; fold-change of 3.17 (p≤0.05) and 6.97 (p≤0.05) respectively. Dual inhibition resulted in reduced cellular ROS production compared to negative control (p≤0.05) and single blockade of miR-24-3p (p≤0.01) or miR-145-5p (p≤0.05).Conclusion: Dual blockade of two miRNAs can act synergistically to increase the expression of shared gene targets. Dual blockade of miR-24-3p and miR-145-5p represents a novel therapeutic option worthy of further research.
Author(s): Tingle SJ, Sewpaul A, Bates L, Thompson ER, Shuttleworth V, Figueiredo R, Ibrahim IK, Ali S, Wilson C, Sheerin NS
Publication type: Article
Publication status: Published
Journal: Transplantation
Year: 2020
Volume: 104
Issue: 9
Pages: 1853-1861
Print publication date: 01/09/2020
Online publication date: 02/03/2020
Acceptance date: 04/02/2020
Date deposited: 15/02/2020
ISSN (print): 0041-1337
ISSN (electronic): 1534-6080
Publisher: Lippincott Williams & Wilkins
URL: https://doi.org/10.1097/TP.0000000000003215
DOI: 10.1097/TP.0000000000003215
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