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Dual microRNA blockade increases expression of antioxidant protective proteins; implications for ischaemia reperfusion injury

Lookup NU author(s): Dr Sam Tingle, Dr Avinash Sewpaul, Lucy Bates, Dr Emily Thompson, Victoria Shuttleworth, Rodrigo Figueiredo, Dr Ibrahim Ibrahim, Professor Simi Ali, Colin Wilson, Professor Neil Sheerin

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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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Abstract

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.


Publication metadata

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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