Dual microRNA blockade increases expression of antioxidant protective proteins; implications for ischaemia reperfusion injury
2020
BACKGROUND: MicroRNAs (miRNA) are short noncoding 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 2 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.
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