Samuel Tingle, Avinash Sewpaul, Lucy Bates, Rodrigo Figueiredo, Simi Ali, Neil Sheerin, Colin Wilson
Ischaemia reperfusion injury (IRI) causes significant damage to organs from deceased donors. microRNAs are short non-coding RNAs which each cause repression of many target genes. miR-24-3p and miR-145-5p are two microRNAs with potential detrimental roles in IRI. Our group has used antisense oligonucleotides (ASO) to block miR-24-3p during ex vivo normothermic perfusion (EVNP) of human kidney grafts. This project aimed to model changes in miR-24-3p and miR-145-5p with hypoxia and assess whether ASO therapy can alter the expression of the genes which they regulate.
IRI was modelled by placing Human Umbilical Vein Endothelial Cells (HUVECs) into a hypoxic incubator for 24hrs, followed by reoxygenation for 6hrs. Lipofectamine was used to transfect cells with ASO to block miRNAs. microRNA and mRNA expression was quantified with reverse-transcription qPCR. One-way ANOVA was used for statistical analysis.
Hypoxia caused significant upregulation of miR-24-3p (1.51;p≤0.001) and miR-145-5p (1.95;p≤0.001), and significant downregulation of miR-24-3p target HMOX1 (0.165;p≤0.001) and shared target SOD2 (0.502;p≤0.001) in HUVECs. Using ASO transfection to inhibit miR-24-3p and miR-145-5p in combination caused synergistic upregulation of HMOX1 (2.62;p<0.05) and SOD2 (1.61;p<0.05) following hypoxia and reoxygenation (Figure 1).
Dual blockade of miR-24-3p and miR-145-5p significantly increased expression antioxidant genes HMOX1 and SOD2, which both have established protective roles in renal IRI. Dual blockade of miR-24-3p and miR-145-5p during EVNP represents a novel therapeutic option worthy of further research.