Mitochondrial miR-23b-5p is a new biomarker of warm ischemic injury in donor livers and a candidate for graft evaluation: experimental studies

Abstract

Background and aims: Warm ischaemic injury (WII) stems from incorrect energy metabolism and is the main cause of graft dysfunction. Mitochondria, as the centre of cellular metabolic activities, may be the key in identifying accurate indicators for evaluating the quality of grafts. Our research focuses on the screening, clinical application, and mechanism of the optimal WII mitochondrion biomarker. Approach and results: Using a 100% hepatic warm ischaemia mouse model, without reperfusion, transmission electron microscopy demonstrated evident morphological changes of hepatic mitochondria at 15 min of ischaemia. However, all 13 mt-mRNAs could not display continuously upregulated consistency at 0–15–30–60 min during WII. High-throughput analysis of miRNA expression in both purified mitochondria and liver tissues suggested miR-23b-5p was a potential mitochondrial microRNA (mitomiR) biomarker with high sensitivity and 0-15-30-60 min change consistency. Fluorescence in-situ hybridization and reverse transcription quantitative polymerase chain reaction (RT-qPCR) further confirmed the results. Through overexpression and inhibition, the functionality of this mitomiR during WII was identified as a protective regulator in vitro and then verified in Dicer1 fl/fl Alb Cre mice by downregulation of other miRNAs and supplementation of mature mitomiR-23b-5p. Dual-luciferase reporter assay and the Seahorse XF analyzer determined that mitomiR-23b-5p reduced mitochondrial respiratory function by silencing mt-RNR2 (16S). Clinically, mitomiR-23b-5p was positively correlated with serum alanine aminotransferase levels 3 days after the operation ( P =0.032), and the C-statistic for 90-day graft survival rate was 0.698. Conclusions: MitomiR-23b-5p plays a protective regulatory role and implements a special mitochondrial regulation mechanism not yet reported in WII. These clinical results further support the experimental result that the expression of MitomiR-23b-5p is closely related to the prognosis of clinical liver transplantation patients. This is a promising new biomarker for WII evaluation of donor livers.