The MicroRNA Expression Profile in Donation After Cardiac Death (DCD) Liver Grafts and Its Ability to Identify Primary Non Function.

Abstract

Purpose DCD grafts are a marginal organ and usage has to be balanced against the risk of primary non function (PNF) or initial poor graft function (IPGF). To give insight into pathophysiology and potentially rationalise DCD usage, the aim of this work was to determine if a microRNA expression profile was able to discriminate between DCD grafts of varying functional quality,. Methods Three DCD groups were studied. PNF group (n=8) retransplanted within a week, good group (n=7) AST ≤ 1000 IU/L and poor group/IPGF (n=8) AST ≥ 2500 IU/L. miRNA was isolated from FFPE trucut post perfusion biopsies. Affymetrix GeneChip® miRNA 2.0 Arrays were used. Expression data analysis was performed using Qlucore Omics Explorer 3.0. A statistical significance level of p<0.05 was used to identify differentially expressed miRNA in comparison of the three groups. To validate microarray results real time quantitative PCR (RT-qPCR) was performed on all samples for miRNA species identified as being significant. Ct (threshold cycle) values were normalised using the average Ct of the endogenous control to generate ΔCT and fold-change to reference sample of normal liver (2ΔΔCT) for relative expression analysis. Data was then analysed by one way non parametric ANOVA to detect significance (p<0.05) between the three groups. If significance was found the Bonferroni correction for multiple comparisons was applied. Results From the array analysis 16 miRNA species (miR-107, miR-378, miR-23b and miR-122_st, miR-103, miR-125b, miR-24, miR-let-7a, miR-191, miR-194, miR-296-5p, miR-455-3p, miR-940, miR-let-7d, miR-22 and miR-155) were identified as being significantly different (p<0.05) and of potential biological interest. miR-155 and miR-940 had the highest relative expression across all samples. The miRNA that was found to have a significant differential expression between the three groups was miR-22. From computational biology analysis miR-22 was predicted to influence signalling pathways that impact protein turnover, metabolism and apoptosis/cell cycle. Conclusion The miRNA species (miR-22), that appeared to define whether a poor quality graft had the potential to recover (IPGF) to one that would not (PNF), are those that contribute to the ‘survival response' by minimising cellular replacement processes and halting apoptotic initiation of proliferation to conserve energy.