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Background MicroRNAs (miRNAs) have emerged as important regulators of cellular functions in response to injury. Recent studies have shown that specific hepatocyte and cholangiocyte-derived miRNAs (respectively HDmiRs and CDmiRs) are released during liver injury into the circulation and are predictive markers for graft function after liver transplantation. The cellular mechanism of miRNA release in relation to liver injury however remains largely unknown. The aim of this study was to investigate bidirectional release of HDmiRs to bile and serum in patients with liver injury and acute rejection after liver transplantation. Methods Paired bile and serum samples (n=62) from liver transplant recipients were collected at different time-points following transplantation. Cell-free solutions were analyzed for HDmiRs and CDmiRs by quantitative qRT-PCR. Cell-free bile samples obtained from donor gall bladders were used for stability and fractionation experiments. Results Hepatocyte abundance of HDmiR-122 and HDmiR-148a was confirmed by comparing expression levels in n=24 liver biopsies and n=8 extrahepatic bile duct biopsies (respectively 140-fold and 6-fold enriched).Fractionation of fresh bile showed that HDmiRs are mostly present in non-pelletable molecular complexes. Less than 6% of HDmiRs and CDmiRs were found in pelletable cell fragments or microvesicles. Despite the toxic environment of bile, HDmiRS and CDmiRs were stable and protected from degradation for at least 1 hour at 37°C. In patients experiencing liver injury and biopsy-proven acute rejection, serum levels of HDmiR-122 were significantly higher (P<0.001), while levels in bile were decreased (P=0.011). This opposite or polarized release of HDmiRs into bile and blood is shown even stronger when liver function is compared based on conjugated bilirubin levels in bile. Liver grafts with good conjugation showed an increased release of all HDmiRs into bile (P<0.001) but decreased levels into serum (P<0.01), while the opposite was found in liver grafts with poor conjugating function. Levels of CDmiRs in bile were significantly decreased with high bilirubin secretion. 

Conclusion This study demonstrates that HDmiRs are directional released into bile and blood, influenced by the conjugating function of the transplanted liver graft. These findings shed new light on HDmiRs as biomarkers for hepatic injury and function after liver transplantation.