Inter-organ signalling by hepatocyte-derived microRNA-122
Matthews, Olivia Fay
MicroRNAs (miRNAs) are small non-protein coding RNA species involved in the regulation of gene expression. Bound to either protein or encapsulated in extracellular vesicles (EVs), miRNA are released from the cell and found within biological fluids. Following release, miRNAs have shown to play a role in cellular communication in vitro and to a lesser extent in vivo. The studies described in this thesis aimed to investigate the organ uptake of the hepatocyte enriched miRNA, miR-122, in vivo during in the absence and presence of paracetamol-related drug induced liver injury (DILI). As it is known that kidney tubules can internalise EVs containing miRNA, it was hypothesised that miR-122 released from the liver during paracetamol-related DILI, enters the kidney and has a protective role against subsequent injury. The production of miRNA is reliant on the activity of the cytoplasmic enzyme Dicer. To induced Dicer1 knockdown, Dicer1 flox/flox mice were treated with a hepatotropic Cre-AAV8 or its negative control Null-AAV8. In this model, investigation of Dicer expression via western blotting and RT-qPCR indicated a significant loss of Dicer in the liver one week following Cre-AAV8 treatment. Consequently, a time dependent loss of total hepatic miRNA was observed using RT-qPCR and in situ hybridisation. Cre-recombinase delivery was confirmed to be liver-specific with stable Dicer and organ-enriched miRNA expression in the spleen, kidney, heart, lung and brain. In the absence of DILI, It was found that the loss of total miRNA in the liver resulted in a significant reduction of miR-122 expression in the spleen and kidney. During paracetamol-induced liver injury, the well described increase of miR-122 in the circulation was not observed in the Cre-AAV8 treated mice. Subsequently, the increase of miR-122 expression in the spleen, kidney and heart was significantly diminished with the loss of hepatic miRNA production. Depletion of miR-122 resulted in an increase of CYP2E1 expression and activity in the liver and kidney following 300mg/kg paracetamol. Therefore, these findings show that under normal physiological conditions, expression of miR-122 in the spleen and kidney is reliant on miRNA transfer from the liver. During paracetamol induced hepatotoxicity, the mechanism of miR-122 signalling is upregulated and extends to the heart. Lastly, the observed upregulation of CYP2E1 following a loss of miR-122 in the kidneys indicates hepatic miRNA plays a role in regulating drug metabolism in the kidney.