MicroRNA-122: release, uptake and isomiRs in drug-induced liver injury
Creators: name: Tranter, John David
Publisher: name: The University of Edinburgh
Year: 2021
Subjects: microRNAs
IsomiRs
miR-122
Extracellular vesicles
3'-Dumbbell-PCR
Drug-induced liver injury
Paracetamol
Acetaminophen
Biomarkers

description: microRNAs (miRNAs) are small, non-protein coding RNAs which post-transcriptionally downregulate gene expression. Circulating miRNAs are promising clinical biomarkers, and circulate either within extracellular vesicles (ECVs) or bound to proteins, both of which protect miRNAs against degradation. Recently, small RNA sequencing has demonstrated the existence of isomiRs, of which vary in length and/or sequence when compared to the parent (canonical) miRNA. microRNA-122 (miR-122) is a liver-specific, circulating biomarker of drug-induced liver injury (DILI), notably that caused by paracetamol (APAP; acetaminophen). It has been previously demonstrated by our group that miR-122 is present in circulating exosomes, a type of ECV. IsomiRs of miR-122 were identified in serum samples obtained from patients with paracetamol-DILI by small RNA sequencing. We observed that certain isomiRs are DILI-specific. Clinically relevant isomiRs of miR-122 underwent reverse transcription quantitative PCR (RT-qPCR) using two commercially available assays for miR-122. We demonstrated that these systems are not specific for canonical miR-122, and additionally that the presence of different isomiRs interferes with the accurate quantification of miR-122. A previously published PCR-based assay termed 3’-Dumbbell-PCR (3’-Db-PCR) was adapted and optimised for the selective detection of a DILI-specific miR-122 isomiR. The 3’-Db-PCR assay was demonstrated to be more selective for the DILI-specific isomiR than standard RT-qPCR, and was applied in the subsequent in vivo study. Using a wild-type mouse model of paracetamol-DILI, we demonstrated that liver miR-122 decreased and, concurrently, circulating miR-122 increased. Over time, miR-122 increased in the renal cortex, renal medulla, and spleen. Fluorescence-activated cell sorting (FACS) of the kidney revealed that levels of miR-122 increased specifically in renal tubular cells. 3’-Db-PCR produced results which correlated to standard RT-qPCR of blood plasma, but these correlations were found to be less significant or completely insignificant in the tissues. Validation of PCR products by Sanger sequencing was inconclusive. Following this study, we utilised a genetically-modified mouse model containing a double-fluorescent Cre reporter allele in order to investigate the release of miR-122 in ECVs from the liver. These mice were administered a liver specific adeno-associated viral (AAV) vector expressing Cre recombinase, with the aim of causing the liver to produce enhanced green fluorescent protein (eGFP) tagged ECVs which could be identified in the blood, kidney, and spleen. Additionally, some of these mice were also administered paracetamol to increase ECV release. After paracetamol administration, miR-122 decreased in the liver and increased in the blood, kidney and spleen, as previously observed. Western blotting and confocal microscopy confirmed the expression of eGFP in the liver, however the expression of eGFP in the blood, kidney and spleen, was not detected. These results therefore confirm that certain isomiRs of miR-122 are DILI-specific, but current ‘gold standard’ RT-qPCR assays are unable to discriminate between canonical miR-122 and its isomiRs. Therefore, miR-122 cannot be quantified by RT-qPCR due to isomiR interference, which may also translate to other miRNAs under clinical investigation. 3’-Db-PCR is more selective than RT-qPCR assays for miR-122, but further investigations are required to validate its PCR products and therefore what the assay actually detects. miR-122 is released from the liver in paracetamol-DILI into the circulation, and is then taken up by both the spleen and renal tubular cells. The confirmation of ECV-dependent or independent miR-122 release and uptake into these organs in liver-derived ECVs must be investigated further by refining our current model, using a different model, and/or by use of different techniques.