Serum microRNA signatures as "liquid biopsies" for interrogating hepatotoxic mechanisms and liver pathogenesis in human

Abstract

MicroRNAs (miRNAs) released into the peripheral circulation upon cellular injury have shown a promise as a new class of tissue-specific biomarkers. We were first to demonstrate that next-generation sequencing analysis of serum from human subjects with acetaminophen-induced liver injury revealed a specific signature of circulating miRNAs. We consequently hypothesized that different types of hepatic liver impairments might feature distinct signatures of circulating miRNAs and that this approach might be useful as minimally invasive diagnostic “liquid biopsies” enabling the interrogation of underlying molecular mechanisms of injury in distant tissues. Therefore we examined serum circulating miRNAs in a total of 72 serum samples from a group of 53 subjects that included patients with accidental acetaminophen overdose, hepatitis B infection, liver cirrhosis and type 2 diabetes as well as gender- and age-matched healthy subjects with no evidence of liver disease. The miRNA signatures were identified using next-generation sequencing that provided analysis for the whole miRNome, including miRNA isoforms. Compared to the healthy subjects, a total of 179 miRNAs showed altered serum levels across the diseased subjects. Although many subjects have elevated alanine aminotransferase suggesting liver impairments, we identified distinct miRNA signatures for different impairments with minimum overlap. Furthermore, the bioinformatics analysis of miRNA signatures revealed relevant molecular pathways associated with the mechanisms of toxicity and or pathogenesis of disease. Interestingly, the high proportion of miRNA isoforms present in the respective signatures indicated a new level of complexity in cellular response to stress or disease. Our study demonstrates for the first time that signatures of circulating miRNAs show specificity for liver injury phenotypes and, once validated, might become useful for diagnosis of organ pathologies as “liquid biopsies”.

Acetaminophen Overdose as a Potential Risk Factor for Parkinson's Disease

“…To determine whether acetaminophen overdose might increase PD risk, the levels of circulating miRNAs in serum from both patients with PD and subjects who were hospitalized with acetaminophen overdose were compared ( Table ). Serum levels of three circulating miRNAs, hsa‐miR‐192, hsa‐miR‐24, and hsa‐miR‐30c, showed the same directional change (increase or decrease when compared with healthy subjects) in patients with PD and in subjects with acetaminophen overdose.…”

“…Circulating miRNAs were found to be stable even under conditions as harsh as boiling, extreme pH, long‐time storage at room temperature, and multiple freeze‐thaw cycles . Details regarding sample collection, sample preparation, sequencing, and data processing can be found in Krauskopf et al ., and see for patient characteristics and levels of liver damage markers. Circulating miRNAs characteristically present in the serum of patients with PD were gathered from Khoo et al .…”

Direct detection of circulating microRNA-122 using dynamic chemical labelling with single molecule detection overcomes stability and isomiR challenges for biomarker qualification

“…24 Krauskopf et al also used RNA sequencing and reported substantially increased circulating isomiRs with DILI in humans. 25 In mice, Chowdhary et al performed Northern blotting to demonstrate miR--122 isomiRs in the circulation following paracetamol toxicity. 26 In our study, the concentration of isomiRs increased with sample degradation and, importantly, microRNA degradation was substantially increased with DILI.…”

Accessible miRNAs as Novel Toxicity Biomarkers

“…42,43 With RNA-sequencing technologies allowing for cheaper and efficient analyses of the full panel of miRNAs, rather than a targeted qPCR assay, researchers have started to employ miRNA profiles or "liquid biopsy" approaches in the patients with tissue injury, as well as in disease populations. 37,[43][44][45] These approaches not only help identify additional miRNA candidates but also provide a pattern of miRNAs associated with both certain disease states and types of injury.…”