Abstract
Micro-RNA (miR)-122 is a promising exploratory biomarker for detecting liver injury in preclinical and clinical studies. Elevations in serum or plasma have been associated with viral and autoimmune hepatitis, non-alcoholic steatohepatitis (NASH), hepatocellular carcinoma, and drug-induced liver injury (DILI). However, these associations were primarily based upon population differences between the disease state and the controls. Thus, little is known about the variability and subsequent variance components of circulating miR-122 in healthy humans, which has implications for the practical use of the biomarker clinically. To address this, we set out to perform variance components analysis of miR-122 in a cohort of 40 healthy volunteers. Employing a quantitative real-time polymerase chain reaction (qRT-PCR) assay to detect miR-122 and other circulating miRNAs in human serum, the relative expression of miR-122 was determined using two different normalization approaches: to the mean expression of a panel of several endogenous miRNAs identified using an adaptive algorithm (miRA-Norm) and to the expression of an exogenous miRNA control (Caenorhabditis elegans miR-39). Results from a longitudinal study in healthy volunteers (N = 40) demonstrated high variability with 117- and 111-fold 95% confidence reference interval, respectively. This high variability of miR-122 in serum appeared to be due in part to ethnicity, as 95% confidence reference intervals were approximately three-fold lower in volunteers that identified as Caucasian relative to those that identified as Non-Caucasian. Variance analysis revealed equivalent contributions of intra- and inter-donor variability to miR-122. Surprisingly, miR-122 exhibited the highest variability compared to other 36 abundant miRNAs in circulation; the next variable miRNA, miR-133a, demonstrated a 45- to 62-fold reference interval depending on normalization approaches. In contrast, alanine aminotransferase (ALT) activity levels in this population exhibited a 5-fold total variance, with 80% of this variance due to inter-donor sources. In conclusion, miR-122 demonstrated higher than expected variability in serum from healthy volunteers, which has implications for its potential utility as a prospective biomarker of liver damage or injury.
Highlights
Twenty-five years ago, Ambros and colleagues discovered and characterized the first microRNA [1]
Samples were excluded from subsequent analysis based on review of standard clinical chemistry parameters: two subjects were removed from our analysis due to two-fold or greater elevations in either alanine aminotransferase (ALT) activities (Donor 8) compared to the upper limit of normal (ULN) of the testing laboratory’s reported reference intervals, or multiple, atypically elevated blood glucose
A depiction of the movement of the expression of miRNAs, including the 5-selected endogenous miRNAs, and the mean values of these are represented in S1 Fig. The selection of these five miRNAs for normalization was consistent over 4 other human serum sample sets from patients in a controlled clinical setting analyzed by identical quantitative real-time polymerase chain reaction (qRT-PCR) methods and the panel of miRNAs outlined here
Summary
Twenty-five years ago, Ambros and colleagues discovered and characterized the first microRNA [1] Since these small noncoding 22-nucleotide miRNAs have been shown to be important mediators in epigenetic, transcriptional, and post-transcriptional gene regulation [2, 3], as well as cellular proliferation, differentiation, metabolism, infectious and non-infectious disease progression, apoptosis and metastasis [2, 4,5,6]. Circulating miRNAs are actively being pursued as both therapeutic targets as well as biomarkers of disease and toxicities In circulation, they exist as two predominant species as either protein bound (in particular, bound to Argonaut-2 [11]), or encapsulated in extracellular vesicles or exosomes [4]. These different circulating species are believed to be leakage markers from necrotic or damaged cells and tissues, or they may originate from active secretion processes [5]
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