Opioids are the most widely used analgesics for the treatment of pain and related disorders. Four distinct opioid receptors have been reported, namely, μ, κ and δ receptors and opioid receptor-like-1 receptor, all of which are classified as G-protein coupled proteins [1
]. μ-opioid receptor (MOR) is primarily responsible for opioid analgesia and is encoded by the OPRM1 gene, which can produce various alternatively spliced variants in humans [2
]. In the past decade, opioids have been increasingly used for pain management; however, their widespread use has been accompanied by increased drug abuse, overdose, and even death [3
]. Although opioids are generally effective analgesics for chronic pain, individual variability exists among patients in terms of efficacy and side effects, thereby increasing the demand for personalized medication strategies to maximize patient benefit and cost-effectiveness [3
]. However, in the clinical setting, very limited options are currently available for effective patient stratification to identify individuals who are expected to optimally benefit from opioid treatment. Imaging technology represents one of the most promising tools that can be applied to investigate the functional dynamics in the central nervous system (CNS) in response to opioid treatment in males [4
] and is particularly expected to be useful for analgesic drug development. However, measurable biomarkers using easily accessible biological specimens, such as blood, are desired for practical medical use.
MicroRNAs (miRNAs) represent a class of non-coding small RNAs with average lengths of 22-nucleotide and are produced by the RNase III proteins Drosha and Dicer [6
]. Mature single-strand miRNA is incorporated into RNA-induced silencing complex, which guides the complex into complementary 3′- or 5′-untranslated regions (UTRs), open reading frames, or promoter regions of target genes [7
]. miRNAs generally act as negative regulators of target gene products via mRNA cleavage or translational repression, but in some cases, miRNAs can also upregulate target genes [8
]. It is now widely recognized that miRNAs play crucial roles in almost all aspects of developmental and pathological processes, and disruptions in miRNA regulation are associated with certain types of cancers, liver and cardiovascular diseases, and CNS impairments, including schizophrenia and drug addiction [9
Besides its functional importance, miRNAs also exhibit advantageous characteristics as biomarkers for clinical use [11
]. In particular, miRNAs are remarkably stable in body fluids, such as blood, and even under varying pH conditions, boiling, multiple freeze-thaw cycles, and extended storage [12
]. The high stability of miRNAs in the blood is attributed to binding to Argonaute 2 protein or incorporation to microvesicles, such as exosomes, which protect miRNAs from degradation under RNase-rich circulation conditions [13
]. Exosomes are small vesicles with diameters ranging from 30–120 nm that are present in most biological fluids. Exosomes act as carriers of various molecules, such as proteins, lipids, mRNAs, and miRNAs [14
] and can cross the blood-brain barrier [15
]. A number of reports have revealed that certain miRNAs can be secreted by tumor cells [7
]. Studies have also demonstrated that corresponding exosome miRNA profiles of tumor cells can reflect the intracellular miRNA content, although whether the selectivity of miRNAs embedded into exosomes is strictly regulated remains to be clarified [16
Previous studies have demonstrated that various miRNAs, such as let-7 family of miRNAs and miR-23b, are functionally associated with the endogenous MOR system and pain signaling [2
]. Neurons and glial cells actively release microvesicles into the extracellular space, thereby providing evidence that exosome-mediated intracellular communication may be a common mechanism in CNS [17
]. Thus, we hypothesized that the pharmacological response of CNS cells against MOR activation can alter plasma miRNA profiles, which can in turn act as potential blood biomarkers that can be used to evaluate MOR activation.
The objective of the present study was to identify plasma miRNAs that are differentially regulated upon treatment with the MOR activators hydromorphone and oxycodone [18
]. Measurements were performed on blood samples previously collected from two independent clinical studies, in which extended-release (ER) tablets of hydromorphone or oxycodone were administered to healthy male volunteers [19
Emerging evidence has revealed the functional involvement of miRNAs in MOR signaling. However, these findings are mostly limited to intracellular regulatory mechanisms. Thus, the present study aimed to identify candidate circulating biomarkers to evaluate MOR stimulation using plasma samples collected from two previously conducted independent clinical studies, in which either hydromorphone or oxycodone were administered to healthy subjects [20
Nine miRNAs were commonly upregulated by both hydromorphone and oxycodone treatments (Table 1
), including let 7a-5p, miR-146a-5p and miR-23b-3p. Binding of the let-7 family of miRNAs to the 3′-UTR of OPRM1 suggests their involvement in opioid tolerance [22
], and therefore the observed upregulation of let-7a-5p may reflect negative feedback response against MOR stimulation. miR-146a-5p was observed to be upregulated upon morphine treatment in human monocyte-derived macrophages [23
] and was reported to play a role in pain signaling [24
]. A previous study demonstrated that miR-23b-3p is upregulated by morphine and is involved in the modulation of MOR signaling [25
]. On the other hand, seventeen miRNAs were downregulated in both hydromorphone- and oxycodone-treated subjects (Table 2
). However, their corresponding roles in MOR signaling remain to be elucidated.
In addition, several miRNAs that were reported to be functionally associated with MOR signaling were found to be differentially regulated by either hydromorphone or oxycodone. Six miRNAs, namely, let-7f-5p, let-7d-5p, let-7c, let-7e-5p, miR-181a-5p and miR-103a-3p, were upregulated upon hydromorphone treatment (Table 1
). let-7 Family miRNAs (i.e., let-7f-5p, let-7d-5p, let-7c and let-7e-5p) potentially modulate MOR function as previously mentioned [22
]. miR-181a-5p was found to be upregulated by morphine treatment in mouse hippocampal progenitor cell lineages, suggesting its potential role in neurogenesis [26
] and drug addiction [27
]. miR-103a-3p was also reported to be upregulated following chronic exposure to morphine, which is known to downregulate MOR [28
]. On the other hand, oxycodone treatment resulted in the upregulation of two miRNAs, namely, miR-339-3p and miR-23a-3p (Table 1
), and miR-339-3p was reported to inhibit MOR function [29
]. Thus, the upregulation of these miRNAs provide evidence of negative feedback responses against MOR stimulation by hydromorphone or oxycodone. Taken together, all the aforementioned differentially regulated miRNAs are promising candidates as circulating biomarkers for evaluating MOR stimulation (Figure 6
Our current findings provided valuable insights into MOR signaling-associated miRNA regulation. However, certain limitations need to be addressed. First, the multiplex qRT-PCR panel system used in the study does not cover a sufficient number of circulating miRNA species, considering the existence of a large number of miRNAs [30
]. In future studies, miRNA-Seq analysis using next-generation sequencing technology can be used to obtain more comprehensive miRNA expression profiles; Second, additional observational time points after treatment with multiple opioids are required for a more detailed characterization of miRNA expression profiles following opioid treatment; Third, additional experiments must be conducted to validate the present findings and establish reproducibility. Although the use of miRNA-based biomarkers in diagnosis has long been expected, specificity and reproducibility in previous experiments have been generally found to be poor [10
]. Indeed, the number of commonly upregulated miRNAs represented only 26% to 41% of all miRNAs upregulated in response to treatment with hydromorphone and oxycodone. One possible explanation for the low overlap is that hydromorphone and oxycodone may elicit agonist-specific pharmacological effects similar to the mechanisms by which morphine and fentanyl activate the protein kinase C and β-arrestin2 pathways following MOR stimulation, respectively [31
]. Although hydromorphone acts primarily on MOR and to a lesser extent on the δ-opioid receptor, oxycodone exerts action on multiple receptors including the κ-opioid receptor [18
]. Thus, although the general profiles of differentially regulated miRNAs represent the molecular dynamics associated with MOR stimulation, hydromorphone and oxycodone can also elicit drug-specific responses that can influence miRNA expression regulation. In addition, the experimental procedure and analytical methods for measurement of concentrations of circulating miRNAs have yet to be optimized in general; e.g., potential confounding factors such as variation in the results of RNA extraction and PCR and the normalization method for qRT-PCR data.
Lastly, findings from the present study may not be directly applicable to diseased subjects, since pathological conditions, such as cancer or drug addiction, are likely to considerably affect plasma miRNA profiles. For instance, let-7 family miRNAs were identified as candidate biomarkers to evaluate MOR signaling in the present study. However, let-7 miRNAs are also known to act as tumor suppressors by targeting various oncogenes [32
]; let-7 miRNAs can be selectively incorporated into exosomes and secreted from tumor cells [33
], resulting in elevated circulation levels. Increased circulating let-7 miRNAs can confound the interpretation of plasma let-7 expression profiles following opioid treatment. Indeed, although the present study was conducted on healthy volunteers without disease-related confounding factors, changes in the plasma let-7 level could not clearly separate subjects into the groups before and after drug treatment. These data suggest that let-7 miRNA alone will not be a practical clinical biomarker, and no other miRNAs quantified in the present study can serve as such an ideal biomarker. One potential solution is to investigate larger sets of miRNAs, such as those shown in Figure 6
, instead of more specific miRNAs (e.g., let-7 family miRNAs) to capture systems-level effects (e.g., MOR stimulation). Previous studies have applied similar concepts for interpreting mRNA transcriptome data and have demonstrated that analysis using pre-specified gene sets, and not individual genes, can facilitate understanding systems-level biological impacts following drug treatment [34
]. The accumulation of more miRNA profiling data in various diseased conditions will advance our knowledge of circulating miRNA profiles and explore their potential use in evaluating MOR signaling.
In conclusion, we identified circulating miRNAs that were differentially regulated upon hydromorphone and/or oxycodone treatment. These miRNAs serve as promising candidate biomarkers to evaluate MOR activation in the body. Further investigation will be required to better characterize these miRNAs and can facilitate the development of novel diagnostic tools to support personalized opioid medication and to improve clinical benefit and maximize cost-effectiveness of the drugs.