The most important miRNAs known in the context of sepsis so far and their functions in immune cells, as well as the respective literature, are summarized in Table 1. It has to be kept in mind, however, that neither mouse models nor in vitro experiments with cell lines or primary cells fully cover the functional networks of the human organism.66,67 Thus, miRNAs in the context of human sepsis can only be considered “guilty by association,” and the exact impact of these miRNAs on the inflammatory responses during the different stages of sepsis needs to be fully elucidated.
ALI is orchestrated by activated immune cells and by excess cytokine and protease release into the alveolar space.68 Given these conditions, immunomodulatory miRNAs proposed as biomarkers in sepsis might also be of diagnostic value in ALI. Additionally, miRNAs affecting epithelial and endothelial cells might play a role. Surprisingly, unlike in sepsis, clinical studies evaluating miRNAs as possible biomarkers in ALI are scarce. One study analyzing blood samples of 45 patients with ARDS induced by cardiopulmonary bypass found differential expression of a set consisting of 6 upregulated and 5 downregulated miRNAs.69 If these miRNAs might reveal as suitable biomarkers still needs to be determined.
There exist a variety of animal studies investigating the role of miRNAs in the pathogenesis of ALI. Data were mostly derived from rodents subjected to ALI induced by intratracheal injection of lipopolysaccharide (LPS), acid, or bacteria, or by ventilator trauma. In these studies, a large number of different miRNAs was found to be differentially regulated (extensively reviewed in the study by Rajasekaran et al70); however, a consensus regarding the value of these miRNAs in the development and resolution of ALI has not yet been achieved. ALI animal models have also been used to investigate miRNA treatment approaches in a surprisingly high number of studies, which is most likely due to the fact that an easy-to-handle and specific application of miR-mimics or anti-miRs via the airways is possible. In these studies, several miRNAs have been evaluated with respect to their capacity to influence the course of ALI.70 Interestingly, 3 of those inflammation-related miRNAs relevant in sepsis revealed also here as promising therapeutic targets: in LPS-mediated injury, anti-miR-155 application significantly reduced the numbers of inflammatory cells and the levels of proinflammatory cytokines in bronchoalveolar lavage,71 which was further corroborated in miR-155−/− mice, thus suggesting a role of miR-155 as a driver of lung inflammation.72 miR-146a and miR-125b, on the other hand, were found to ameliorate lung injury. miR-146a-mimic application suppressed both LPS- and acid-induced expression of proinflammatory cytokines and inducible nitric oxide synthase.73,74 Overexpression of miR-125b reduced lung permeability and expression of proinflammatory mediators and improved mice survival.75 Both miRNAs thus have been suggested as potential therapeutic targets for ALI. Similar to sepsis, miRNAs hold promise to become valuable biomarkers and therapy tools in the future; however, additional studies will be required to assess these issues.
Acute failure of liver, kidney, and heart are important clinical complications in intensive care medicine, with high mortality rates. Also here, miRNAs are increasingly gaining attention because biomarkers enabling an early and exact diagnosis and prognostic estimation, as well as innovative therapy approaches, are strongly needed.
Several miRNAs are specifically expressed or enriched in the liver, with miR-122 being the most abundant liver-specific miRNA. Both acute and chronic liver damage are associated with hepatocyte cell death, which leads to the release of liver-specific miRNAs. Starkey Lewis et al found substantially elevated plasma levels of miR-122 and miR-192 in acetaminophen-induced acute liver injury.76 Determination of miR-122 significantly outperformed alanine aminotransferase (ALT), international normalized ratio (INR), and acetaminophen plasma concentrations for the prediction of this type of liver injury.77 In a study evaluating miRNAs in liver steatosis, miR-122 was found to correlate with the severity of the disease.78 In chronic hepatitis C, the typical inflammation-related miRNAs miR-155, miR-125b, and miR-146a were increased in patients’ plasma.79 As liver-specific delivery of nucleic acids by microparticles has successfully been demonstrated by Press et al,80 therapeutic approaches using miRNA mimics or antagonists are conceivable in the near future.
The intestine is a unique organ where multiple communications between the immune system, gut epithelium, and commensal microbiota take place. A breakdown of homeostasis can lead to inflammatory disorders as frequently seen in the perioperative context. Biomarkers indicating the onset of acute gut injury are scarce, and miRNAs are currently one of the most promising molecules in this field. To date, however, available data are mainly derived from models of inflammatory bowel disease (IBD). For example, a very recent study using a murine model of dextrane sodium sulfate-induced colitis shed light on the pivotal function of miR-223 in IBD: administration of miR-223 mimics inhibited the NLRP3 inflammasome, thereby reducing interleukin-1β-mediated dextrane sodium sulfate-induced colitis.81 These findings are consistent with clinical data reporting miR-223 to be elevated in a subset of patients experiencing IBD,82 thus suggesting miR-223 as a potent new biomarker for gut inflammation. Further studies are needed to clarify whether these findings can be transferred into the acute perioperative setting.
In acute kidney injury (AKI), many miRNAs have been shown to be involved in the amplification or reduction of acute injury processes. While molecular mechanisms have only been investigated in animal studies so far (extensively reviewed in the article by Fan et al83), a considerable number of clinical studies have provided data on the potential of certain miRNAs to serve as markers of early AKI. Specifically, miR-21 has been revealed as stable biomarker: urine and plasma miR-21 levels have been shown to correlate with AKI severity and hospital mortality and to predict the probability of postoperative renal replacement therapy. Also, lower baseline plasma levels of miR-21 have been shown to predict AKI after cardiac surgery.84,85 In animal models of AKI, overexpression of miR-21 provided renoprotection, thus suggesting this miRNA as a therapeutic target.86,87 Further, decreased serum levels of the kidney-enriched miRNAs miR-29a, miR-101-3p, and miR-127a have been shown to predict AKI in intensive care unit patients.88 Even in AKI, anti-inflammatory miR-146a plays an important role, as decreased blood levels have been shown to be a predictor of AKI in the intensive care unit.88
Research on the role of miRNAs as biomarkers for different cardiovascular disease entities has exponentially expanded during the last few years, and miRNAs have been suggested as new biomarkers providing additional information to established protein-based markers such as cardiac troponins and natriuretic peptide. A large number of encouraging results have been obtained so far, which have extensively been reviewed before.89,90 Here, we will focus on the description of the most striking miRNAs in myocardial infarction (MI) and heart failure.
In acute MI, miRNAs with high myocardial expression are released into the peripheral circulation, which opens up new opportunities of improving diagnostic discriminatory power and/or accelerate diagnosis by determination of specific miRNAs in the peripheral blood of patients suspicious of MI.91 For example, the cardiac-specific miR-208b is detectable within 3 hours after MI and may persist elevated for as long as 90 days. In several studies, miR-208b was revealed as a useful early biomarker for MI.92,93 Also, a signature consisting of 6 miRNAs was revealed as a reliable predictor of MI, with an AUC significantly exceeding troponin C and creatine kinase-MB.94 In MI, miRNAs were also shown to exert predictive impact: in 2 large cohorts, an miRNA set consisting of miR-126, miR-197, and miR-233 was identified to reliably predict MI in persons with coronary artery disease.95,96
In heart failure, miRNAs miR-558, mR122*, and miR-520-d-5p were identified in a cohort of 53 patients as a stable biomarker set to predict the diagnosis “nonischemic heart failure.”97 In another study comprising 42 patients experiencing heart failure, miR-182 was identified to predict mortality with higher prognostic power than NT-proBNP and high-sensitive C-reactive protein.98 Taken together, miRNAs are clearly on the verge of implementation in the prediction and diagnosis of AKI, MI, and heart failure and may be a valuable future tool in intensive care medicine.
In animal models, commonly used anesthetic drugs (eg, propofol, sevoflurane, and ketamine) have been found to induce neurotoxic effects such as neurodegeneration, neural apoptosis, and impairment of neural stem-cell self-renewal.99–101 Recent research identified miRNAs as one of the key players mediating neurotoxic or protective effects.102 In 2014, Goto et al103 discovered in rodents that propofol and sevoflurane administration substantially altered miRNA expression profiles. These results made the pace for further rodent studies in this area. For example, it was shown that administration of propofol induces downregulation of miR-21 and induction of miR-665, leading to impairment of neuronal differentiation and induction of apoptosis.104,105 For isoflurane, downregulation of miR-214 and let-7d was reported, leading to an increase of apoptosis via induction of Bax,106,107 and in ketamine anesthesia, miRNA expression patterns could be associated with hippocampal neurodegeneration and memory impairment.108,109
Taken together, a large body of animal studies suggests that commonly used drugs for induction and maintenance of anesthesia induce alterations in miRNA expression, which might deteriorate neuronal integrity and neurocognitive processes such as memory and learning. Whether these experimental findings may also be true for the human organism needs to be investigated in the near future.
In postoperative care, reliable biomarkers that allow for prediction or at least timely detection of complications are needed. Generally, miRNA markers of acute organ injury as described above may also be of considerable predictive value in this setting. With regard to specific postoperative complications, only a few studies addressing miRNAs as possible biomarkers are available so far: for example, a study investigating 30 children after heart surgery revealed a set of 3 miRNAs (208a, 208b, and 499) as possible biomarkers for early detection of postoperative myocardial damage.110 Also, miRNA-499 was identified as a marker for postoperative MI in 30 patients undergoing coronary artery bypass grafting.111 Several ongoing studies are evaluating the suitability of miRNAs as biomarkers in postoperative care, for example, as predictors of postoperative delirium (ClinicalTrials.gov). Taken together, miRNAs may serve as valuable biomarkers in the postoperative setting in the near future.
Pain plays a central role in perioperative care. Several classifications of pain exist, the broadest one being the distinction between acute and chronic pain, with a subclassification of the latter into inflammatory versus neuropathic. Chronic pain syndromes greatly contribute to the overall cost for the medical system,112,113 and both diagnostic and treatment options are limited, not at least due to lack of understanding of its pathophysiology. In 2007, a first study reported the downregulation of 7 miRNAs in the trigeminal rat ganglion after inducing inflammatory pain in the masseter muscle.114 Since then, noncoding RNA molecules have been acknowledged to play a critical part in especially chronic pain pathophysiology.
The dorsal root ganglion (DRG) has been identified as a key structure involved in the pathophysiology of neuropathic pain processing,115,116 and spinal nerve ligation leads to changes of both the proteome117 and the transcriptome118 of this structure. Evidence for miRNA involvement was presented by Zhao et al119 in 2010, who reported that miRNA function seems to primarily impact inflammatory pain. A global reduction of miRNA expression levels via Dicer knockdown leads to the downregulation of several nociceptor-associated proteins crucial for development and maintenance of hyperalgesia.120,121 Shortly after, a number of proteins connected to pain recognition, such as CACNA2D1, SCN11A, and P2RX4, were found to be regulated by miRNAs.115,119,122,123
miRNAs can also directly trigger pain sensation. Based on the observation that miRNAs circulating in blood and cerebrospinal fluid can aggravate neurodegeneration,124 Park et al125 found that extracellular miRNAs could induce rapid onset of pain via induction of rapid inward currents in DRG neurons. This effect was mainly mediated by toll-like receptor-7 that recognized the single-strand RNA motif GUUGUGU in the mature sequences of hsa-let-7b and hsa-miR-599.125 Interestingly, let-7b is highly enriched in DRG neurons and is released upon neuronal activity125 and has also been linked to complex regional pain syndrome.126 The ultimate location for pain processing is the central nervous system, and it is hardly astonishing that miRNAs related to central pain processing have been described. Pohl et al127 focused on the investigation of the effects of inflammatory pain on the prefrontal cortex that is activated in acute as well as chronic pain, finding significantly increased levels of miR-155 and miR-223. In a more functional approach, Imai et al128 combined functional MRI, in silico analyses, and laboratory methods to draw a connection between neuropathic pain that decreases the expression of miR-200b/miR-449 and the mesolimbic circuitry via unleashed expression of DNA methyltransferase 3a.
In summary, miRNAs are evidently involved in major known pathways relevant to pain development and maintenance113 and may support therapeutic decisions someday as exemplified by hsa-miR-124 expression in CD4 T cells that has been found to be predictive of treatment response in chronic lower back pain.129
Despite the multitude of miRNAs that have been proposed as possible biomarkers, determination of miRNAs has not made its way into clinical practice so far. This is, indeed, surprising and mainly due to the fact that a universal measuring method enabling an easy-to-handle, fast, reliable, and inexpensive determination of miRNAs does not exist to date. miRNA expression profiling is a technical challenge: miRNAs are tiny molecules, miRNA family members exhibit a high degree of homology, and absolute miRNA concentrations in body fluids are rather low. Several measurement platforms are currently available to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription quantitative polymerase chain reaction, and microarray hybridization. Each method has its strengths and weaknesses, and selection of the measuring method depends on the specific scientific questions to be addressed.130 The different detection platforms currently used in miRNA diagnostics and research are briefly summarized in Table 2.
Moreover, to ensure reliable miRNA measurement, it is necessary to carefully choose the compartment most suitable for measuring the miRNAs of interest (eg, serum, plasma, blood cells, tissue specimens, or body fluids such as urine or liquor) and to select an appropriate normalization strategy.140 Also, it has to be taken into account that miRNA expression profiles are influenced by genetic heterogeneity and exogenous influences, such as medication, nutrition, or exposure to certain environmental conditions.141,142
In the field of perioperative medicine, multi-institutional studies adhering to standardized protocols for sample preparation, miRNA detection, and data analysis are required to clearly make out those miRNAs qualifying as valid biomarkers for future clinical use (possible miRNA candidates are summarized in Figure 3). Actually, we are very close to an implementation of miRNAs into the daily clinical use, which will provide valuable complementary data on our roads toward a personalized medicine.
The concept of inhibiting or overexpressing miRNAs for therapeutic purposes represents a new frontier in modern medicine. To date, first approaches—either using miRNA mimics or miR-inhibitors—have made their way into clinical studies so far.
Synthetic miRNAs that mimic natural miRNAs are supposed to exert therapeutic effects by reconstituting miRNAs that are downregulated during disease or by downregulating signaling pathways involved in disease pathology. miRNA mimics are double-stranded oligonucleotides that require liposomes, lipoprotein-based carriers, or nanoparticles as vehicles for their delivery.143,144 The first miRNA mimic to enter a clinical study in the field of oncology was MRX34. This substance was designed to deliver a mimic of the naturally occurring tumor suppressor miR-34, which is underexpressed in a wide variety of cancers. MRX34 was tested in a multicenter phase 1 clinical trial starting 2013, which included patients with primary liver cancer, other solid cancers, and hematological malignancies. Results of this study are elusive, as it was stopped in 2016 due to multiple immune-related severe adverse events. Very recently, a phase 1 study evaluating the miRNA mimic MRG-201 was initiated. This substance is designed to mimic miR-29b, thereby decreasing the expression of collagen and other proteins that are involved in fibrous scar formation, and is applied in healthy volunteers by intradermal injection.
Pharmacologic approaches of miRNA inhibition exert therapeutic effects by use of anti-miRs or miRNA sponges. Both classes of molecules block natural miRNAs and thus are supposed to silence miRNAs that are elevated during disease or to disinhibit signaling pathways involved in disease pathology. Anti-miRs are single-stranded oligonucleotides that are chemically modified to enhance target affinity, stability, and tissue uptake.143,144 Unlike double-stranded miRNA mimics, anti-miRs can be administered dissolved in saline solution. Once entering the circulation, they are easily taken up by multiple tissues and organs, where they specifically bind to endogenous miRNAs thus reducing their availability. Sponges are RNA molecules that contain multiple seed sites of a specific miRNA that act as competitive inhibitors by “hoovering” endogenous miRNAs.
The prime example of a successful therapeutic anti-miRNA approach is miravirsen, an LNA-modified anti-miR-122 that effectively combats a hepatitis C virus infection.145 Miravirsen targets the liver-specific miR-122, which is “hijacked” by the hepatitis C virus to bind to sequences in the 5′-UTR of the viral RNA, thereby enhancing virus replication. In a first phase 2a clinical trial enrolling 36 patients with chronic hepatitis C, miravirsen treatment showed a dose-dependent antiviral activity clearly exceeding the time of therapy. Notably, in 4 of 9 patients receiving the highest doses, stable seroconversion was achieved. In this clinical trial, no adverse side effects were reported. Further evaluation of miravirsen is a topic of several ongoing studies.
Another substance that very recently entered clinical phase 1 evaluation is MRG-106, an LNA anti-miR of miRNA-155. In hematological malignancies, miRNA-155 plays a key role in differentiation, function, and proliferation of blood and lymphoid cells, and inhibition of miRNA-155 in lymphoma cells reduced proliferation in vitro. The phase 1 trial of MRG-106 enrolls patients experiencing cutaneous T-cell lymphoma and aims at assessing safety, tolerability, and molecular effects of MRG-106 in the lesions of MF patients. Trials for miRNA mimics or LNA inhibitors that have made their way into clinical evaluation so far are summarized in Table 3.
Currently, miRNA-based therapy still is in its infancy and a number of problems have to be addressed until a broad, reliable, and safe clinical use will be a feasible objective. The development of delivery systems enabling cell-specific uptake and the design of therapeutical molecules without toxic side effects remain a major challenge. Moreover, unwanted off-target effects have to be minimized. After taking these hurdles, miRNA-based therapy strategies will open up one of the most innovative and promising perspectives in current medicine.
It is to be expected that miRNAs will find their way as very helpful new biomarkers and as effective therapy tools into the clinical routine in the near future. This will help to strike out on new paths, which—not least in perioperative medicine—will entail significant medical improvements.
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