MicroRNAs (miRNAs) are a class of 21∼25-ribonucleotide single-strand non-coding RNA molecules which is endogenously expressed in eukaryote cells (Guo et al., 2014). It has been well recognized that miRNAs play an important role in the post-transcriptional regulation of genes by targeting mRNA molecules. Through base-pairing with the 3′untranslated regions (3′UTRs), miRNAs modulate direct cleavage and/or translational repression of target mRNAs (Bartel, 2009), thus regulating wide spectrum of processes in physiological and pathological conditions. Their regulatory networks are very complex, partially because miRNA expression varies dramatically in different tissues in response to different cell stimulations.

Hypoxic/ischemic injury is a process in which blood flow cessation and oxygen deprivation occur in the body, organs, or cells. Metabolic waste products are accumulated due to the starvation of oxygen and nutrients, leading to profound tissue dysfunction and damage (Zhao et al., 2018). More specifically, hypoxia is a state of low oxygen supply, resulted from a decrease in partial pressure of oxygen, inadequate oxygen transport, or the inability of the tissues/organs to uptake oxygen from the blood. In contrast, ischemia is defined as inadequate blood flow to the tissues, resulting in the deprivation of oxygen and glucose, as well as other substances in the blood (Chao and Xia, 2010; Yang et al., 2015). In this review, the term of “H/I injury” is used to describe the general influences of hypoxic and/or ischemic insult, because hypoxia and ischemia processes often occur one after another. A wide range of pathophysiological processes have been reported to be associated with H/I injury, including ATP depletion, calcium overload, mitochondrial dysfunction, oxidative stress, and etc. (Eltzschig and Eckle, 2011; Chen et al., 2019). More recently, emerging evidence suggests that H/I inflammation, a quick pathological response to oxygen/blood flow depletion, has a major impact on H/I injury through pro-inflammatory cytokines and their signaling pathways (Chen et al., 2020c; Sikora et al., 2021; Troscher et al., 2021).

Because of energy imbalance, H/I insult induces pro-inflammatory events including microglia activation, cytokines production, and immune cell development via various biological processes such as apoptosis and oxidative reaction (Chen et al., 2020c). Different organs may suffer from H/I inflammation in different ways, thus leading to various H/I-related diseases. H/I neuroinflammation induces brain injury in ischemic stroke and post-stroke epilepsy (Li X. et al., 2020; Troscher et al., 2021; Xue Y. et al., 2021), while H/I inflammation leads to other pathophysiological events in peripheral organs, including heart failure, acute kidney injury, and hepatocellular damages (Nangaku and Eckardt, 2007; Klune and Tsung, 2010; Frohlich et al., 2013; Yang et al., 2015). On the other side, some beneficial reaction may go through the regulation of inflammatory events. For example, long term limb remote ischemic conditioning could decrease blood pressure via inflammation regulation (Gao et al., 2021). Studies have demonstrated the vital roles of miRNAs in the regulation of gene expression under H/I condition, not only in H/I-sensitive organs such as kidney, heart, and brain, but also in the H/I-insensitive organs such as liver and muscle (Nallamshetty et al., 2013; Bertero et al., 2017). H/I inflammation is mediated, at least partially, by the miRNA-induced repression of gene expressions. The alternations of miRNA expression in response to H/I injury differ widely in different organs due to their differential sensitivities to the reduction of oxygen and blood flow. Numerous studies have shown the changes of miRNA expression profiles along with the potential targets and inflammatory pathways upon H/I insult in specific organs (Tables 1, 2). However, few studies have compared the common changes and the differences of miRNA profiles between H/I-sensitive and -insensitive organs.

Opioid receptors belong to the large family of seven-transmembrane G protein-coupled receptors with three major sub-types known as MOR, KOR, and DOR (Xia, 2015). It is well demonstrated that DOR is protective against hypoxic, ischemic, and excitotoxic insults. There exists a differential distribution of DOR in different parts of the brain with a higher density in the cortex, striatum and dorsal root ganglion (Xia, 2015; Tan et al., 2016; Li X. et al., 2020; Gao et al., 2021). DOR is present not only in the nervous system but also in other organs such as the heart, lungs, liver, and gastrointestinal and reproductive tracts (Feng et al., 2012). Our recent studies as well as those of others present strong evidence of the DOR-mediated cyto-protection in different organs, including brain, kidney, heart, and liver (Zhang et al., 2000, 2002, 2006; Ma et al., 2005; Chao et al., 2007, 2008, 2009, 2012; Kang et al., 2009; Chao and Xia, 2010; Feng et al., 2011, 2012; He et al., 2013a; Luo et al., 2019). Moreover, there is accumulating evidence that DOR activation can achieve a protective role against H/I injury by modulating miRNA expression in multiple organs (Yang et al., 2012; He et al., 2013b; Zhi et al., 2016, 2017), especially in the process of neuroinflammation (Chen et al., 2020c). It is therefore possible for DOR signaling, directly or indirectly, to protect organs against H/I inflammation by targeting specific miRNA molecules.

In this review, we highlight some of the recent updates about the effects of H/I and DOR activation on miRNA expression in the current literature, along with our recent work in this field. In particular, we intend to summarize and compare differential regulation of miRNA expression in different organs and discuss potential clinical significances in terms of specific DOR treatment against H/I inflammation.

δ-Opioid receptor is neuroprotective against H/I injury in the brain (Zhang et al., 2002; Chao et al., 2008, 2009; Kang et al., 2009; He et al., 2013a). The administration of DOR agonists can prolong survival of peripheral organs, such as lung, heart, liver, and kidney (Peart et al., 2005; Patel et al., 2006). As a protective molecule, DOR can tonically regulate miRNA expression even in the normoxic condition. We measured miRNA expression profiles in different organs such as the kidney, heart, brain, and liver after DOR activation with a specific and potent DOR agonist UFP-512 applied in Sprague Dawley rats (Yang et al., 2012; He et al., 2013b; Zhi et al., 2016, 2017). DOR activation can influence the expression of many miRNAs in different organs. As summarized in Table 5, the brain had the most dramatic changes in miRNAs after DOR activation. Conversely, miRNAs in the heart and liver kept relatively stable during the observation (DOR activation for 1, 5, and 10 days), suggesting a differential regulation of miRNAs among organs in response to DOR signals under normoxic condition.

Among hypoxia-sensitive organs, seven common miRNA changes were observed in the kidney and brain, including miR-21, miR-29b, miR-298, miR-347, miR-351, miR-466b, and miR-511 (Table 5; Yang et al., 2012; He et al., 2013b). MicroRNAs in the kidney displayed the similar expression tendency with those of the brain. For instance, miR-29b was significantly down-regulated in the brain and kidney at 1 day after DOR activation with UFP-512 and maintained at a relatively stable level after 5–10 days DOR activation. Reduced expression level of miR-347 was observed after DOR activation at day 1 in the brain, and at days 5 and 10 in the kidney. Similarly, miR-511 was significantly down-regulated after DOR activation in rat brain and kidney at day 10 (Tables 6, 7). In contrast, there were few common miRNA changes among hypoxia-insensitive organ (liver) and hypoxia-sensitive organs. In our work, only the alternation of miR-107-3p expression was seen in both the heart and liver in normoxic condition upon DOR activation (Table 5). Further investigating their functions and elucidating the mechanistic differences between the H/I sensitive and -insensitive organs will yield valuable information for better understand the DOR-mediated regulation of miRNAs in physiological conditions.

Since either DOR activation or hypoxic condition has a profound impact on miRNA expression profiles in different organs, it will be interesting to learn the possible effect of DOR activation on hypoxia-induced miRNA changes. Our studies have shown that miRNA expression profiles can be significantly altered when DOR activation was applied to hypoxic organs (Yang et al., 2012; He et al., 2013b; Zhi et al., 2016, 2017). We noticed that the differential alternations in the miRNAs largely depended on the duration of hypoxia, and DOR activation led to diverse outcomes in response to short-term or prolonged hypoxia in different organs.

As one of the hypoxia-sensitive organs, the hypoxic brain showed the most dramatic changes in miRNA expression in response to DOR activation. Some miRNAs were largely altered at the earliest time point (1 day), including miR-347, miR-101b, miR-466b, miR-29a, miR-363*, and miR-31 (Yang et al., 2012). The majority of miRNAs, especially those that mediates cyto-protective function such as anti-neuroinflammation, were down-regulated compared with those in normal condition. Among them, miR-29a alleviated cerebral ischemia/reperfusion injury via down-regulating target gene TP53INP1 and the NF-κB/NLRP3 pathway (Liu X. et al., 2021). In the brain exposed to a mid-term hypoxia (5 days), DOR activation provoked even more miRNA reduction as compared to that in short-term hypoxia (1 day), whereas the expression of miRNAs turned to the baseline level upon DOR activation under prolonged hypoxia (Table 6; Yang et al., 2012). These findings implicated that brain miRNA expression responded quickly after the hypoxia insult, and possibly involved in anti-hypoxic injury by modulating the expression of target genes.

δ-Opioid receptor activation can also modify hypoxia-induced changes of miRNA expression in the kidney. The changes in several miRNAs could be detected upon DOR activation in the kidney exposed to a continuous hypoxia for a prolonged period. For instance, the down-regulation of miR-511 and miR-298 expression was observed upon DOR activation followed by a continuous exposure to hypoxia for 5 to 10 days (He et al., 2013b). In the case of miR-370, miR-20b-5p, and miR-29a/b, although hypoxia alone did not alter their expression levels at certain time points, the administration of UFP-512 to the hypoxic kidney significantly down-regulated the expression of these miRNAs. MiR-21 was reported to induce macrophage M1 inflammatory phenotype, cytokine production, endothelial-mesenchymal transition, and fibrosis in ischemic kidney (Pushpakumar et al., 2021). DOR activation could lower miR-21 expression in prolonged hypoxia (5 days and 10 days). Apparently, DOR activation continuously modulated renal miRNA expression during the whole hypoxic period (Table 7; He et al., 2013b).

Unlike other hypoxia-sensitive organs, the majority of altered miRNAs in the heart was up-regulated under 1 day hypoxia. The administration of UFP-512 to the hypoxic heart induced a further upregulation in terms of the expression of miR-141-3p, miR-376a-3p and miR-134-5p. Moreover, the expression of miR-134-5p and miR-7b was increased throughout the entire time course after DOR activation under hypoxia, suggesting their important roles in the regulation of renal adaptation to hypoxic stress (Table 8; Zhi et al., 2016).

Similar to that in hypoxia-sensitive organs, miRNA expression in hypoxia-insensitive organs can also be influenced by DOR activation in hypoxic condition. The expression of miR-34a-5p, miR-142-5p, miR-145-5p, miR-146a-5p, and miR-204-5p were significantly increased in the liver after DOR activation and hypoxic stress (Zhi et al., 2017). MiR-192-5p was the only miRNA whose expression level started to decrease after DOR activation under hypoxia (Table 9). MiR-146a was reported to ameliorate ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro by directly suppressing IRAK1 and TRAF6 in the liver (Jiang et al., 2014). Prolonged hypoxia could down-regulate miR-146a level, whereas DOR activation restored miR-146a expression, which might inhibit pro-inflammatory cytokine release and cellular apoptosis. Therefore, DOR signaling likely functions to upregulate hepatic tolerance to hypoxic stress by differentially modulating the expression of different miRNAs.

Collectively, among hypoxia-sensitive organs, the kidney and brain had a common change in the miRNAs, i.e., a significant alternation in nine miRNAs in both organs (Yang et al., 2012; He et al., 2013b), whereas hypoxia-sensitive and -insensitive organs had no common change at all (Table 10).

It seems that hypoxia comprehensively modifies miRNA profiles with a major difference among organs, while DOR signaling is able to modulate such regulation in most of these organs (Feng et al., 2012; He et al., 2013a; Nallamshetty et al., 2013; Yang et al., 2015). DOR activation may induce cyto-protection against H/I insult in both hypoxia-sensitive and -insensitive organs, at least partially, through modulating miRNA expression.

Hypoxic/ischemic-induced inflammatory injury to different organs is a frequently encountered clinical problem and the common cause of various diseases with limited therapeutic options. Because the role of miRNAs in controlling H/I inflammation, recent studies on miRNA expression under H/I condition have drawn much attention from clinicians and scientists worldwide. Many experiments have been conducted in cell models, animal models, and patients to investigate the potential targets and signaling pathways of miRNAs involved in H/I pathology. Some of the miRNAs are regarded as injury factors under hypoxic condition by promoting cellular inflammation, mitochondrial dysfunction, oxidative stress and apoptosis, while others play protective roles against H/I insult by inhibiting pro-inflammatory cytokines release and NLRP3 inflammasome (Tables 1–4). Our summary of increased or decreased miRNAs in response to DOR activation (Tables 5, 10) provides a guide for future clarifications of their functions in controlling H/I inflammation.

Hypoxic/ischemic stress comprehensively alters miRNA expression in H/I-sensitive and -insensitive organs, largely depending on the duration of hypoxia. MicroRNA expression in H/I-sensitive organs such as brain and kidney often respond quickly to relatively short-term hypoxia (1 day). After prolonged hypoxia (10 days), the expression of some miRNAs turned back to the baseline level. There is evidence suggesting that a mild/moderate H/I stress may induce pro-inflammatory cytokine release and causes a quick inflammatory response, whereas a severe/prolonged stress eventually causes cell apoptosis and necrosis (Krock et al., 2011; Hadjipanayi and Schilling, 2013). The differential cellular signaling is partially mediated by the miRNA-induced repression of gene expression.

Although various miRNAs can exhibit very different responses even in the same organ under similar H/I condition, there are several H/I sensitive miRNAs that display common changes among different organs. These miRNAs were named “hypoxamiRs,” including miR-21, miR-210, and miR-199a (Nallamshetty et al., 2013; Greco et al., 2014; Azzouzi et al., 2015; Bertero et al., 2017). Accumulating evidence shows that the expression of miR-21 can be directly induced by hypoxia stimulation in several cell types due to the consensus hypoxia-response-element (HRE) sequence in its promoter region (Kulshreshtha et al., 2007; Parikh et al., 2012). Similarly, the expression of miR-210 could be up-regulated by either HIF-dependent mechanism via HRE binding (Lee et al., 2009; Pocock, 2011), or HIF-independent transcriptional regulations (Bertero et al., 2017). One of the most important functions mediated by these “hypoxamiRs” is cellular inflammation (Tables 1–4).

Aging is a key factor affecting H/I inflammation and miRNA expression. In contrast to the immature and young organs, the aged ones are more sensitive to H/I stress. The oxygen-deprived conditions (hypoxia and ischemia) lead to oxidative stress, cellular damage and protein modifications (Adav and Sze, 2020). H/I inflammation and abnormal miRNA expression have been proposed as risk factors for aging and neurodegenerative diseases. Some circulating inflamma-miRs, e.g., miR-21-5p and miR-126-3p, are even thought as potential biomarkers of cognitive impairment AD patients (Giuliani et al., 2021). MicroRNAs implicated in pathological aging such as miR-92a-5p and miR-532-5p are also regarded as potential biomarkers and putative molecular effectors of cognitive frailty (Carini et al., 2021). Serum miR-214 as well as salivary miR-874 and miR-145-3p might serve as auxiliary biomarkers for PD (Chen et al., 2020d; Li L. et al., 2021). In aged rats, transcription factor HIF could protect against ischemic brain injury by reducing inflammatory responses via the Akt signaling pathway (Du et al., 2020). All these facts suggest that an elevated level of inflammation exists in various aging-related chronic diseases, and modulating miRNA expression is a promising avenue for the prevention and treatment of aging and chronic diseases.

The miRNA expression can be modulated by sequence-specific miRNA mimics (or agomirs) and inhibitors (or antagomirs), both in vitro and in vivo. A recent study showed that miR-363-3p treatment attenuates brain ischemia-induced long-term cognitive deficits in rats (Panta et al., 2020), suggesting the potential applications of miRNA mimics. The process of miRNA biogenesis involves transcription, pre-miRNA splicing, exporting, and stability of mature miRNAs. Therefore, the miRNA expression can be modulated by specific molecular signals.

The information in this review suggests that DOR activation is effective in protecting organs against H/I injury, with the capability of modulating miRNAome in both normoxic and hypoxic conditions. Although the direct regulatory mode of DOR on miRNA expression is still unknown, DOR signaling may affect miRNA biogenesis by modulating some key transcriptional factors. For instance, ERK is found to suppress pre-miRNA export from the nucleus to cytoplasm through phosphorylation of Exportin-5, resulting in a global reduction of pre-miRNA loading and miRNA synthesis (Xie et al., 2020; Zhang et al., 2020d). Since ERK activity could be upregulated by DOR activation (Ma et al., 2005; Cai et al., 2021), DOR may regulate miRNA expressions via this signaling pathway.

Different kinds of DOR agonists have been developed in the past. Many of them displayed analgesic, antidepressant, anxiolytic and other opioid effects. For instance, Deltorphin I is an opioid peptide with relatively high affinity and selectivity to DOR and produce centrally mediated analgesic effects in animals (Thomas et al., 1997). Another commonly used one is delta opioid peptide DADLE ([D-Ala2, D-Leu5]-Enkephalin), which was used for pre-conditioning and post-conditioning to induce neuroprotection against hippocampal injury resulted from transient forebrain ischemia in rats (Wang S. et al., 2011). There are also other non-peptide DOR agonists used for antidepressant, anxiolytic or anti-inflammatory properties, including SNC-80 (Bilsky et al., 1995; Perrine et al., 2006), AZD2327 (Hudzik et al., 2014), and ADL5859 (Nozaki et al., 2012). In addition, AR-M 1000390 (Wei et al., 2000) and DPI-3290 (Ananthan, 2006) were also used for DOR agonists. However, UFP-512 that we commonly used in our studies might yield more reliable data for DOR activation because of its specificity and potent binding affinity (Balboni et al., 2002; He et al., 2013b; Xia, 2015). Future development of more specific DOR agonists with lower side-effects may facilitate the application of DOR for the treatment of H/I inflammation.

In summary, it is possible to develop a new protective strategy against H/I injury by activating DOR signaling and targeting certain miRNAs to suppress H/I inflammation in both hypoxia/ischemia-sensitive and -insensitive organs. However, controversies and ambiguity still persist in the literature, especially regarding the up-regulation versus down-regulation of miRNAs under different conditions and their downstream targets in the different organs. Moreover, the molecular mechanisms involved in the DOR-mediated regulation of miRNAs are largely unknown at present. The controversies may be partially attributed to the differences in the models, species, and experimental approaches among different studies. Nevertheless, the solution of the above-mentioned fundamental issues depends on more reliable and in-depth studies in future.

YX initiated the project and made the outline. YC searched the literature and prepare the manuscript. YH, SZ, and XH participated in writing the manuscript. YX and DX supervised the project and revised the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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