Role of Micro-RNA for Pain After Surgery: Narrative Review of Animal and Human Studies

Abstract

One of the most prevalent symptoms after major surgery is pain. When postoperative pain treatment is unsatisfactory, it can lead to poor surgical recovery, decreased quality of life, and increased health care costs. Current analgesics, single or in combination, have limited efficacy due to low potency, limited duration of action, toxicities, and risk of addiction. The lack of nonaddictive strong analgesics along with the over prescription of opioids has led to an opioid epidemic in the United States. Therefore, there is an urgent need for the development of newer analgesics. Microribonucleic acids (miRNAs) are small noncoding RNA molecules that modulate protein synthesis in neurons and supporting cells (glia, leukocytes, and Schwann cells). The literature indicates that miRNA regulation is important in nociception. Here, we summarize the current evidence on the role of miRNAs on mechanisms involved in incisional, inflammatory, neuropathic, and cancer pain. We also discuss the role of modulating miRNA functions as potential therapeutic targets for analgesic use and opioid tolerance. Finally, we propose how the delivery of analog miRNAs (mimic-miRNAs or antago-miRNAs) could be introduced into clinical practice to provide analgesia in the perioperative period.

Despite the substantial use of prescription opioids and other less potent analgesics during and after major surgery, pain is still undertreated. Three unwanted consequences related to poorly managed pain and administration of high dosages of opioids include the risks of developing postoperative persistent pain, acute opioid-related adverse events, and addiction to persistent opioid use.^1,2 Clinical studies indicate that 10%–50% of patients undergoing major procedures can develop postoperative persistent pain and may use opioids for 6–12 months after surgery.^1,3 Both postoperative persistent pain and opioid use are risk factors for opioid addiction. Therefore, finding alternatives to opioids for treating perioperative pain could result in a decreased risk of long-term opioid addiction.

With the goal of reducing opioid consumption perioperatively, the recent consensus statements and guidelines from experts in the field of perioperative medicine emphasize the use of multimodal analgesic regimens.^4,5 These strategies include a combination of regional anesthesia techniques with systemic nonopioid analgesics, such as oral gabapentinoids, intravenous lidocaine and ketamine, and nonsteroidal anti-inflammatory drugs.^4,6 However, these systemic analgesics also have their own adverse events, such as sedation, bleeding, anastomotic gastrointestinal leaks, cardiovascular toxicities, and renal injury, that make them unsuitable for many patients.^7,8 Furthermore, the actual duration of action of these analgesics is still limited to a few hours or days after major surgery, which ultimately results in the use of opioids. This notion highlights the importance of developing novel analgesic modalities that can effectively treat postoperative pain and decrease or eliminate the overprescription of opioids or high dosages of nonopioid analgesics.

In 1993, Ambros’s laboratory reported for the first time gene silencing by ribonucleic acid interference (RNAi) in Caenorhabditis elegans.⁹ RNAi has evolved as an ancient defense system against viruses and transposons and a mechanism to regulate endogenous gene expression. As a result of this interference, messenger RNA (mRNA) is prevented from translating into a protein.¹⁰ Notably, it is estimated that >60% of the mammalian genes are microribonucleic acid (miRNA) targets.¹¹ There are 2 major mechanisms of short-stranded RNAi that have been studied extensively: small interference RNA (siRNA) and miRNA. Of these 2, mounting evidence indicates that miRNA-based treatments can be used to restore or repress miRNA expression and thus protein synthesis in different human conditions.^12–14 In perioperative medicine, miRNAs regulate inflammation and immune responses, ischemia–reperfusion mechanisms, and the action of anesthetics and analgesics.^15–17 Furthermore, miRNAs can serve as a means of cell-to-cell communication. As an example, Simeoli et al¹⁸ indicated that, after nerve injury, dorsal root ganglia (DRG) sensory neurons release extracellular vesicles containing miR-21, which then promotes M1 differentiation in macrophages.¹⁹ After lipopolysaccharide (LPS) stimulation, human-derived monocytic cells can release exosomes containing miR-532–3p. Remarkably, the intraplantar injection of these exosomes reduced thermal hyperalgesia.²⁰ There is evidence to support the idea that the delivery of miRNAs could be used to modulate the signaling pathways and cellular elements involved in nociception.

In this narrative review, we will summarize the importance of miRNAs in the context of postsurgical pain and how “analog miRNAs” could be introduced to provide adequate and sustained pain relief after major surgery.

The Search for the “Ideal” Analgesic for Major Surgery

The current armamentarium of drugs and techniques available to provide analgesia after major surgery can be grouped into opioids and nonopioids and systemic or nonsystemic (local or regional analgesia). These currently available analgesics have limited efficacy and can cause significant adverse events. In the search for the “ideal” analgesic, we consider 4 criteria that must be fulfilled: (1) have ease of administration, (2) have long-lasting effects, (3) be well-tolerated (low toxicity), and (4) be nontolerant or nonaddictive. In that search, novel analgesic therapeutics (Table 1) are being investigated, including long-lasting local anesthetics (ie, liposomal bupivacaine), fatty acid amide hydrolase inhibitors, nitric oxide synthase inhibitors, cannabinoids, kappa and delta opioid agonists, biased opioid agonists that can preferentially activate G protein-coupled protein (GPCR) pathways, opioid vaccines, selective reversible inhibitor of microsomal prostaglandin synthase enzyme, and interleukin-6 antagonists.^21–23 While some of these therapies have shown promising results in humans, others are still in experimental stages.^21–23

Table 1.

Newer analgesics entering clinical trials

Therapeutic agent	Mechanism of action	Comment
Local anesthetics Liposomal loaded local anesthetics HXT-011: bupivacaine plus meloxican Neosaxitoxin	Sodium channel blocker Sodium channel blocker + COX inhibition Sodium channel blocker	These newer local anesthetics formulations are clinically used or in clinical trials to test safety and efficacy.
Fatty acid amide hydrolase inhibitors Pf-04457845	Inhibitor of the fatty acid amide hydrolase	Used in humans only in clinical studies for osteoarthritis pain, epilepsy and cannabis dependence.
Nitric oxide synthase inhibitors L-NG-methylarginine hydrochloride NXN-188	Inhibition of the production of nitric oxide Non-selective inhibitor Selective inhibitor	Only used in clinical trials.
MK8825, ubrogepant, olcegepant, telcagepant and rimegepant	CGRP receptor antagonist	Only used in clinical trials.
Tonabersat (SB-220453)	Gap junction modulator, anticonvulsant	Only used in clinical trials.
Tocilizumab	Interleukin 6 antagonist	Approved for rheumatoid arthritis, Castleman’s disease, giant cell arteritis, polyarticular and systemic juvenile idiopathic arthritis and chimeric antigen receptor T cell therapy-induced cytokine release syndrome.
Brivoligide	EGR1 - Transcription factor decoy	Only used in clinical trials.

Abbreviations: COX, cyclooxygenase; EGR1, early growth factor gene; GPCR, G protein-coupled receptor.

One of these newer analgesic modalities is focused on RNAi technologies.¹⁰ In fact, a recent initiative by the US National Institutes of Health (https://grants.nih.gov/grants/guide/pa-files/PAR-18-742.html) is focused on understanding the role of epigenomics and noncoding RNAs on chronic pain, opioid use disorders, and opioid-induced hyperalgesia.

Acute and Persistent Postoperative Pain: Mechanisms

Surgical pain is evoked by tissue damage, has the goal of protecting the injured area, and plays an important role in initiating the healing process. Surgical pain after major procedures shares components of somatic, neuropathic, and visceral pain depending on patients’ predisposing factors and the location and extent of the tissue trauma.²⁴ Somatic pain arises from trauma (ie, surgical incision) to tissues, such as abdominal wall structures (ie, muscle and parietal peritoneum), bones, or intra-articular structures (ie, capsule, ligaments, or tendons). Neuropathic pain is from nerve damage (ie, distention, ischemia, or transection) occurring during surgery, and visceral pain results from damage (ie, distention or ischemia) to organs such as the small or large intestine. As an example, postamputation pain has an element of acute somatic pain that can transition to chronic neuropathic pain (ie, postthoracotomy pain and postamputation phantom pain).^25,26 On the other hand, pelvic exenterations share components of visceral, neuropathic, and somatic pain.^25,26 The mixed component of different types of pain makes its treatment difficult, mainly after extensive surgical procedures.

Peripheral and central sensitization occur after activation and changes in the expression of nociceptors and secondary mediators located in sensory terminal, DRG cells, spinal neurons, and glial cells. At the level of the tissue injury (ie, skin and muscle), there is a significant increase in levels of inflammatory cytokines (ie, interleukin [IL]-1β and leukemia inhibitor factor [LIF]), complement mediators (C5a), growth factors (ie, nerve growth factor [NGF]), caspase 1, and the cyclooxygenase (COX) enzyme and a decrease in pH caused by accumulation of lactate.²⁷ Inflammatory mediators such as IL-1β are released by invading leucocytes (ie, neutrophils and mastocytes), which are also critical cells in the resolution process of inflammation.²⁸ At the DRG level, changes in activity and expression of nociceptive molecules and its receptors also occur and result in peripheral sensitization. The dorsal horn of the spinal cord is also a site of change in the activity and expression of mediators. Studies in animals demonstrate that a non-N-methyl-D-asparate (NMDA)/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, in particular, the GluR 1 subunit of the AMPA receptor, is critical in incisional pain. However, other molecules, such as proinflammatory cytokines, Toll-like receptor (TLR) 4, inducible nitric oxide synthase (iNOS), secondary messengers (PI3K, mitogen-activated protein kinase and mitogen activated protein protein [MEK], p38 kinase [p38], nuclear factor κ-light-chain-enhancer of activated B cells [NFκB]), and ion channels (acid sensing ion channel 3 [ASIC3], a2b subunit voltage-gated channels, and P2XR), also play a significant role in the development and maintenance of central sensitization.²⁹ Epigenetic changes have also been implicated in surgical pain. DNA methylation, histone acetylation, and alterations in noncoding RNA expression occur after incision.^30–32 For instance, in 24-hour incision, the miR206 levels are maximally reduced in rats, and it correlates with peaked expression of phospholipase A2 activating protein (PLAA).³¹

There are several considerations to make when studying the mechanisms of incisional pain. First, while most of the molecular changes occur acutely, they also persist days after surgery. For instance, the spinal levels of the enzyme COX2 acutely peak 4–6 hours after plantar incision, but they can remain elevated up to 3 days.³³ Interestingly, this coincides as well with the duration of spinal cord glial cell activation after skin or muscle injury.³⁴ Notably, in rats, some of the central changes in neurotransmission can be observed up to 3 weeks postoperatively in areas of nociceptive of processing in the brain.³⁵ These time-related changes highlight the importance of developing strategies that can modulate nociceptive molecules beyond the few hours that a surgical input can last during surgery. Second, animal studies indicate that activation of different nociceptors may be responsible for evoked versus spontaneous pain or different modalities of hyperalgesia (mechanical versus thermal). As an example, mechanical hyperalgesia is effectively reduced by gabapentinoids, while inhibition of P2X purinoceptors has been shown to ameliorate thermal hyperalgesia.^36,37 Furthermore, the ongoing spontaneous activity observed in sensory neurons after incision is responsible for nonevoked pain (pain at rest). This suggests that analgesics targeting nociceptors (ie, transient receptor potential cation channel subfamily V member 1 [TRPV1]) responsible for the increased spontaneous firing in small sensory neurons can effectively blockade pain at rest.²⁷ Finally, the multiple changes occurring at expressional levels of different pronociceptive molecules explain why effective postoperative pain management strategies rely on the concept of multimodal analgesia.²⁹

Mechanisms of RNAi: MiRNAs

In 1993, a seminal study from Ambros’s laboratory led to the discovery of miRNAs.³⁸ MiRNAs are endogenous, single-strand, noncoding RNA molecules that act as posttranscriptional inhibitors or regulators of mRNA.¹⁵ An miRNA cluster is a polycistronic gene in which several miRNAs are encoded in a single primary transcript. MiRNAs are encoded by a complex network of genes and can regulate hundreds of genes.¹¹

MiRNA transcription takes place at the nuclear level by RNA polymerase II. In the nucleus, the primary or pri-miRNA is processed by the Drosha/DGCR8 complex to give origin to the precursor miRNA (pre-miRNA) hairpins. After the cleavage, ~65- to 70-nucleotide-long pre-miRNAs are actively transported from the nucleus to cytoplasm by RAs related nuclear protein-guanosine triphosphate (Ran-GTP) and the export receptor exportin-5.³⁹ Once the pre-miRNA is located in the cytoplasm, the double stranded RNA (dsRNA) is ready for loading onto RNA-induced silencing complex (RISC), which includes dsRNA-binding protein (dsRBP), Dicer, and Argonaute (Ago). The core components of RISC are the Ago protein family members. The Ago-2 possesses an active catalytic domain for cleavage activity in humans among the 8 members of this family. Translational repression and/or target mRNA destabilization occurs in multiprotein complexes that include GW182 proteins, deadenylases, and poly(A) binding proteins. Alteration in the Drosha, Dicer, and Ago protein family can modify the function of miRNAs.⁴⁰ Then, a single-strand miRNA is formed by the activity of the RNA polymerase III.⁴⁰

Depending on the number, type, and position of mismatches in the miRNA/mRNA pairing, it will trigger degradation or translation arrest.⁴⁰ There are 2 ways to alter the functions of miRNAs: miRNA inhibition (antago-miRs) and miRNA replacement (miRNA mimics).^11,41 Synthetic miRNAs, also called miRNA mimics, mimic the function of target endogenous mRNAs. In the case of antagonist miRNA or anti-microRNA (anti-miR), single-stranded synthetic miRNAs have antisense base pairs complementary with the target mRNA to inhibit the action of endogenous miRNA.⁴² These 2 approaches result in the inhibition/degradation of mRNA.^11,43,44

The identification of miRNA targets is vital to develop safe and successful analgesic strategies. Validation studies to elucidate the target genes modulated by miRNA can be done in vitro or in silico. For validation in silico, there are many bioinformatic algorithms, including miRanda (http://microrna.sanger.ac.uk), TargetScan (http://www.targetscan.org), and PicTar (http://pictar.bio.nyu.edu), that can be queried to predict miRNA target sites. However, it is worth mentioning that in the search for the miRNA-binding sites, these sites should be carefully used due to possible discrepancies between results. These algorithms have been constructed to recognize elements in the 3′-untranslated region (UTR) of the miRNA and the complementary 3′-UTRs of orthologous genes. Computational analysis can also be used to predict off-target sites and the levels of inhibition of each target.^11,45

Functional determination of the predicted miRNA/mRNA interaction can be performed in in vitro experiments using a reporter system.⁴⁵ This assay has basis on the principle that the binding of the studied miRNA to its target mRNA will reduce the synthesis of the reporter protein, which is quantified and compared to a control. Once in silico and in vitro validation studies have been completed, signaling and cell function studies are needed to determine if the predicted miRNA/mRNA interaction translates into biological changes.⁴⁵

MiRNAs in Surgical Pain.

Single miRNAs or miRNA clusters participate in mechanisms related to nociception and the response to analgesic drugs in animals and humans with different pain syndromes (Figure 1).^12–14 Significant abnormalities in the expression of miRNAs at the peripheral (nerve endings and dorsal root ganglia) and central (spinal cord) levels have been described in surgical pain models, as well as in experimental paradigms of opioid-induced hyperalgesia and tolerance.^31,46–53

Figure 1.

MiRNAs regulate several mechanisms of postoperative pain. Postoperative pain results from an accumulation of multiple sources, including central and peripheral nervous systems and inflammatory contributions. At several of these sites, miRNAs have demonstrated important roles in regulating mechanisms that can both promote (highlighted in red) and limit (highlighted in blue) pain. In the setting of somatic pain, such as pain occurring at the incisional site, miRNAs play regulatory roles in the central nervous system, such as miR-150 targeting of TLR5 and miR-93 targeting of STAT3, that result in decreased proinflammatory cytokine expression. In a similar fashion, miRNAs also regulate proinflammatory signaling from immune cells that contribute to inflammatory pain. These miRNAs include miR-15a and miR-21, which promote inflammation, and miR-124, which has an inhibitory role. Visceral pain has also been shown to be regulated by miRNAs in both positive and negative manners. At the level of sensory nerves, genetic studies and experimental siRNA-based therapies have implicated sodium and potassium ion channels as potential targets for analgesic treatments. Altogether, there is a growing accumulation of evidence implicating miRNA-mediated regulation of pain. COX indicates cyclooxygenase; DRG, dorsal root ganglia; IL, interleukin; miR, microRNA; miRNA, microribonucleic acid; NAV, voltage-gated sodium channel; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B cells; NPY, neuropeptide Y; PLAA, phospholipase A2 activating protein; siRNA, small interference RNA; STAT3, signal transducer and activator of transcription 3; TLR, Toll-like receptor; TNF, tumor necrosis factor; TRPV, transient receptor potential cation channel subfamily V member.

Several miRNAs, mainly those regulating inflammation, appear to play a key role in incisional pain (Table 2).^47,54–62 Low levels of miR-203 were found in the hind paw tissue of animals with incisional pain.³¹ The expressional change in that miRNA occurred acutely (2 hours) after surgery, lasted for 48 hours, and was inversely correlated with PLAA expression. High levels of PLAA were reversed after intraplanar injection of miR-203.³¹ A study by Li et al,⁶³ demonstrated that the expression of miR-146 and the miR-183 cluster in the DRG and spinal cord was significantly reduced in a surgical model of osteoarthritis. Similarly, miR-16, miR-124–3p, and miR-141 were downregulated in the spinal cord of rats with inflammatory pain and correlated with mRNA upregulation of inflammatory mediators including member RAS oncogene family (RAB23), IL-6, IL-6R, IL-1B, and tumor necrosis factor (TNF)-α.^60,64,65 Notably, the intrathecal administration of miR-16, miR-124–3p, and miR-141 mimics ameliorated inflammatory pain after complete Freund’s adjuvant (CFA) injection.^60,64

Table 2.

miRNAs and miRNAs targets participating in incision and inflammatory pain

Neuropathic pain		Incisional pain		Inflammatory pain		Visceral pain		Cancer bone pain
miRNA	Target	miRNA	Target	miRNA	Target	miRNA	Target	miRNA	Target
• miR-let-7d	Igfr1	• miR-146a	Traf6	• miR16	Rab23	• miR-21	Rasgrp1	• miR-34c-5p	Cav2.3
• miR-7a	Nefl	• miR-146a	Nfkb	• miR34	Pomc	• miR-21	Klf5	• miR-124	Synpo
• miR-15a	Fgf2/Fgfr1	• miR-146a	Tlr2	• miR-124	Rankl	• miR-21	Sc5d	• miR-132	Creb
• miR-15b	Bace1	• miR-146a	Ngf	• miR-124–3p	Il6r	• miR-24	Sert	• miRNA-140–3p	MYMK
• miR-19	Socs1	• miR-146a	Nav1.3	• miR-143–3p	Mrgpre	• miR-29a	Htr7
• miR-23	Nox4	• miR-146a	Trpv1	• miR-143–3p	Ptgs2	• miR-29	Nfg
• miR-26a-5p	Mapk6	• miR-183	Nav1.3	• miR-143–3p	Tnf	• miR-29	Il6
• miR-28–5p	Zeb1	• miR-183	Trpv1	• miR-141–3p	Hmgb1	• miR-29	Ptges3
• miR-30b	Scn9a	• miR-203	Plaa	• miR-155	p38	• miR92b-3p	Vgat
• miR-30c-5p	Tfg11			• miR-219	Camkiiγ	• miR92b-3p	Kcc-2
• miR-32–5p	Dusp5			• miR-187–3p	P2x7r	• miR-144	Zo1
• miR-34c	Nlrp3			• miR-451	TLR4	• miR-144	Ocln
• miR-93	Stat3			• miR-584d	Ugt11a1	• miR-146a-5p	Ccl8
• miR-98	Hmga2					• miR-181a	Gabra1
• miR-124a	Sirt					• miR-199	Trpv1
• miR-124–3p	Ezh2					• miR-199a-5p	Arhgap12
• miR -132	Mecp2					• miR-211–5p	Erk
• miR-132–3p	GluA1					• miR-325–5p	Ccl2
• miR-133–3p	Sn2b					• miR-449	Nk1r
• miR-133–3p	Piezo2					• miR-484–5p	TARGET?
• miR-134–5p	Twist1					• miR-520D-3p	TARGET?
• miR-136	Zeb1					• miR- 520f	TARGET?
• miR-137	Tnfaip1					• miR-1294	TARGET?
• miR-141	Hmgb1
• miR-142–3p	Hmgb1
• miR-143–3p	Sn2b
• miR-143–3p	Trpm8
• miR-145	Rreb1
• miR-145	Akt1
• miR-146a-5p	Traf6
• miR-146a-5p	Irak1
• miR-150	Tlr5
• miR-155	Sirt
• miR-155	Sgk3
• miR-155	Soc1
• miR-182–5p	Ephb1
• miR-183	Bbnf
• miR-183	Cacna2d1
• miR-183	Mtor
• miR-183–5p	Trek-1
• miR-195	Patch1
• miR-206	Bdnf
• miR-217	Tlr5
• miR-221	Socs
• miR-381	Hmgb1
• miR-381	Cxcr4
• miR-449	Trpa1
• miR-449	Kcnma1
• miR-449	Tpte
• miR-539	Nr2b

Ifgr1: insulin like growth factor 1 receptor. Nefl: neurofilament, light polypeptide. Fgf2: fibroblast growth factor 2. Fgfr1: fibroblast growth factor receptor 1. Bace1: beta-secretase 1. Socs1: suppressor of cytokine signaling 1. Nox4: NADPH oxidase 4. Mapk6: Mitogen-activated protein 6. Zeb1: Zinc finger E-box binding homeobox 1. Scn9a: Sodium voltage-gated channel alpha subunit 9. Tgfb1: Transforming growth factor beta 1. Dusp5: Dual specificity phosphatase 5. Nlrp3: NLR family, pyrin domain containing 3. Stat3: Signal transducer and activator of transcription 3. Hmga2: High mobility group AT-hook 2. Sirt: Sirtuin 1. Ezh2: Enhancer of zeste 2 polycomb repressive complex 2 subunit. Mecp2: Methyl-CpG binding protein 2. Glua1: Glutamate ionotropic receptor AMPA type subunit 1. Piezo2: piezo type mechanosensitive ion channel component 2. Twist1: Twist family bHLH transcrpitor factor 1. TNFAIP1: Tumor necrosis factor, alpha-induced protein 1. Hmgb1: high mobility group box 1. Rrbe1: Ras responsive element binding protein 1. Akt1: Thymoma viral proto-oncogene 1. Traf: Tumor necrosis factor receptor associated factor 6. Irak1: Interleukin 1 receptor associated kinase 1. Tlr5: Toll-like receptor 5. Sgk3: serum/glucocorticoid regulated kinase family member 3. Ephb1: Eph receptor 1. Cacna2d1: calcium voltage-gated channel auxiliary subunit alpha2delta 1. Mtor: Mechanistic target of rapamycin kinase. TREK-1 (Kcnk2): Potassium channel, subfamily K, member 2. Cxcr4: chemokine (C-X-C motif) receptor 4. Ocln: Occludin. Nk1r: Tachykinin receptor 1. Rasgrp1: RAS guanyl releasing protein 1. Klf5: Kruppel like factor 5. Sc5d: sterol-C5-desaturase. Sert: Serotonin transporter. Htr7: 5-hydroxytryptamine (serotonin) receptor 7 Nfg: Nerve growth factor. Il6: Interleukin 6. Ptges3: Prostaglandin E synthase 3. Vgat: Vesicular GABA Transporter. Kcc-2: K+/Cl-Cotransporter. Zo1: Tight junction protein 1. Ccl8: C-C motif chemokine ligand 8. Grabra1: gamma-aminobutyric acid (GABA) A receptor, subunit alpha 1. Trpv1: Transient receptor potential cation channel subfamily V member. Erk: Extracellular regulated MAP kinase. CCL2: C-C motif chemokine ligand 2. Cav2.3: calcium channel, voltage-dependent, R type, alpha 1E. Creb: cAMP responsive element binding protein. Arhgap12: Rho GTPase activating protein 12. MYMK: myomaker, myoblast fusion factor. Synpo: Synaptopodyn. Traf6: Tumor necrosis factor receptor associated factor 6. Nfkb: Nuclear factor kappa-light-chain-enhancer of activated B cells. Tlr2: Toll-like receptor. Rankl: Receptor activator of nuclear factor kappa-B Ligand. Nav1.3: Sodium voltage-gated channel. Trpv1: Transient receptor potential cation channel subfamily V member 1. Plaa: Phospholipase A-2-activating protein. Rab23: Member RAS oncogene family. Pomc: Proopiomelanocortin. Il6r: Interleukin 6 receptor. Mrgpre: MAS-related GPR, member E. Ptgs2: Prostaglandin-endoperoxide synthase 2. Tnf: Tumor necrosis factor. Hmgb1: high mobility group box 1. P38: p38 kinase. Camkiiγ: Calcium/calmodulin-dependent protein kinase II gamma. P2x7r: purinergic receptor P2X, ligand-gated ion channel, 7. Ugt1a1: UDP glucuronosyltransferase family 1 member A1.

Analgesics commonly used to provide analgesia in the context of surgery can alter the expression of miRNAs. The daily intraperitoneal administration of gabapentin reduced miR-15a in DRG and partially reversed mechanical hyperalgesia in rodents with arthritic pain after CFA injection.⁶⁶ In a similar animal model, the local coadministration of miR-124 plus ketoprofen-loaded particles was more effective than ketoprofen alone in reducing inflammation assessed by paw and ankle thickness.⁵⁶ Western blot analysis indicated that animals treated with miR-124 plus ketoprofen-loaded particles had lower levels of the receptor activator of NFκB protein in the synovial tissue than those treated with either miR-124 or ketoprofen-loaded particles alone.⁵⁶

Complex regional pain syndrome (CRPS) has been reported as a postoperative complication after major orthopedic surgery. The current evidence indicates that a deregulated inflammatory response linked to miRNA expressional changes is implicated in CRPS.^54,55 TaqMan low-density array in the blood of CRPS patients demonstrated that 18 miRNAs were significantly altered. Among them, miR-532–3p was associated with CRPS type 2, pain intensity, IL1Ra, and vascular endothelial growth factor (VEGF). Pain also correlated with miR-296–5p, miR-361–3p, and miR-30d.⁴⁷ McDonald et al²⁰ conducted a study demonstrating the presence of exosomes containing miRNAs (miR-21–3p, miR-126–3p, and miR-212) in the blood of patients with CRPS. These miRNAs also increased after RAXW 264.7 cells were stimulated with LPS.

Opioid Tolerance and miRNAs.

The efficacy of opioids to treat surgical pain may vary from patient to patient, and its use can induce tolerance or opioid-induced persistent sensitization. Tapocik et al⁶⁷ and other investigators have shown that multiple miRNAs are involved in opioid tolerance and sensitization.^68–73 In the spinal cord or DRG of rodents, an increase in the expression of miR93–5p and a decrease in the levels of miR-365, miR-219–5p, and miR-338 or upregulation of miR-223–3p have been linked to induction of morphine tolerance in the presence or absence of pain.^70,74,75 Wang et al⁷⁰ measured the expression of miR-365 in the spinal cord of rats treated with 10 μg of morphine or saline intrathecally for 7 consecutive days. Downregulation of miR-365 was observed in morphine-tolerant animals compared to saline-treated rats. Notably, the intrathecal delivery of miR-365 using a lentivirus as a vector, partially reversed morphine tolerance. Similarly, overexpression of miR-219–5p in the spinal cord also reversed morphine tolerance by reducing the expression of calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ) and the NR1 subunit of the NDMA receptor.⁶⁹ Changes in the expression of miRNAs also occurred in the DRG of mice acutely tolerant to morphine.⁷⁶ Mice injected with a single dose of 100 mg of morphine subcutaneously showed downregulation of miR-375 in their DRG. Such change in miRNA expression inversely correlated with JAK2 levels and increased expression of brain-derived neurotrophic factor (BDNF). Interestingly, the intrathecal injection of miR-375 agomir reversed the tolerance.⁷⁶

Neuropathic Pain.

Surgical pain with a neuropathic component is a common complication after mastectomy, thoracotomy, or hernia repairs. Laboratory studies indicate that miRNAs participate in mechanisms of peripheral and central sensitization after nerve injury (ie, spinal nerve ligation, chronic constriction injury, diabetic neuropathy, and chemotherapy-induced neuropathy).^{14,19,77–106} Furthermore, nerve damage triggers changes in the expression of different miRNAs that are observed at spinal and supraspinal levels. Liu et al¹⁰⁷ demonstrated significant expressional changes in miR-3573–5p and miR-3074 in the spinal cord in animals with avulsion of the brachial plexus model while the expression of miR-30c-1–3p, miR-702–3p, miR-184, miR-25–5p, miR-873–5p, miR-93–3p, miR-455–3p, and miR-32–3p was altered in the thalamus and the anterior cingulate.¹⁰⁷

Animal studies have also indicated that, after chronic injury of peripheral nerves, there is upregulation of spinal cord miRNAs (ie, miR-15b, miR-155, miR-196, and miR-30c-5p), while others are downregulated (ie, miR-96, miR-217, miR-34c, or miR-206–3p; Table 2).^{77–82,108–112} It is worth mentioning that the level of expression of the same miRNA can vary in different models of neuropathic pain. As an example, Chen et al¹¹³ demonstrated downregulation of miR-96 in the DRG of animals with sciatic nerve constriction injury, while it was upregulated in animals with spinal nerve ligation injury. This is evidence to illustrate the complexity of the epigenetic changes governing nociception after nerve injury. However, as shown in the next sections, 2 miRNAs that are significantly modulated during incisional and neuropathic pain are miR-146 and miR-183.

MiRNAs also modulate genes implicated in the production of inflammatory cytokines after the damage of peripheral nerves (Table 2). The intrathecal administration of lentivirus-miR-mimics for miR-98, miR-142–3p, miR-190a-5p, miR-145, and miR-150, partially attenuated mechanical and thermal hyperalgesia in animals with neuropathic pain due to chronic constriction injury and diabetic neuropathy. A common mechanism for this finding was the downregulation of IL-6, TNF-α, COX 2, and IL-1β by directly targeting the mRNAs of high mobility group adenine-thymine hook2 (HMGA2), high mobility group box1 (HMGB1), protein kinase B (AKT3), voltage-glutamate transporter 1 (also known as SCL17A6), signal transducer and activator of transcription 3 (STAT3), and Toll-like receptor 5 (TLR5).^48–53 In similar studies, Hori et al¹¹⁴ reported that miR-21, miR-431, and miR-511–3p induced hyperalgesia by increasing the production of IL-6 in the spinal dorsal horn, whereas the intrathecal administration (3 injections) of miR-30c-5p inhibitor and miR-32-p knockdown reversed hyperalgesia and allodynia in rats with spinal nerve ligation by suppressing the production of inflammatory cytokines.¹¹⁵

Human studies also demonstrate the aberrant expression of miRNAs in subjects with painful neuropathies and fibromyalgia syndrome.¹⁰⁴ For instance, increased levels of miR-30c-5p in blood and cerebrospinal fluid (CSF) were a predictor of neuropathic pain in patients with diabetes mellitus.¹¹⁶ In a mixed cohort of patients with painful neuropathies, the expression of miR-21 and miR-132–3p was increased in the sural nerve and leucocytes, but they showed reduced levels of miR-146 and miR-155 in the affected skin.^99,117 Heyn et al¹¹⁸ studied the expression of miR-124a and miR-155 in CD4+ T cells of 11 patients with neuropathic pain and 9 healthy subjects. The expression of both miRNAs was significantly higher in patients with pain than controls and downregulation of the expression of sirtuin 1 (SIRT). Notably, SIRT is a key controller of T regulatory cells differentiation, which has been shown to reduce pain sensitization.¹¹⁸

Visceral and Cancer Pain.

Visceral pain is common in certain conditions that might require surgery, such as endometriosis, or in patients with irritable and inflammatory bowel syndromes, chronic prostatitis, and bladder pain syndromes. Extensive research has been conducted to i dentify how miRNA modulates visceral nociception (Table 2).^55,119–125 Increased expression of miR-146a-5p and downregulation of miR-211–5p and miR-325–5p were detected in the spinal cord and DRG of rats with chronic peritoneal and colonic inflammation, respectively.^126–128 Notably, 4 consecutive intrathecal injections of miR-325-agomir and the systemic treatment with miR-211–5p reversed hypersensitivity to colonic distention and mechanical and thermal hyperalgesia.^126,127 Li et al¹²⁹ investigated the role of miR-187–3p in ischemia-reperfusion (IR) pain using an aortic cross-clamping model in rats. In IR animals, downregulation of miR-187–3p in the spinal cord was inversely correlated with the expression of purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7R). Intrathecal treatment with miR-187–3p agomir reversed mechanical and thermal hyperalgesia and decreased inflammation at the level of the spinal cord.¹²⁹

In men with chronic prostatitis/chronic pelvic pain syndrome, the expression of miR21–5p in their prostate fluid is higher than in healthy men.¹³⁰ Similarly, in patients with irritable bowel syndrome, the colonic expression of miR-29a is increased and negatively correlated with the expression of 5-hydroxytryptamine (serotonin) receptor 7 (Htr7) mRNA. When the expression of miR-29a was in knockdown mice, the expression of HTR7 was increased and animals showed less visceral pain.¹³¹ The expression of miR-199a-5p is increased in patients with bladder pain syndrome. Monastyrskaya et al¹²³ showed that miR-199a-5p downregulated the expression of LIN7C, ARH-GAP12, PALS1, RND1, and PVRL1, which in turn was associated with an increase in bladder epithelial permeability. Ciszek et al¹³² measured blood miRNA in women with and without chronic abdominal/pelvic pain. Blood levels of miR-484–5p, miR-1294, and miR-520f were significantly downregulated in those with pain, while blood levels of miR-520D-3p were increased compared to healthy controls. The authors linked the observed changes to deregulation in proinflammatory cytokines such as IL-8.

The most commonly used experimental model to study cancer pain consists of inoculating malignant cells into the long bones (ie, tibia) of rodents. Using this animal paradigm, Elramah et al¹³³ showed a significant downregulation of miR-124 and upregulation of synaptopodin in the spinal dorsal horn ipsilateral to the tumors. Then, these authors elegantly treated animals with 2 μg of miR-124 mimic intrathecally using i-Fect reagent as a carrier for 3 consecutive days and found that the mimic miRNA significantly ameliorated pain in comparison to a C. elegans–specific miRNA.¹³³ Cyclic adenosine monophosphate-responsive element-binding protein (CREB) is an important transcription factor that is activated after nociceptive inputs reach the spine dorsal horn. One of the mechanisms by which CREB participates in nociception is by promoting miR-132 upregulation as it was shown in the spinal cord of animals inoculated with NCTNC 2472 fibrosarcoma cells.¹³⁴ MiR-34c-5p and its target calcium channel, voltage-dependent (Cav2.3), were observed in the DRG neurons of mice inoculated with the same fibrosarcoma cells.¹³⁵

Transient musculoskeletal (MSK) pain is a common (approximately 30%) complication in chronic myeloid leukemia patients taking tyrosine kinase inhibitors (TKIs) such as imatinib. A recent study by Asano et al¹³⁶ demonstrated the presence of circulating exosomes loaded with miRNA-140–3p in patients with MSK pain taking TKIs. The authors suggested that elevated miR-140–3p can reduce the expression of Myomarker (a critical gene for muscle regeneration), and this may be responsible for MSK symptoms.¹³⁶

Strategies to Utilize miRNAs as Novel Postoperative Analgesic Therapies in Humans.

The interest in RNA-focused therapies has grown since the Fire and Mello discovery of RNAi.¹³⁷ Since then, monogenic and polygenic targeting therapies have been proposed to cure different human pathologic conditions. To date, several biotechnology companies have been created to develop miRNA-based therapies in different medical fields, including cancer, cardiology, and inflammatory, infectious, and congenital diseases. As a result, multiple studies are ongoing to evaluate the efficacy and toxicokinetics of systemic miRNA delivery in humans.¹³⁸ In 2016, Neudecker et al¹⁵ reviewed the role of miRNAs in the context of perioperative medicine. However, no study was tested in humans to determine the safety or efficacy of miRNAs in the context of surgery and pain treatment.

Several important questions should be considered for “analgesic-miRNA” or “analgo-miRNA” in the context of surgery (Figure 2). First, which miRNA should be targeted or administered? To answer this question, we should consider mimics of downregulated miRNA (synthetic double-standard miRNAs) or antagonists (chemically modified anti-miR oligo-nucleotides or sponges) of overexpressed miRNA to return miRNA to its physiological state along with the trajectory and characteristics of surgical pain. In rodents, miR-203, miR-146, and miR183 are all decreased postoperatively. If similar changes were occurring in humans, then the administration of analgo-miRs to restore the levels at the site of injury or centrally should be considered.^31,63 In results from animal studies using miRNAs in models of neuropathic pain, one could consider examining these miRNAs in clinical studies targeting postoperative pain after procedures with high rates of postsurgical neuropathic pain (ie, mastectomies or thoracotomies). Monogenic targeting molecules might have a limited efficacy in the treatment of surgical pain because during peripheral and central sensitization, there is activation of redundant cellular pathways. Therefore, polygenic therapy regulation based on miRNAs (multitargeting therapy) is an attractive strategy to ameliorate surgical pain. Furthermore, miRNAs can be used to target “nondrugable genes” that also play a role in surgical pain. However, it is worth mentioning that miRNAs can activate genes or even bring pseudoactivity by themselves; therefore, careful selection of “analog miRNAs” is key to avoid unwanted adverse effects.

Figure 2.

Analgo-miRNAs for surgical pain management. On the left, the current status of pain management (multimodal analgesia) for major surgery is displayed. On the right, a novel proposed concept of RNAi-based analgesia is depicted, which is currently being tested in the laboratory. miRNAs are single-stranded, noncoding RNA molecules that act as posttranscriptional inhibitors or regulators of the mRNA. miRNA therapies are in clinical trials for the treatment of some diseases. Here, we propose the administration of miRNAs perioperatively to provide pain relief. i.t. indicates intrathecal; i.v., intravenously; miRNA, microribonucleic acid; RNA, ribonucleic acid; RNAi, ribonucleic acid interference.

The second question is when and where “analog miRNAs” should be delivered. miRNA “mimics” or “antago-miRNAs” have a short half-life and rapidly degrade by nucleases, which limits their duration of action. The concentration of nucleases in the CSF is lower than in blood, thus making the intrathecal administration more attractive for the use of “analog miRNAs.” In addition, nuclease-resistant delivery systems have been developed for patient treatment, which could further increase the duration of action of “analog miRNAs.”¹³⁹ Preclinical studies have shown that both anti-miRs and miRNA mimetics can effectively be delivered intrathecally. However, the current evidence is not clear regarding the differences in efficacy between anti-miRs and miRNA mimetics, which can be explained by the fact that most animal studies have focused on interventions modulating single miRNAs. While the intrathecal delivery is clearly the most studied route of administration of miRNAs in animals, it should be taken into consideration that it might not be suitable for everyday use in many patients. Therefore, new studies should focus on other alternatives for miRNA delivery, such as local. In fact, the intraplanar injection of miR-203 reversed surgical-induced pain in animals with plantar incision.³¹

Time of miRNA delivery is also a clinically relevant consideration. There are no clinical studies demonstrating the efficacy of preoperative versus postoperative administration of “analog miRNAs” for surgical pain control. Also, the available preclinical evidence is not clear on whether there are differences in the duration of action between intrathecally delivered anti-miRs and miRNA mimetics. However, animal studies suggest that patients could require the administration of multiple injections of “analog miRNAs” hours or days before surgery to facilitate the silencing or modulation of preformed or highly expressed protein before surgery. Contrarily, silencing of molecules only expressed after surgical insult could be achieved with single injections of miRNAs. Finally, the coadministration miRNA antagonists or miRNA mimics with local anesthetics could be an attractive alternative.

The last question is how miRNAs should be delivered to provide effective analgesia. Several barriers exist to successfully deploy miRNA to target cells.⁴¹ There are 2 main methods of carrying miRNAs: viral and nonviral vectors.^10,140 Nonviral vectors can also be grouped into inorganics, organic (peptides, chitosan, lipids/liposomes, and aptamers), and polymer-based nanomaterials. Among the nonviral carriers, cationic lipids are used due to their biocompatibility, biodegradability, enhanced cellular entry of miRNAs, easy production, moderate toxicity, and immunogenicity.¹⁴¹ Inorganic carriers include gold, grapheme, quantum dot, and silica nanoparticles.¹⁴¹ Except for quantum dots (highly toxic), low toxicity and high biocompatibility are features of these inorganic nanoparticles. Finally, polymers such as polyethyleneimine, dendrimers, and polylactic-coglycolic acid have been extensively investigated to deliver miRNAs. One of the main advantages of polymer is high stability in body fluids, in addition to low cytotoxicity.¹⁴¹ The type of vector or carrier is of particular importance, since the spread of the “analog miRNAs” in the CSF will depend on the density, specific gravity, and baricity of the delivered mimic or antago-miR and the vector solution, which ultimately will determine the dermatomal level of analgesia.

SiRNAs could be considered as an alternative to analog miRNAs. SiRNAs are short (21–23 base pairs), double-stranded RNAs with multiple biological functions, including posttranscriptional repression and heterochromatin formation. While miRNA and siRNA cause RNA-induced translational silencing, there are major differences among them. First, siRNAs are considered exogenous, while miRNAs are naturally occurring. Second, siRNAs bind perfectly to their mRNA targets, while miRNAs can attach to multiple mRNAs.¹⁴² Effector phases of posttranscriptional siRNA silencing occur primarily in the cytoplasm.⁴⁰ Chemical synthesis of siRNA results in purer and more stable siRNAs than those generated by gene expression. Synthetic siRNAs are typically 19–21 base pairs in length with 2-nucleotide single-stranded overhangs at their 3′ ends. After incorporation into the cytoplasm, synthetic siRNAs are incorporated into the RNAi machinery to initiate protein inhibition. However, there are “off-target” effects of siRNAs. This appears to occur when there is an imperfect match between the siRNA and the target mRNAs. siRNA has been used to repress synthesis of a wide variety of proteins involved in nociceptive mechanisms. For instance, Li et al¹⁴³ demonstrated that spinal downregulation of the Cavβ3 subunit of the voltage-activated calcium channel via siRNA reversed hypersensitivity to mechanical stimulation in animals with spinal nerve ligation. The available literature suggests that effective downregulation of nociceptors can be achieved after multiple injections, after which the duration of changes in nociceptor protein levels appears to be long lasting (days). There is no evidence of siRNA use in humans with the goal of providing analgesia in the context of surgery. Contrarily, siRNAs have been effectively administered in humans with malignancies.¹⁴⁴

CONCLUSIONS AND FUTURE PERSPECTIVES

Peripheral and central sensitization are associated with epigenetic alterations occurring in the DRG and spinal cord and mediated by activation of leucocytes, myeloid cells, neurons, and glial cells. Among those epigenetic aberrations, research has demonstrated downregulation and upregulation of miRNAs. Animals studies convincingly demonstrate that mimic-miR/antago-miRs administered before a surgical insult can ameliorate nociceptive behaviors. This exciting research using RNAi technologies based on miRNA mimics or antago-miRNAs has shown promising results and could bring new hope in the care of thousands of patients undergoing major surgery. However, there is a need for the development of better animal models that can reproduce pain related to specific procedures, such as craniotomies or labor.¹⁴⁵

New discoveries in delivery systems will allow clinicians to safely administer mimic-miR/antago-miRs. Single or multiple analog miRNAs could be used to suppress proteins involved in surgical pain. Therefore, it can be theorized that this novel analgesic modality could (1) be administered preoperatively, (2) provide long-lasting analgesia, (3) diminish or prevent the chronification of pain, and (4) avoid or spare the use of other analgesics such as opioids. In addition, when given intrathecally, analog miRNAs could avoid systemic adverse events. On the other hand, off-target effects and still unclear rational selection of best miRs/anti-miRs can be considered as disadvantages. Based on the premises presented above, we hope to see future clinical studies evaluating the safety and efficacy of analog miRNAs as newer analgesics emerge in the context of major surgery.

GLOSSARY

Ago

Argonaute

Akt1

thymoma viral proto-oncogene

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

anti-miR

anti-microRNA

Arhgap12

Rho GTPase activating protein 12

ASIC3

acid sensing ion channel 3

Bace1

beta-secretase 1

BDNF

brain-derived neurotrophic factor

Cacna2d1

calcium voltage-gated channel auxiliary subunit alpha2delta 1

Camkiiγ

calcium/calmodulin-dependent protein kinase II gamma

cAMP

cyclic adenosine monophosphate

Cav2.3

calcium channel, voltage-dependent

CCL

C-C motif chemokine ligand

CFA

complete Freund’s adjuvant

COX

cyclooxygenase

Creb

cyclic adenosine monophosphate-responsive element-binding protein

CRPS

complex regional pain syndrome

CSF

cerebrospinal fluid

Cxcr4

chemokine (C-X-C motif) receptor 4

DRG

dorsal root ganglia

dsRBP

dsRNA-binding protein

dsRNA

double stranded RNA

Dusp5

dual specificity phosphatase 5

Ephb1

Eph receptor

Erk

extracellular-regulated MAP kinase

Ezh2

enhancer of zeste 2 polycomb repressive complex 2 subunit

Fgf2

fibroblast growth factor 2

Fgfr1

fibroblast growth factor receptor 1

GluA1

glutamate ionotropic receptor AMPA type subunit 1

GPCR

G protein-coupled receptor

GTP

guanosine triphosphate

Hmga2

high mobility group adenine-thymine-hook 2

HMGB1

high mobility group box 1

Htr7

5-hydroxytryptamine (serotonin) receptor 7

IL

interleukin

Il6r

interleukin 6 receptor

iNOS

inducible nitric oxide synthase

IR

ischemia-reperfusion

Irak1

interleukin 1 receptor–associated kinase 1

i.v.

intravenous

Kcc-2

K+/Cl-cotransporter

Kcnma1

potassium calcium-activated channel subfamily M alpha 1

Klf5

Kruppel like factor 5

LIF

leukemia inhibitor factor

LPS

lipopolysaccharide

MAP

mitogen-activated protein

Mapk6

mitogen-activated protein 6

Mecp2

methyl-CpG binding protein 2

MEK

mitogen activated protein kinase kinase

miR

miRNA

miRNA

microRNA

MRGPRE

mastocyte-related G protein-coupled receptors, member E

mRNA

messenger ribonucleic acid

MSK

musculoskeletal

Mtor

mechanistic target of rapamycin kinase

MYMK

myomaker, myoblast fusion factor

Nav

voltage-gated sodium channel

Nefl

neurofilament, light polypeptide

NFκB

nuclear factor kappa-light-chain-enhancer of activated B cells

Nfg

nerve growth factor

NGF

nerve growth factor

Nk1r

tachykinin receptor 1

Nlrp3

nod-like receptor family, pyrin domain containing 3

Nox4

nicotinamide adenine dinucleotide phosphate oxidase 4

NPY

neuropeptide Y

NR1

subunit NR1 NMDA receptor

OCLN

occludin

P2X7R

purinergic receptor P2X, ligand-gated ion channel, 7

P38

p38 kinase

PI3K

phosphoinositide 3-kinase

Piezo2

piezo type mechanosensitive ion channel component 2

PLAA

phospholipase A2 activating protein

POMC

proopiomelanocortin

pre-miRNA

precursor miRNA

PTGES2

prostaglandin-endoperoxide synthase 2

Ptgs2

prostaglandin-endoperoxide synthase 2

RAB23

member RAS oncogene family

Ran-GTP

RAs related nuclear protein-guanosine triphosphate

Rankl

receptor activator of nuclear factor kappa-B ligand

RANL

receptor activator of nuclear factor kappa-B ligand

Rasgrp1

RAS guanyl releasing protein 1

RISC

RNA-induced silencing complex

RNA

ribonucleic acid

RNAi

ribonucleic acid interference

SC5D

sterol-C5-desaturase

Scn9a

sodium voltage-gated channel alpha subunit 9

Sert

serotonin transporter

Sgk3

serum/glucocorticoid regulated kinase family member 3

SIRT

sirtuin 1

siRNA

small interference RNA

Socs1

suppressor of cytokine signaling 1

Stat3

signal transducer and activator of transcription 3

Synpo

synaptopodyn

Tgfb1

transforming growth factor beta 1

TKIs

tyrosine kinase inhibitors

TLR

Toll-like receptor

TNF

tumor necrosis factor

Tnfaip1

tumor necrosis factor, alpha-induced protein 1

Tpte

transmembrane phosphatase with tensin homology

TRAF6

tumor necrosis factor receptor associated factor 6

TREK-1 (Kcnk2)

potassium channel, subfamily K, member

TRK

tropomyosin receptor kinase

Trpa1

transient receptor potential cation channel, subfamily A, member 1

TRPV1

transient receptor potential cation channel subfamily V member 1

Twist1

twist family basic helix-loop-helix transcrpitor factor 1

Ugt1a1

Uridine diphosphate glucuronosyltransferase family 1 member A1

UTR

untranslated region

VEGF

vascular endothelial growth factor

Vgat

vesicular gamma aminobutyric acid transporter

Zeb1

zinc finger E-box binding homeobox 1

Zo1

tight junction protein 1