Abstract

Chronic pain, a common clinical symptom, is often treated inadequately or ineffectively in part due to the incomplete understanding of molecular mechanisms that initiate and maintain this disorder. Newly identified noncoding RNAs govern gene expression. Recent studies have shown that peripheral noxious stimuli drive expressional changes in noncoding RNAs and that these changes are associated with pain hypersensitivity under chronic pain conditions. This review first presents current evidence for the peripheral inflammation/nerve injury–induced change in the expression of two types of noncoding RNAs, microRNAs, and Kcna2 antisense RNA, in pain-related regions, particularly in the dorsal root ganglion. The authors then discuss how peripheral noxious stimuli induce such changes. The authors finally explore potential mechanisms of how expressional changes in dorsal root ganglion microRNAs and Kcna2 antisense RNA contribute to the development and maintenance of chronic pain. An understanding of these mechanisms may propose novel therapeutic strategies for preventing and/or treating chronic pain.

CHRONIC pain usually caused by inflammation and tissue or nerve injury is a major public health problem worldwide. It is characterized by ongoing or intermittent burning pain, an enhanced response to noxious stimuli (hyperalgesia), and pain in response to normally innocuous stimuli (allodynia). Current treatment for this disorder has had restricted success due to our inadequate understanding of the mechanisms that lead to the initiation and maintenance of chronic pain. It is known that inflammation and nerve injury produce changes in the expression of receptors, enzymes, ion channels, neurotransmitters, neuromodulators, and structural proteins in primary sensory neurons of the dorsal root ganglion (DRG) at both transcriptional and translational levels.1–3  Such changes are considered to contribute to chronic pain development and maintenance.1–3  However, it is unclear how peripheral inflammation or nerve injury alters the expression of these genes and/or proteins in DRG. Understanding this mechanism may enable the development of new strategies to prevent and/or treat chronic pain.

Recent studies suggest that the mechanism for gene regulation involves widespread noncoding (nc) RNAs.4–6  RNA had long been thought to be a simple and intermediary component of gene expression, as it is transcribed from DNA and then translated into proteins in cells. However, it has become increasingly clear that mammalian genomes encode not only protein-coding RNAs but also a vast number of ncRNA transcripts.7  Because the function of each newly identified ncRNA has not been fully elucidated, the common practice is to group ncRNA transcripts based on transcript size: small/short ncRNAs (e.g., microRNAs [miRNAs]; 18 to 200 nucleotides) and long ncRNAs (e.g., native Kcna2 antisense [AS] RNA; >200 nucleotides).7–9  ncRNAs have been systematically identified in the mammalian nervous system, including in pain-related regions.10  They can be regulated and may govern the expression of both protein-coding and noncoding genes. An intriguing association between aberrant expression of ncRNAs and the development of diseases has been demonstrated recently.11,12  Previous studies have shown that peripheral inflammation and nerve injury drive changes in the expression of some miRNAs and Kcna2 AS RNA in DRG.13–16  These changes might be responsible for inflammation/nerve injury–induced alterations of some pain-associated genes, an increase in DRG neuronal excitability, and behavioral pain hypersensitivity.14,16  The evidence indicates that ncRNAs might be new key players in the mechanisms that underlie the development and maintenance of chronic pain.

In this article, we first review current evidence for the changes of two types of ncRNAs, miRNAs and Kcna2 AS RNA, in pain-related regions, particularly in DRG, after peripheral inflammation and nerve injury. We then discuss how peripheral noxious stimuli induce such changes. We finally explore potential mechanisms of how expressional changes in DRG miRNAs and Kcna2 AS RNA contribute to the development and maintenance of chronic pain. This review provides more up-to-date knowledge regarding the role of ncRNAs in the mechanisms of chronic pain.17,18 

miRNAs in Chronic Pain

Formation of miRNAs

Since the discovery of the first miRNA, lin-4 in Caenorhabditis elegans, hundreds of miRNAs have been identified in the nervous system.19–21  These miRNAs are coded by specific genes. Generally, a miRNA molecule is synthesized from a long RNA primary transcript known as a pri-miRNA (fig. 1). In the cellular nucleus, pri-miRNA is cleaved by Drosha, an RNAIII endonuclease, to produce a characteristic stem-loop structure of approximately 60 to 70 nucleotides in length, known as pre-miRNA (fig. 1). After pre-miRNA is exported from the nucleus into the cytoplasm, it is cleaved by Dicer, another RNAIII endonuclease, to produce double-stranded mature miRNA (fig. 1). The latter is either unwound via an unknown helicase or cleaved by the enzyme Ago2 to lead to a single-stranded miRNA (fig. 1).22  The single strands completely or incompletely bind to specific messenger RNA (mRNA) sequences, resulting in degradation or translational repression of target mRNAs.23  A recent link between miRNA-mediated poly(A)-tail length shortening and mRNA destabilization has been reported, suggesting another potential mechanism of ncRNA-mediated gene regulation.24 

Fig. 1.

Formation of mature microRNA (miRNA). miRNA is transcribed from the genome (DNA) via RNA polymerase II (Pol II). The resulting pri-miRNA transcript is then cleaved via the endonuclease Drosha to create a 60–70 nucleotide long pre-miRNA. This transcript is then removed from the nucleus via exportin-5 to the cytoplasm where it is cleaved by Dicer, another endonuclease. The resulting double-stranded mature miRNA is unwound by a helicase or cleaved by Ago2. The single-stranded mature miRNA then acts as the core of RNA-induced silencing complex. This complex guides the miRNA to its target sequence located within the 3’-untranslated region (3’-UTR) of the target messenger RNA (mRNA). Incomplete or complete base-pairing results in degradation of the mRNA or inhibition of translation.

Fig. 1.

Formation of mature microRNA (miRNA). miRNA is transcribed from the genome (DNA) via RNA polymerase II (Pol II). The resulting pri-miRNA transcript is then cleaved via the endonuclease Drosha to create a 60–70 nucleotide long pre-miRNA. This transcript is then removed from the nucleus via exportin-5 to the cytoplasm where it is cleaved by Dicer, another endonuclease. The resulting double-stranded mature miRNA is unwound by a helicase or cleaved by Ago2. The single-stranded mature miRNA then acts as the core of RNA-induced silencing complex. This complex guides the miRNA to its target sequence located within the 3’-untranslated region (3’-UTR) of the target messenger RNA (mRNA). Incomplete or complete base-pairing results in degradation of the mRNA or inhibition of translation.

Expressional Changes of miRNAs after Noxious Stimulation

Changes in the expression of miRNAs in response to noxious stimulation have been reported. Bai et al.13  reported a significant down-regulation of mature miR-10a, -29a, -98, -99a, -124a, -134, and -183 in the mandibular division of trigeminal ganglion ipsilateral to complete Freund’s adjuvant (CFA)–injected rat masseter muscle in a model of peripheral inflammation (table 1). Such down-regulated miRNAs were observed 4 h after injection and recovered differentially to a normal level or higher than normal level.13  Expression and down-regulation of miRNAs occurred in all sizes of trigeminal ganglion neurons (but not in glial cells and other nonneuronal cells) that innervate the inflamed muscle although the miRNA signals varied among neurons (table 1).13  Injection of CFA into a hind paw also reduced expression levels of miR-1, -16, -206, and -143 in the ipsilateral DRG neurons,25,26  but increased miR-1, -16, and -206 levels in the ipsilateral spinal dorsal horn neurons (table 1).25  These changes clearly correlate to CFA-induced peripheral inflammation. It should be noted that CFA-induced changes in miRNAs may be immune related as CFA also causes immune response. Interestingly, peripheral injection of formalin led to a significant down-regulation of miRNA-124a expression in the neurons of dorsal horn ipsilateral to injection (table 1).27  These studies provide promising evidence of miRNA changes in pain-related regions under inflammatory conditions.

Table 1.

miRNAs Associated with Peripheral Inflammation

miRNAs Associated with Peripheral Inflammation
miRNAs Associated with Peripheral Inflammation

In addition to peripheral inflammation, expressional changes of miRNAs were observed after peripheral nerve injury. L5 spinal nerve ligation (SNL) induced a drastic decrease in the expression of miR-1, -7a, -96, -103, -182, -183, and -206 in the injured DRG14,25,28,29  and in the expression of miR-200b and -429 in the nucleus accumbens (table 2).30  L5 SNL also down-regulated the expression of 59 miRNAs in the uninjured L4 DRG (table 2).31  Consistently, in the sciatic nerve transection or chronic constriction injury model of neuropathic pain, the injured DRG showed reduced expression of several miRNAs, including miR-10a, -30b, -99a, -100, -143, -582-3p, and -720 (table 2).26,32  In contrast, miR-21 in the injured DRG was up-regulated after L5 SNL (table 2).14,15  Although these changes in expression may not be ruled out to be related to regeneration, the evidence indicates that the expression of miRNAs is differentially and spatially regulated in pain-related regions after peripheral nerve injury.

Table 2.

Noncoding RNAs Associated with Peripheral Nerve Injury

Noncoding RNAs Associated with Peripheral Nerve Injury
Noncoding RNAs Associated with Peripheral Nerve Injury

Furthermore, expressional changes of miRNAs have also been observed in patients with painful diseases. In bladder biopsies from patients with bladder pain syndrome (also known as interstitial cystitis), 31 miRNAs were differentially expressed (table 1).33  An inverse relation was observed in which neurokinin-1 mRNA/protein was down-regulated and four miRNAs (miR-449b, -500, -328, and -320) were up-regulated.33  Differential expression of 18 miRNAs was reported in blood from patients with complex regional pain syndrome (table 1).34  In addition, miR-146a, -199a, and -558 may be linked to pain-related pathophysiology of osteoarthritis through regulation of the expression of cyclooxygenase-2 (table 1).35–37  It seems that miRNA profiles have the potential to serve as biomarkers of pain.

miRNAs Regulated by Inflammatory Mediators in Chronic Pain

How peripheral noxious stimulation causes the alternations of miRNA expression in pain-related regions is incompletely understood, but it is very likely that miRNA expression may be controlled by inflammatory mediators (fig. 2). Administration of resolvin D1, an anti-inflammatory lipid mediator, counter-regulated the expression of miR-21, -142, -146b, -203, -208a, -219, and -302d in a murine peritonitis model of self-limiting acute inflammation (table 1),38  suggesting at least partial involvement of inflammatory mediators in inflammation-induced changes in miRNA expression. A recent study revealed that stimulation with interleukin (IL)-1β, an inflammatory mediator, produced a significant reduction in miR-558 in normal and osteoarthritis chondrocytes possibly through IL-1β–induced activation of mitogen-activated protein kinase and nuclear factor-κB (table 1).37  IL-1β also increased the expression of miR-21 in DRG neurons,15  the insulinoma cell line MIN6,39  and human pancreatic islets.39  Activator protein 1 (AP-1), a transcription factor, may participate in this effect of IL-1β as the promoter region of miR-21 contains the binding site of AP-1,40  and IL-1β triggers AP-1 activation in DRG neurons.15  Given that peripheral inflammation and nerve injury increase DRG IL-1β expression, IL-1β may be responsible for inflammation-induced down-regulation of miR-558 and nerve injury–induced up-regulation of miR-21 in the injured DRG (fig. 2).15,37 

Fig. 2.

Proposed model for the mechanism by which microRNAs (miRNAs) contribute to chronic inflammatory and neuropathic pain. After peripheral inflammation or nerve injury, the increase in inflammatory mediators such as interleukin (IL)-1β causes a change in the expression of miRNAs in dorsal root ganglion (DRG) neurons. This change includes up-regulation of some miRNAs (e.g., miR-7a) and down-regulation of other miRNAs (e.g., miR-21), resulting in an alteration in pain-related genes, such as an increase in β-subunit of voltage-gated sodium channels (Nav), in DRG. Such an alteration leads to an increase in DRG neuronal excitability, spinal central sensitization, and pain hypersensitivity (hyperalgesia and allodynia).

Fig. 2.

Proposed model for the mechanism by which microRNAs (miRNAs) contribute to chronic inflammatory and neuropathic pain. After peripheral inflammation or nerve injury, the increase in inflammatory mediators such as interleukin (IL)-1β causes a change in the expression of miRNAs in dorsal root ganglion (DRG) neurons. This change includes up-regulation of some miRNAs (e.g., miR-7a) and down-regulation of other miRNAs (e.g., miR-21), resulting in an alteration in pain-related genes, such as an increase in β-subunit of voltage-gated sodium channels (Nav), in DRG. Such an alteration leads to an increase in DRG neuronal excitability, spinal central sensitization, and pain hypersensitivity (hyperalgesia and allodynia).

Potential Mechanisms of miRNAs’ Effects on Chronic Pain

It has been demonstrated that miRNAs exert their functions through their complete or incomplete sequence homology to the 3’-untranslated region of target mRNAs, resulting in a block in translation or mRNA degradation (fig. 1).23  Studies on inflammatory pain suggest that miRNAs specifically target pain-related genes (fig. 2). When a miRNA-124a inhibitor was intravenously administered after formalin injection, the down-regulation of miR-124a in the spinal cord was enhanced. This resulted in exaggerated formalin-induced nociceptive behaviors associated with an up-regulation of the pain-relevant miRNA-124a target methyl CpG-binding protein 2 and proinflammatory marker genes in the spinal cord.27  In contrast, blocking formalin-induced down-regulation of spinal cord miRNA-124a through pre-miRNA-124a administration counteracted these effects and reduced nociception by down-regulating these target genes.27  miRNA-181a possesses multiple complementary binding sites for the γ-aminobutyric acid (GABA)A receptor subunit GABAAα-1 gene, GABRA1, suggesting a possible target for this miRNA. A neonatally zymosan-induced increase in miR-181a resulted in down-regulation of the GABAAα-1 mRNA and protein in the spinal cord.41  This effect may contribute to neonatal cystitis-induced chronic visceral pain.41  Identification of the target genes of miRNAs with specific changes in chronic pain may provide insight into the role of miRNAs in chronic pain development and maintenance.

The importance of miRNAs in pain is further validated in a study in which the activity of Dicer, a key enzyme in mature miRNA formation (fig. 1), is eliminated.23  Conditional knockout of Dicer in DRG Nav1.8 neurons resulted in not only the loss of all mature miRNAs but also the reduced pain-related transcripts including voltage sodium channel (Nav) 1.7, Nav1.8, and Ca2+/calmodulin-dependent protein kinase II in the primary sensory neurons.42  The conditional null mice failed to display inflammatory mediator-induced enhancement in excitability of Nav1.8 sensory neurons and formalin-induced c-FOS expression in spinal cord.42  These mice also exhibited significant inhibition of inflammatory pain after formalin, CFA, and carrageenan injection.42  In contrast, Dicer null mice displayed an intact acute nociceptive behavior in response to electrical, mechanical, and thermal stimuli,42  indicating that the loss of mature miRNAs in the nociceptors does not affect acute pain transmission to the spinal cord and brain. Therefore, miRNAs may be potential targets for the prevention and/or treatment of chronic inflammatory pain.

The functional role of miRNAs in neuropathic pain has also been observed (fig. 2). Although Dicer null mice exhibited intact SNL-induced pain hypersensitivity,42  the role of miRNAs in neuropathic pain cannot be ruled out as deletion of DRG Nav1.8 or most DRG nociceptors had no effect on neuropathic pain.43–45  Moreover, nerve injury–induced increases in abnormal ectopic discharges were found primarily in injured myelinated afferents and the corresponding large and medium DRG neurons.46,47  Thus, miRNAs expressed in large and medium DRG neurons may be involved in the production of abnormal spontaneous activity and neuropathic pain initiation. Indeed, miR-7a is expressed in small, medium, and large DRG neurons and robustly decreased in the injured DRG in the late phase of neuropathic pain14  (table 2). Blocking this decrease through miR-7a overexpression in the injured DRG suppressed up-regulation of the β2 subunit protein of voltage-gated sodium channels in the DRG, normalized long-lasting hyperexcitability in nociceptive neurons, and attenuated established neuropathic pain without affecting acute pain and inflammatory pain.14  Furthermore, mimicking nerve injury–induced down-regulation of DRG miR-7a through intrathecal administration of a specific miR-7a inhibitor increased β2 subunit protein levels in the DRG and led to pain-related behaviors in intact rats.14  Another miRNA, miR-21, is persistently up-regulated in the injured DRG neurons during the late phase of neuropathic pain15  (table 2). The intrathecal administration of a miR-21 inhibitor (a single-stranded RNA with chemical modifications) alleviated nerve injury–induced mechanical and thermal hyperalgesia.15  miR-21 may participate in neuropathic pain conditions by down-regulating multiple targets including negative regulators of matrix metalloproteinases (which exhibit increased activity after nerve injury),48  an endogenous inhibitor of phosphatidylinositol 3-kinase (that is decreased after nerve injury),49  and negative regulators of extracellular signal–regulated kinase.50  miRNAs may also be therapeutic targets for intractable chronic neuropathic pain.

Taken together, it is very likely that inflammatory mediators produced by peripheral inflammation or nerve injury act on peripheral nociceptors and then change the expression of DRG miRNAs. These changes may alter pain-related gene expression and lead to an increase in neuronal excitability in DRG, resulting in spinal cord central sensitization and pain hypersensitivity in response to peripheral stimulation (fig. 2).

Native Kcna2 AS RNA in Chronic Neuropathic Pain

Identification of Native Kcna2 AS RNA and Its Expression in DRG

Long ncRNAs include AS RNA, double-stranded RNA, and long RNA species. Unlike the study of miRNAs, the study of long ncRNAs is still in its infancy. Although long ncRNAs may be implicated in gene-regulatory roles such as chromosome dosage compensation, imprinting, epigenetic regulation, cell cycle control, nuclear and cytoplasmic trafficking, transcription, translation, splicing, and cell differentiation,7,51,52  most long ncRNAs remain uncharacterized and their biological significance underestimated.7,52,53  We recently identified a new native RNA that is 2.52 kb in size and contains no apparent open reading frame,16,54  indicating that it is a long ncRNA. We named it Kcna2 AS RNA because most of its sequence is complementary to the voltage-gated K+ channel Kcna2 RNA (also known as Kv1.2 RNA). This AS RNA seems to be transcribed from the opposing DNA strands of the Kcna2 RNA gene at the same genomic locus.

Under normal conditions, Kcna2 AS RNA was expressed in pain-related areas, including DRG, from rats, although the signals were weak. It is also observed in DRGs from mouse, monkey, and human specimens.16  Using in situ hybridization histochemistry, we found that Kcna2 AS RNA was detected exclusively in DRG neurons. Approximately one fifth of neurons are labeled in the DRG of normal rats. Most are medium-sized although some are small and a few are large.16  Consistent with this subpopulation distribution pattern, the double-labeling observations showed that the majority of Kcna2 AS RNA–labeled neurons are positive for neurofilament-200 protein, a marker for myelinated A-fibers and large and medium DRG neurons. Some were positive for P2X3/isolectin B4, the markers for small DRG nonpeptidergic neurons, or for calcitonin gene–related peptide, a marker for small DRG peptidergic neurons. Compared with Kcna2 AS RNA, Kcna2 mRNA and protein are highly expressed in DRG. Approximately 70% of the DRG neurons were positive for Kcna2 protein.16,54,55  Most of these positive neurons were large in size.16,54,55  Double labeling of Kcna2 AS RNA with Kcna2 protein showed a tiny overlap between them.16  It seems that Kcna2 AS RNA and Kcna2 protein have opposing expression and distinct subpopulation distribution in normal DRG neurons.

Myeloid Zinc Finger Gene 1–Mediated Increase of Kcna2 AS RNA after Nerve Injury

The data from our laboratory16,54  and those of others55–60  revealed that peripheral nerve injury time-dependently down-regulated Kcna2 mRNA and protein in the injured DRG. In contrast, the level of Kcna2 AS RNA was time-dependently increased in the injured DRG after peripheral nerve injury (fig. 3).16,54  Such an increase occurred predominantly in large DRG neurons. No changes in the amount of Kcna2 AS RNA were observed in intact DRG, spinal cord, and other pain-related brain regions. Furthermore, using single-cell quantitative reverse-transcription polymerase chain reaction, we demonstrated that the ratios of Kcna2 mRNA to Kcna2 AS RNA were decreased, particularly in individual medium and large DRG neurons after SNL (fig. 3).16  These results indicate that expression of Kcna2 AS RNA, like that of miRNAs, can be induced in the injured DRG after peripheral nerve injury (table 2).

Fig. 3.

Kcna2 antisense (AS) RNA up-regulation specifically and selectively targets Kcna2 expression in neuropathic pain. (A) Under normal conditions, due to highly low expression of Kcna2 AS RNA, Kcna2 messenger RNA (mRNA) that is transcribed from the genome is translated into protein, resulting in the expression of the Kcna2 channel at the cell membrane. (B) Under neuropathic pain conditions, peripheral nerve injury promotes the expression of Kcna2 AS RNA that is transcribed from the opposing strand of the Kcna2 gene. Increased expression of Kcna2 AS RNA specifically and selectively inhibits expression of Kcna2 mRNA via extensive overlap of their complementary regions, including the transcription and translation inhibition sites, leading to reduced expression levels of the membrane Kcna2 channel only, not other Kcna family members (e.g., Kcna1).

Fig. 3.

Kcna2 antisense (AS) RNA up-regulation specifically and selectively targets Kcna2 expression in neuropathic pain. (A) Under normal conditions, due to highly low expression of Kcna2 AS RNA, Kcna2 messenger RNA (mRNA) that is transcribed from the genome is translated into protein, resulting in the expression of the Kcna2 channel at the cell membrane. (B) Under neuropathic pain conditions, peripheral nerve injury promotes the expression of Kcna2 AS RNA that is transcribed from the opposing strand of the Kcna2 gene. Increased expression of Kcna2 AS RNA specifically and selectively inhibits expression of Kcna2 mRNA via extensive overlap of their complementary regions, including the transcription and translation inhibition sites, leading to reduced expression levels of the membrane Kcna2 channel only, not other Kcna family members (e.g., Kcna1).

Nerve injury–induced up-regulation of Kcna2 AS RNA is triggered by myeloid zinc finger gene 1 (MZF1), a transcription factor belonging to the family of zinc finger proteins. The Kcna2 AS gene promoter contains the consensus MZF1-binding motif. Once bound to this motif, MZF1 promotes transcription of target genes.61,62  We found that MZF1 binds to this motif on the Kcna2 AS gene promoter in the DRG,16  and SNL time-dependently increases MZF1 expression and its binding activity in the injured DRG.16  Moreover, MZF1 directly promotes Kcna2 AS gene transcription and is coexpressed with Kcna2 AS RNA in DRG neurons.16  It is very likely that nerve injury–induced up-regulation of DRG Kcna2 AS RNA occurs specifically in response to the increased MZF1. It is worth noting that the increase in Kcna2 AS RNA might be induced by other transcription factors and/or caused by increases in RNA stability and other epigenetic modifications. These possibilities will be addressed in future studies.

Kcna2 RNA Specifically and Selectively Targeted by Kcna2 AS RNA

Nerve injury–induced opposing changes in the expression of Kcna2 AS RNA and Kcna2 mRNA/protein in individual DRG neurons suggest that the increased Kcna2 AS RNA may be responsible for the decreased Kcna2 mRNA and protein under neuropathic pain conditions (fig. 3). Consistent with this speculation, overexpression of full-length Kcna2 AS RNA in cultured HEK-293T cells or in cultured DRG neurons markedly knocked down Kcna2 mRNA, but not Kcna1 mRNA, Kcna4 mRNA, Scn10a (Nav1.8), and their proteins.16  In in vivo experiments, Kcna2 AS RNA overexpression time-dependently reduced Kcna2 mRNA in the DRG.16  No changes were observed in the expression of Kcna1, Kcna4, and Scn10a at the levels of mRNA and protein in the DRGs injected with AAV-Kcna2 AS RNA.16  These results suggest that nerve injury–induced DRG Kcna2 down-regulation is likely caused by a nerve injury–induced increase in DRG Kcna2 AS RNA (fig. 3). Kcna2 AS RNA functions as a biologically active regulator of Kcna2 mRNA and specifically and selectively targets Kcna2 in primary sensory neurons in neuropathic pain. This effect may be related to the extensive overlap of their complementary regions, including the transcription and translation initiation sites (fig. 3).16 

DRG Kcna2 AS RNA as a Trigger in Neuropathic Pain Genesis

Although the detailed mechanisms by which nerve injury leads to neuropathic pain are still elusive, it is generally believed that neuropathic pain is induced by abnormal spontaneous activity that arises in neuromas and the medium and large DRG cell bodies.1–3  Voltage-dependent potassium channels (Kv) govern cell excitability. Application of Kv antagonists to sensory axons and to sites of ectopic afferent discharge facilitates ectopic firing.63–66  Injection of these antagonists into nerve-end neuromas provokes intense pain in humans.67  We found that selective reduction of Kcna2 expression in DRG by Kcna2 AS RNA decreased total Kv current, depolarized the resting membrane potential, decreased current threshold for activation of action potentials, increased the number of action potentials in large and medium DRG neurons, and produced neuropathic pain symptoms.16  Rescuing nerve injury–induced down-regulation of DRG Kcna2 by blocking nerve injury–induced up-regulation of DRG Kcna2 AS RNA attenuated induction and maintenance of nerve injury–induced mechanical, cold, and heat pain hypersensitivities.16 

Given that nociceptive neurotransmitters and/or modulators (substance P and calcitonin gene–related peptide) in the injured myelinated fibers and in large and medium DRG neurons are dramatically increased at the early stage after nerve injury,68,69  it is conceivable that peripheral nerve injury up-regulates the expression of native Kcna2 AS RNA through activation of the MZF1 transcription factor in the injured DRG. This up-regulation silences the expression of DRG Kcna2 mRNA and protein, resulting in a decrease of total Kv current and an increase of ectopic discharge in large and medium DRG neurons. Ectopic discharge triggers the release of nociceptive transmitters and/or modulators in primary afferent terminals, leading to central sensitization in the dorsal horn and major symptoms of neuropathic pain (fig. 4). Thus, Kcna2 AS RNA may be an endogenous trigger in neuropathic pain development and maintenance. Kcna2 AS RNA may be a potential target for the prevention and/or treatment of neuropathic pain.

Fig. 4.

Proposed model for the mechanism of how Kcna2 AS RNA is involved in neuropathic pain. Nerve injury leads to an increase in myeloid zinc finger gene 1 (MZF1), a transcription factor that enhances the transcription of Kcna2 AS RNA, in dorsal root ganglion (DRG). The Kcan2 AS RNA silences expression of the Kcna2 messenger RNA (mRNA) and protein. The reduced Kcna2 protein expression at DRG neuronal membrane results in reduced K+ current (Kv), increases number of action potentials (AP) and neuronal excitability in DRG neurons, and produces spinal cord central sensitization and neuropathic pain symptoms (hyperalgesia and allodynia).

Fig. 4.

Proposed model for the mechanism of how Kcna2 AS RNA is involved in neuropathic pain. Nerve injury leads to an increase in myeloid zinc finger gene 1 (MZF1), a transcription factor that enhances the transcription of Kcna2 AS RNA, in dorsal root ganglion (DRG). The Kcan2 AS RNA silences expression of the Kcna2 messenger RNA (mRNA) and protein. The reduced Kcna2 protein expression at DRG neuronal membrane results in reduced K+ current (Kv), increases number of action potentials (AP) and neuronal excitability in DRG neurons, and produces spinal cord central sensitization and neuropathic pain symptoms (hyperalgesia and allodynia).

Conclusion

The lines of evidence described above indicate that ncRNAs including miRNAs and Kcna2 AS RNA in peripheral and central nervous systems are endogenous instigators of chronic pain. miRNAs have been extensively studied in the past decade and may be used as prognostic and diagnostic biomarkers and potential new drug targets for chronic inflammatory pain and neuropathic pain17,18 ; however, miRNAs have multiple and specific downstream targets due to their small size.48–50  This characterization may result in the limited use of miRNAs in chronic pain treatment because they might interfere with other physiological functions and produce potential side effects. Compared with the previous reviews on miRNAs in pain processing,17,18  this review updates current knowledge on miRNAs in chronic pain. More importantly, this review summarizes the latest finding on a long ncRNA Kcna2 AS RNA in chronic pain,16,54  which has not been discussed in previous reviews.17,18  Although the studies on long ncRNAs are still at the early stage, accumulating evidence indicates that they specifically and selectively target their corresponding gene’s expression.16,54  As peripheral inflammation and nerve injury alter the expression of many other genes in addition to Kcna2 in pain-related regions,1–3  it is very likely that those genes, like Kcna2, are regulated by a corresponding long ncRNAs. Significant regulations of long ncRNA transcription may be a general cellular response to peripheral inflammation and nerve injury and participate in the induction and maintenance of chronic pain. Given that long ncRNAs have the characterization of specifically and selectively targeting the corresponding genes, it is conceivable that the significance of long ncRNAs in chronic pain will become even more apparent in the coming years.

Acknowledgments

This work was supported by grants from the National Institutes of Health, Bethesda, Maryland (grant nos. NS072206, HL117684, and DA033390), and the Rita Allen Foundation, Princeton, New Jersey.

Competing Interests

The authors declare no competing interests.

References

1.
Campbell
JN
,
Meyer
RA
:
Mechanisms of neuropathic pain.
Neuron
2006
;
52
:
77
92
2.
Latremoliere
A
,
Woolf
CJ
:
Central sensitization: A generator of pain hypersensitivity by central neural plasticity.
J Pain
2009
;
10
:
895
26
3.
Wang
W
,
Gu
J
,
Li
YQ
,
Tao
YX
:
Are voltage-gated sodium channels on the dorsal root ganglion involved in the development of neuropathic pain?
Mol Pain
2011
;
7
:
16
4.
Novina
CD
,
Sharp
PA
:
The RNAi revolution.
Nature
2004
;
430
:
161
4
5.
Hannon
GJ
:
RNA interference.
Nature
2002
;
418
:
244
51
6.
Willingham
AT
,
Gingeras
TR
:
TUF love for “junk” DNA.
Cell
2006
;
125
:
1215
20
7.
Wapinski
O
,
Chang
HY
:
Long noncoding RNAs and human disease.
Trends Cell Biol
2011
;
21
:
354
61
8.
Gibb
EA
,
Brown
CJ
,
Lam
WL
:
The functional role of long non-coding RNA in human carcinomas.
Mol Cancer
2011
;
10
:
38
9.
Ørom
UA
,
Derrien
T
,
Beringer
M
,
Gumireddy
K
,
Gardini
A
,
Bussotti
G
,
Lai
F
,
Zytnicki
M
,
Notredame
C
,
Huang
Q
,
Guigo
R
,
Shiekhattar
R
:
Long noncoding RNAs with enhancer-like function in human cells.
Cell
2010
;
143
:
46
58
10.
Røsok
Ø
,
Sioud
M
:
Systematic identification of sense-antisense transcripts in mammalian cells.
Nat Biotechnol
2004
;
22
:
104
8
11.
Fabbri
M
,
Calin
GA
:
Epigenetics and miRNAs in human cancer.
Adv Genet
2010
;
70
:
87
99
12.
Farazi
TA
,
Spitzer
JI
,
Morozov
P
,
Tuschl
T
:
miRNAs in human cancer.
J Pathol
2011
;
223
:
102
15
13.
Bai
G
,
Ambalavanar
R
,
Wei
D
,
Dessem
D
:
Downregulation of selective microRNAs in trigeminal ganglion neurons following inflammatory muscle pain.
Mol Pain
2007
;
3
:
15
14.
Sakai
A
,
Saitow
F
,
Miyake
N
,
Miyake
K
,
Shimada
T
,
Suzuki
H
:
miR-7a alleviates the maintenance of neuropathic pain through regulation of neuronal excitability.
Brain
2013
;
136
(
Pt 9
):
2738
50
15.
Sakai
A
,
Suzuki
H
:
Nerve injury-induced upregulation of miR-21 in the primary sensory neurons contributes to neuropathic pain in rats.
Biochem Biophys Res Commun
2013
;
435
:
176
81
16.
Zhao
X
,
Tang
Z
,
Zhang
H
,
Atianjoh
FE
,
Zhao
JY
,
Liang
L
,
Wang
W
,
Guan
X
,
Kao
SC
,
Tiwari
V
,
Gao
YJ
,
Hoffman
PN
,
Cui
H
,
Li
M
,
Dong
X
,
Tao
YX
:
A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons.
Nat Neurosci
2013
;
16
:
1024
31
17.
Kress
M
,
Hüttenhofer
A
,
Landry
M
,
Kuner
R
,
Favereaux
A
,
Greenberg
D
,
Bednarik
J
,
Heppenstall
P
,
Kronenberg
F
,
Malcangio
M
,
Rittner
H
,
Uçeyler
N
,
Trajanoski
Z
,
Mouritzen
P
,
Birklein
F
,
Sommer
C
,
Soreq
H
:
microRNAs in nociceptive circuits as predictors of future clinical applications.
Front Mol Neurosci
2013
;
6
:
33
18.
Kynast
KL
,
Russe
OQ
,
Geisslinger
G
,
Niederberger
E
:
Novel findings in pain processing pathways: Implications for miRNAs as future therapeutic targets.
Expert Rev Neurother
2013
;
13
:
515
25
19.
Farh
KK
,
Grimson
A
,
Jan
C
,
Lewis
BP
,
Johnston
WK
,
Lim
LP
,
Burge
CB
,
Bartel
DP
:
The widespread impact of mammalian MicroRNAs on mRNA repression and evolution.
Science
2005
;
310
:
1817
21
20.
Kim
J
,
Krichevsky
A
,
Grad
Y
,
Hayes
GD
,
Kosik
KS
,
Church
GM
,
Ruvkun
G
:
Identification of many microRNAs that copurify with polyribosomes in mammalian neurons.
Proc Natl Acad Sci U S A
2004
;
101
:
360
5
21.
Wheeler
G
,
Ntounia-Fousara
S
,
Granda
B
,
Rathjen
T
,
Dalmay
T
:
Identification of new central nervous system specific mouse microRNAs.
FEBS Lett
2006
;
580
:
2195
200
22.
Muljo
SA
,
Kanellopoulou
C
,
Aravind
L
:
MicroRNA targeting in mammalian genomes: Genes and mechanisms.
Wiley Interdiscip Rev Syst Biol Med
2010
;
2
:
148
61
23.
Bartel
DP
:
MicroRNAs: Target recognition and regulatory functions.
Cell
2009
;
136
:
215
33
24.
Subtelny
AO
,
Eichhorn
SW
,
Chen
GR
,
Sive
H
,
Bartel
DP
:
Poly(A)-tail profiling reveals an embryonic switch in translational control.
Nature
2014
;
508
:
66
71
25.
Kusuda
R
,
Cadetti
F
,
Ravanelli
MI
,
Sousa
TA
,
Zanon
S
,
De Lucca
FL
,
Lucas
G
:
Differential expression of microRNAs in mouse pain models.
Mol Pain
2011
;
7
:
17
26.
Tam Tam
S
,
Bastian
I
,
Zhou
XF
,
Vander Hoek
M
,
Michael
MZ
,
Gibbins
IL
,
Haberberger
RV
:
MicroRNA-143 expression in dorsal root ganglion neurons.
Cell Tissue Res
2011
;
346
:
163
73
27.
Kynast
KL
,
Russe
OQ
,
Möser
CV
,
Geisslinger
G
,
Niederberger
E
:
Modulation of central nervous system-specific microRNA-124a alters the inflammatory response in the formalin test in mice.
Pain
2013
;
154
:
368
76
28.
Favereaux
A
,
Thoumine
O
,
Bouali-Benazzouz
R
,
Roques
V
,
Papon
MA
,
Salam
SA
,
Drutel
G
,
Léger
C
,
Calas
A
,
Nagy
F
,
Landry
M
:
Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: Role in pain.
EMBO J
2011
;
30
:
3830
41
29.
Aldrich
BT
,
Frakes
EP
,
Kasuya
J
,
Hammond
DL
,
Kitamoto
T
:
Changes in expression of sensory organ-specific microRNAs in rat dorsal root ganglia in association with mechanical hypersensitivity induced by spinal nerve ligation.
Neuroscience
2009
;
164
:
711
23
30.
Imai
S
,
Saeki
M
,
Yanase
M
,
Horiuchi
H
,
Abe
M
,
Narita
M
,
Kuzumaki
N
,
Suzuki
T
,
Narita
M
:
Change in microRNAs associated with neuronal adaptive responses in the nucleus accumbens under neuropathic pain.
J Neurosci
2011
;
31
:
15294
9
31.
von Schack
D
,
Agostino
MJ
,
Murray
BS
,
Li
Y
,
Reddy
PS
,
Chen
J
,
Choe
SE
,
Strassle
BW
,
Li
C
,
Bates
B
,
Zhang
L
,
Hu
H
,
Kotnis
S
,
Bingham
B
,
Liu
W
,
Whiteside
GT
,
Samad
TA
,
Kennedy
JD
,
Ajit
SK
:
Dynamic changes in the microRNA expression profile reveal multiple regulatory mechanisms in the spinal nerve ligation model of neuropathic pain.
PLoS One
2011
;
6
:
e17670
32.
Brandenburger
T
,
Castoldi
M
,
Brendel
M
,
Grievink
H
,
Schlösser
L
,
Werdehausen
R
,
Bauer
I
,
Hermanns
H
:
Expression of spinal cord microRNAs in a rat model of chronic neuropathic pain.
Neurosci Lett
2012
;
506
:
281
6
33.
Sanchez
FV
,
Burkhard
FC
,
Kessler
TM
,
Kuhn
A
,
Draeger
A
,
Monastyrskaya
K
:
MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome.
Am J Pathol
2010
;
176
:
288
303
34.
Orlova
IA
,
Alexander
GM
,
Qureshi
RA
,
Sacan
A
,
Graziano
A
,
Barrett
JE
,
Schwartzman
RJ
,
Ajit
SK
:
MicroRNA modulation in complex regional pain syndrome.
J Transl Med
2011
;
9
:
195
35.
Akhtar
N
,
Haqqi
TM
:
MicroRNA-199a* regulates the expression of cyclooxygenase-2 in human chondrocytes.
Ann Rheum Dis
2012
;
71
:
1073
80
36.
Li
X
,
Gibson
G
,
Kim
JS
,
Kroin
J
,
Xu
S
,
van Wijnen
AJ
,
Im
HJ
:
MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis.
Gene
2011
;
480
:
34
41
37.
Park
SJ
,
Cheon
EJ
,
Kim
HA
:
MicroRNA-558 regulates the expression of cyclooxygenase-2 and IL-1β-induced catabolic effects in human articular chondrocytes.
Osteoarthritis Cartilage
2013
;
21
:
981
9
38.
Recchiuti
A
,
Krishnamoorthy
S
,
Fredman
G
,
Chiang
N
,
Serhan
CN
:
MicroRNAs in resolution of acute inflammation: Identification of novel resolvin D1-miRNA circuits.
FASEB J
2011
;
25
:
544
60
39.
Roggli
E
,
Britan
A
,
Gattesco
S
,
Lin-Marq
N
,
Abderrahmani
A
,
Meda
P
,
Regazzi
R
:
Involvement of microRNAs in the cytotoxic effects exerted by proinflammatory cytokines on pancreatic beta-cells.
Diabetes
2010
;
59
:
978
86
40.
Fujita
S
,
Ito
T
,
Mizutani
T
,
Minoguchi
S
,
Yamamichi
N
,
Sakurai
K
,
Iba
H
:
miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism.
J Mol Biol
2008
;
378
:
492
504
41.
Sengupta
JN
,
Pochiraju
S
,
Pochiraju
S
,
Kannampalli
P
,
Bruckert
M
,
Addya
S
,
Yadav
P
,
Miranda
A
,
Shaker
R
,
Banerjee
B
:
MicroRNA-mediated GABA Aα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats.
Pain
2013
;
154
:
59
70
42.
Zhao
J
,
Lee
MC
,
Momin
A
,
Cendan
CM
,
Shepherd
ST
,
Baker
MD
,
Asante
C
,
Bee
L
,
Bethry
A
,
Perkins
JR
,
Nassar
MA
,
Abrahamsen
B
,
Dickenson
A
,
Cobb
BS
,
Merkenschlager
M
,
Wood
JN
:
Small RNAs control sodium channel expression, nociceptor excitability, and pain thresholds.
J Neurosci
2010
;
30
:
10860
71
43.
Abrahamsen
B
,
Zhao
J
,
Asante
CO
,
Cendan
CM
,
Marsh
S
,
Martinez-Barbera
JP
,
Nassar
MA
,
Dickenson
AH
,
Wood
JN
:
The cell and molecular basis of mechanical, cold, and inflammatory pain.
Science
2008
;
321
:
702
5
44.
Akopian
AN
,
Souslova
V
,
England
S
,
Okuse
K
,
Ogata
N
,
Ure
J
,
Smith
A
,
Kerr
BJ
,
McMahon
SB
,
Boyce
S
,
Hill
R
,
Stanfa
LC
,
Dickenson
AH
,
Wood
JN
:
The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways.
Nat Neurosci
1999
;
2
:
541
8
45.
Kerr
BJ
,
Souslova
V
,
McMahon
SB
,
Wood
JN
:
A role for the TTX-resistant sodium channel Nav 1.8 in NGF-induced hyperalgesia, but not neuropathic pain.
Neuroreport
2001
;
12
:
3077
80
46.
Liu
CN
,
Wall
PD
,
Ben-Dor
E
,
Michaelis
M
,
Amir
R
,
Devor
M
:
Tactile allodynia in the absence of C-fiber activation: Altered firing properties of DRG neurons following spinal nerve injury.
Pain
2000
;
85
:
503
21
47.
Tal
M
,
Wall
PD
,
Devor
M
:
Myelinated afferent fiber types that become spontaneously active and mechanosensitive following nerve transection in the rat.
Brain Res
1999
;
824
:
218
23
48.
Gabriely
G
,
Wurdinger
T
,
Kesari
S
,
Esau
CC
,
Burchard
J
,
Linsley
PS
,
Krichevsky
AM
:
MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators.
Mol Cell Biol
2008
;
28
:
5369
80
49.
Meng
F
,
Henson
R
,
Wehbe-Janek
H
,
Ghoshal
K
,
Jacob
ST
,
Patel
T
:
MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer.
Gastroenterology
2007
;
133
:
647
58
50.
Mei
Y
,
Bian
C
,
Li
J
,
Du
Z
,
Zhou
H
,
Yang
Z
,
Zhao
RC
:
miR-21 modulates the ERK-MAPK signaling pathway by regulating SPRY2 expression during human mesenchymal stem cell differentiation.
J Cell Biochem
2013
;
114
:
1374
84
51.
Yu
W
,
Gius
D
,
Onyango
P
,
Muldoon-Jacobs
K
,
Karp
J
,
Feinberg
AP
,
Cui
H
:
Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA.
Nature
2008
;
451
:
202
6
52.
Ponting
CP
,
Belgard
TG
:
Transcribed dark matter: Meaning or myth?
Hum Mol Genet
2010
;
19
(
R2
):
R162
8
53.
Mercer
TR
,
Dinger
ME
,
Mattick
JS
:
Long non-coding RNAs: Insights into functions.
Nat Rev Genet
2009
;
10
:
155
9
54.
Fan
L
,
Guan
X
,
Wang
W
,
Zhao
JY
,
Zhang
H
,
Tiwari
V
,
Hoffman
PN
,
Li
M
,
Tao
YX
:
Impaired neuropathic pain and preserved acute pain in rats overexpressing voltage-gated potassium channel subunit Kv1.2 in primary afferent neurons.
Mol Pain
2014
;
10
:
8
55.
Rasband
MN
,
Park
EW
,
Vanderah
TW
,
Lai
J
,
Porreca
F
,
Trimmer
JS
:
Distinct potassium channels on pain-sensing neurons.
Proc Natl Acad Sci U S A
2001
;
98
:
13373
8
56.
Everill
B
,
Kocsis
JD
:
Nerve growth factor maintains potassium conductance after nerve injury in adult cutaneous afferent dorsal root ganglion neurons.
Neuroscience
2000
;
100
:
417
22
57.
Ishikawa
K
,
Tanaka
M
,
Black
JA
,
Waxman
SG
:
Changes in expression of voltage-gated potassium channels in dorsal root ganglion neurons following axotomy.
Muscle Nerve
1999
;
22
:
502
7
58.
Kim
DS
,
Choi
JO
,
Rim
HD
,
Cho
HJ
:
Downregulation of voltage-gated potassium channel alpha gene expression in dorsal root ganglia following chronic constriction injury of the rat sciatic nerve.
Brain Res Mol Brain Res
2002
;
105
:
146
52
59.
Park
SY
,
Choi
JY
,
Kim
RU
,
Lee
YS
,
Cho
HJ
,
Kim
DS
:
Downregulation of voltage-gated potassium channel alpha gene expression by axotomy and neurotrophins in rat dorsal root ganglia.
Mol Cells
2003
;
16
:
256
9
60.
Yang
EK
,
Takimoto
K
,
Hayashi
Y
,
de Groat
WC
,
Yoshimura
N
:
Altered expression of potassium channel subunit mRNA and alpha-dendrotoxin sensitivity of potassium currents in rat dorsal root ganglion neurons after axotomy.
Neuroscience
2004
;
123
:
867
74
61.
Hsieh
YH
,
Wu
TT
,
Tsai
JH
,
Huang
CY
,
Hsieh
YS
,
Liu
JY
:
PKCalpha expression regulated by Elk-1 and MZF-1 in human HCC cells.
Biochem Biophys Res Commun
2006
;
339
:
217
25
62.
Luo
X
,
Zhang
X
,
Shao
W
,
Yin
Y
,
Zhou
J
:
Crucial roles of MZF-1 in the transcriptional regulation of apomorphine-induced modulation of FGF-2 expression in astrocytic cultures.
J Neurochem
2009
;
108
:
952
61
63.
Devor
M
:
Potassium channels moderate ectopic excitability of nerve-end neuromas in rats.
Neurosci Lett
1983
;
40
:
181
6
64.
Devor
M
,
Govrin-Lippmann
R
:
Axoplasmic transport block reduces ectopic impulse generation in injured peripheral nerves.
Pain
1983
;
16
:
73
85
65.
Targ
EF
,
Kocsis
JD
:
4-Aminopyridine leads to restoration of conduction in demyelinated rat sciatic nerve.
Brain Res
1985
;
328
:
358
61
66.
Targ
EF
,
Kocsis
JD
:
Action potential characteristics of demyelinated rat sciatic nerve following application of 4-aminopyridine.
Brain Res
1986
;
363
:
1
9
67.
Chabal
C
,
Jacobson
L
,
Russell
LC
,
Burchiel
KJ
:
Pain responses to perineuromal injection of normal saline, gallamine, and lidocaine in humans.
Pain
1989
;
36
:
321
5
68.
Devor
M
:
Ectopic discharge in Abeta afferents as a source of neuropathic pain.
Exp Brain Res
2009
;
196
:
115
28
69.
Weissner
W
,
Winterson
BJ
,
Stuart-Tilley
A
,
Devor
M
,
Bove
GM
:
Time course of substance P expression in dorsal root ganglia following complete spinal nerve transection.
J Comp Neurol
2006
;
497
:
78
87