Emerging roles of long non‐coding RNAs in neuropathic pain

Abstract Neuropathic pain, a type of chronic and potentially disabling pain resulting from primary injury/dysfunction of the somatosensory nervous system and spinal cord injury, is one of the most intense types of chronic pain, which incurs a significant economic and public health burden. However, our understanding of its cellular and molecular pathogenesis is still far from complete. Long non‐coding RNAs (lncRNAs) are important regulators of gene expression and have recently been characterized as key modulators of neuronal functions. Emerging evidence suggested that lncRNAs are deregulated and play pivotal roles in the development of neuropathic pain. This review summarizes the current knowledge about the roles of deregulated lncRNAs (eg, KCNA2‐AS, uc.48+, NONRATT021972, MRAK009713, XIST, CCAT1) in the development of neuropathic pain. These studies suggested that specific regulation of lncRNAs or their downstream targets might provide novel therapeutic avenues for this refractory disease.

Strikingly, 40% of lncRNAs are expressed specifically in the brain. 21 There are growing number of studies reporting that lncRNA expression is responsive to neuronal activity and injury, in which lncRNAs modulate nervous system development and synaptic plasticity by regulating neuronal outgrowth, differentiation as well as synapse formation and function. [21][22][23] Importantly, lncRNA deregulation has been reported in different neurological and psychiatric conditions. 24,25 Recently, emerging evidence suggested that lncRNAs could play crucial roles in development of neuropathic pain. [26][27][28] In this article, we review current knowledge about the deregulation of lncRNAs in neuropathic pain in relation to their cellular and molecular functions. The potential utilities of these lncRNAs or their downstream mediators as therapeutic targets for neuropathic pain are also discussed.

| LN CRNA E XPRE SS I ON PROFILING IN NEUROPATHIC PAIN
LncRNA expression profiling by whole transcriptome shotgun sequencing, PCR array or microarray followed by validation of candidate lncRNAs by reverse transcription-quantitative PCR (RT-qPCR) is the most common approach to identify and validate differentially expressed lncRNAs in specific disease states. 29  RNAs and 1066 mRNAs were found to be significantly deregulated on day 14 post-SNI. The deregulation of two selected lncRNAs (XLOC_021333 and Rn50_8_0646.1) was validated by RT-qPCR.
The lncRNA-microRNA-mRNA network was then re-constructed, illustrating the potential involvements of ribosome, phosphatidylinositol 3-kinase-Akt signalling pathway, focal adhesion and extracellular matrix-receptor interactions in the pathogenesis of SNI-induced neuropathic pain. 31 The same group of investigators later published another study using the same Sprague-Dawley rat SNI model to delineate the deregulation of lncRNAs and signalling pathways in the spinal cord in relation to the temporal course of neuropathic pain development. 32 Using the mouse SNI model, Liu and colleagues 33  The above-mentioned animal and bioinformatic studies strongly suggested that lncRNA deregulation is closely linked to neuropathic pain, in which the differentially expressed lncRNAs might take part in the pathogenesis (Table 1).

| FUN C TI ONAL ROLE S OF LN CRNA S IN NEUROPATHIC PAIN
Emerging studies have highlighted the molecular functions of lncR-NAs in the pathogenesis of different neurological diseases. One of the most important functions of lncRNAs is to modulate gene expression. LncRNAs may act as scaffolds of transcriptional and epigenetic protein complex, which can interact with specific genomic loci to modulate gene transcription. LncRNAs may also affect RNA processing by directly interacting with target mRNAs. Alternatively, lncRNAs may "sponge" microRNAs, leading to the disinhibition of their downstream target genes. The functional characterization of differentially expressed lncRNAs in neuropathic pain is just emerging. Herein, specific lncRNAs with their functions and molecular mechanisms characterized in neuropathic pain are discussed in detail.

| KCNA2-AS
Nerve injury is known to reduce the expression of the voltagedependent potassium (Kv) channel subunit Kcna2 through promoter hypermethylation, thereby decreasing total Kv current and enhancing neuronal excitability. 35 Zhao and colleagues 26 identified a novel lncRNA known as KCNA2-AS whose sequence is comple- Kcna2 sense fragment produced opposite effects. 26 These data suggested that KCNA2-AS and its upstream regulator MZF1 represent novel therapeutic targets in neuropathic pain.

| uc.48+
Diabetic peripheral neuropathy is a major cause of neuropathic pain.
Activation of P2X purinoceptors expressed on first-order sensory neurons by ATP released from damaged or inflamed tissues has been shown to mediate pain hypersensitivity in diabetic peripheral neuropathy. 36 A study by Wang and colleagues 27 demonstrated that the expression levels of a lncRNA known as uc.48+ were elevated in the diabetic rat DRG, whose knockdown with small interference RNA (siRNA) reduced diabetes-induced mechanical allodynia and thermal hyperalgesia. Mechanistically, the upregulation of P2X 3 and its downstream ERK pathway in DRG of diabetic rats could be abrogated by uc.48 + siRNA, suggesting that uc.48+ is an upstream positive regulator of P2X 3 . Moreover, knockdown of uc.48+ alleviated the pro-inflammatory signals in DRG of diabetic rats, characterized by the normalized levels of tumour necrosis factor-α (TNF-α). In this connection, TNF-α has been demonstrated to potentiate P2X 3 receptor-mediated nociception. 27 A subsequent study by Xiong and colleagues further demonstrated that intrathecal injection of uc.48 + siRNA attenuated the upregulation of calcitonin gene-related peptide, a peptide whose expression is associated with neuronal sensitization and enhanced pain, 37 in the spinal cord of diabetic rats. These results suggested that targeting uc.48+ could alleviate diabetic neuropathic pain through suppressing the excitatory transmission regulated by P2X 3 receptors in DRG.

| NONRATT021972
NONRATT021972 is another lncRNA recently demonstrated to modulate P2X purinoceptors in diabetic neuropathic pain. It has been reported that the levels of NONRATT021972 were upregulated in the DRG of streptozotocin-induced diabetic rats 38 and Zucker diabetic fatty rats 39 as well as in blood of patients with type 2 diabetes. 28 Interestingly, circulating levels of NONRATT021972 were positively correlated with neuropathic pain scores in the latter study. Functionally, siRNA targeting NONRATT021972 restored sensory nerve conduction velocity and attenuated mechanical allodynia and thermal hyperalgesia in diabetic rats, 28,38,39 indicating this lncRNA is pronociceptive. Similar to the studies on uc.48+, TNF-α, P2X 3 and ERK phosphorylation were all upregulated in DRG of diabetic rats, in which NONRATT021972 siRNA abolished such effects. 38 Aside from neuronal P2X purinoceptors, NONRATT021972 siRNA was shown to attenuate P2X 7 signalling in GFAP-positive satellite glial cells, which presumably reduced TNF-α levels. 39 These findings collectively suggested that NONRATT021972 could mediate neuropathic pain through promoting both neuronal purinoceptor signalling and non-neuronal purinoceptor signalling as well as enhancing the pro-inflammatory state in diabetic neuropathic pain.

| MRAK009713
Chronic constriction injury (CCI) is a common experimental model of neuropathic pain, in which the sciatic nerve is ligated resulting in inflammation and ultimately peripheral pain. 40 Li and colleagues reported that the expression of a novel lncRNA MRAK009713 was upregulated in the DRG of CCI rats, in which siRNA silencing of this lncRNA alleviated mechanical allodynia and thermal hyperalgesia.
Strikingly, overexpression of MRAK009713 was sufficient to induce neuropathic pain-like symptoms. 41 Computational prediction based on the secondary structure, hydrogen bonding and molecular interatomic forces suggested that MRAK009713 could bind to the P2X 3 purinoceptor, in which the physical interaction was further confirmed by RNA immunoprecipitation assay. Enforced expression of MRAK009713 also upregulated the expression of P2X 3 receptors and enhanced the inward current induced by α, β-methylene-adenosine-5′-triphosphate (a P2X 3 agonist) in rat DRG. 41 Collectively, these results indicated that MRAK009713 is a positive regulator of the neuropathic pain by enhancing P2X 3 receptor expression and function.

| XIST
X inactive specific transcript (XIST) is one of the best-characterized lncRNAs to date owing to its heavy involvement in X inactivation. Several studies have demonstrated that XIST expression was upregulated in the spinal cord of CCI rats, in which knockdown of this lncRNA suppressed neuroinflammation and attenuated mechanical allodynia and thermal hyperalgesia. [42][43][44] The pronociceptive action of XIST was found to be mediated through sponging of anti-inflammatory microRNAs, namely miR-137, miR-150 and miR-544, leading to derepression of their pro-inflammatory targets. [42][43][44] In particular, miR-137 was found to target tumour necrosis factor alpha-induced protein 1 (TNFAIP1), which is a crucial inflammation regulator by activating nuclear factor-κB activity. 42 Zinc finger E-box-binding homeobox 1 (ZEB1) and signal transducer and activator of transcription 3 (STAT3) were also identified as the targets of miR-150 and miR-544, respectively. 43 In this regards, knockdown of ZEB1 has been shown to downregulate the expression of pro-inflammatory cytokines, such as interleukin (IL)-6 and IL-8, in breast cancer, 45 whereas STAT3 activation is known to stimulate spinal astrocyte proliferation to perpetuate neuropathic pain in rats. These results indicated that XIST promotes neuroinflammation to maintain neuropathic pain through the miR-137/TNFAIP1, miR-150/ ZEB1 and miR-544/STAT3 axes.

| CCAT1
Colon cancer-associated transcript-1 (CCAT1) is a lncRNA transcribed from a distal enhancer 515-kb upstream of the c-MYC gene. 46 47 These results suggested that restored expression of CCAT1 or inhibition of miR-155 might provide a novel therapeutic approach for the management of neuropathic pain ( Table 2).

| BC168687
Liu and colleagues 48 reported that the levels of the lncRNA BC168687 in the DRG of streptozotocin-induced diabetic rats were significantly higher than that of the control group. In this connection, knockdown of BC168687 attenuated diabetes-induced mechanical allodynia and heat hyperalgesia. 48,49 The increased expression levels of P2X 7 on GFAP-positive satellite glial cells in DRG and the elevated serum concentrations of nitric oxide (an oxidative injury factor released from satellite glial cells) in diabetic rats were also abrogated by BC168687 siRNA. 48

| NEAT1
Nuclear paraspeckle assembly transcript 1 (NEAT1), transcribed from the familial tumour syndrome multiple endocrine neoplasia type 1 locus on chromosome 11, is a lncRNA localized to nuclear paraspeckles, which are subnuclear bodies found in the interchromatin space that regulate gene expression through RNA retention.
Knockdown of NEAT1 has been shown to result in the disintegration of nuclear paraspeckles. 50 Xia and colleagues 51 reported that NEAT1 was significantly upregulated in the spinal cord of rats sustained CCI, in which knockdown of this lncRNA suppressed neuroinflammation (ie, reduced levels of IL-1β, IL-6 and TNF-α) as well as attenuated mechanical allodynia and thermal hyperalgesia. miR-381, whose levels were reduced in the spinal cord of CCI rats, was predicted to be a target of NEAT1. The physical interaction between NEAT1 and miR-381 was further confirmed by luciferase reporter assay and RNA pull-down assay. High-mobility group box 1 (HMGB1), which is known to promote neuroinflammation, 52 was identified as a direct target of miR-381. Additional gain-and loss-of-function analyses indicated that NEAT1 modulated CCI-induced neuropathic pain by regulating the miR-381/HMGB1 axis. 51 These results suggested that NEAT1 and HMGB1 are potential therapeutic targets in neural compression-induced neuropathic pain (Table 2).

| CON CLUS ION
Neuropathic pain is a serious public health issue and an intractable clinical challenge, whose molecular mechanism is still largely elusive.
Emerging studies suggested that lncRNAs play crucial roles in the development of neuropathic pain through regulating ion channels and neuroinflammation, two key features that drive the pathogen-

ACKNOWLEDG EMENTS
This work was supported by Capital's funds for health improvement and research (2016-1-4096).