Differential Expression of Long Non-Coding RNAs and Their Role in Rodent Neuropathic Pain Models

Introduction

Overview of Neuropathic Pain

Pain is a common disease in clinical practice, which has important implications for the human body. Pain perception has evolved as a warning role to respond to organismic danger and tissue damage in a timely manner, then it presents a protective effect by generating related danger signals.¹ Patients are deeply troubled by the continuous generation of pain signals and the dysfunction of the nervous system. As generally known, neuropathic Pain is a common chronic pain in clinical practice. It is generally caused by the dysfunction of somatosensory neurons in the central or peripheral nervous systems.² Recently, neuropathic pain was redefined as “pain initiated or caused by a primary lesion or dysfunction in the nervous system” by the International Association for the Study of Pain (IASP).³ The primary diseases include neurological damage caused by cancer, diabetes, infection, autoimmune diseases, chemotherapy, or trauma.⁴ These conditions usually lead to chronic pain syndrome, manifesting as spontaneous pain, hyperalgesia and allodynia. In addition, chronic pain has been shown to cause sleep disturbances, mental illnesses and difficulty in concentrating.⁵ Nearly 8% of the world’s people suffer from chronic pain, which seriously affects the quality of life.⁶

The primary causes for those symptoms of chronic pain include alteration of ion channels (such as calcium and sodium channels) in neurons,^7,8 activation of immune cells (like microglia, macrophage and CD4+T cell et al) and systemic inflammatory imbalance.^1,9,10 These results ultimately lead to imbalance between the ascending pain pathways and the descending inhibitory pathway. In sensory transduction, dorsal root ganglion (DRG) is the gathering place of the somatosensory primary afferent neuron cell body and it is of vital importance in the signal transmission between peripheral stimuli and central sensory system.¹¹ Admittedly, the pathogenesis of neuropathic pain is complicated. In the beginning, it seems that few neurons were damaged, which led to abnormal electrical activity, synaptic changes and continuous inflammation.^12,13 Further studies have found that the production of chronic pain includes the abnormal production of pro-inflammatory mediators and the interaction between neurons and immune cells as well.^14,15 It contains a complex network system composed of microglia and macrophages.^15,16 For example, colony-stimulating factor 1 (CSF1) induces microglial proliferation and aggravates the pain dramatically.⁹ Reduction of macrophages in DRG can relieve mechanical and thermal hyperalgesia.¹⁷ Besides, Some new studies show that receptors such as melanocortin 4 receptor (MC4R) and Toll-like receptor 4 (TLR4) are also helpful.^18,19 Here, we briefly reviewed recent studies of lncRNAs in neuropathic pain, focusing on different rodent neuropathic pain models.

Biological Functions of Long Non-Coding RNAs

The human transcriptome has pointed that only 1–2% of the human genome is used to encode proteins, and the remaining 98% is non-coding RNA. LncRNAs is one of the non-coding RNA without protein-coding function. However, an important progression has been the emergence of lncRNAs as key post-transcriptional regulators.²⁰ Crucial roles for lncRNAs have been identified in conditions such as cardiovascular diseases, various cancers, autoimmune disorders, kidney and liver system, etc.^21–23 Subsequent studies have shown that lncRNAs play an essential role in gene expression by co-transcriptional regulation, post-transcriptional bridging proteins and chromatin, pairing with other RNA and chromosomal modification,^24,25 especially in the development, proliferation, differentiation, migration and invasion of tumors cells.²⁶ What’s more, some lncRNAs could play a role in an indirect way, which interacts with microRNAs (miRNAs) and proteins to regulate signal transduction pathways downstream, affecting the development of various diseases.^27,28

Bioinformatic analysis and silico inference methods reveal potential interactions between lncRNAs-miRNAs and lncRNAs-RNA binding protein, which have attracted intense interest. MiRNAs, small non-coding RNAs, play an important role in the post-transcriptional regulation.²⁷ The possible mechanisms for lncRNAs to regulate miRNA are as follows: (1) lncRNAs indirectly inhibits negative regulation of miRNA by competitively binding to the 3ʹ-UTR of target mRNA.²⁹ (2) LncRNAs could act as a competitive endogenous RNA to play the “molecular sponge” and inhibit the expression of miRNA.^30,31 (3) LncRNAs is a potential pri-miRNA, which indirectly regulates the expression of target genes.³² RNA-Protein interactions are key aspects in many biological processes.²⁸ The primary concern of the present studies is shifting the attention from RNAs (like tRNAs, rRNAs) involved in translation and regulation towards lncRNAs.²⁰ The Encyclopedia of DNA Elements shows that the correspondence between lncRNAs and RBP is not one-to-one. LncRNAs can interact with multiple RBP and generate biological efficacy via regulating expression of genes or preventing association with DNA.³³ The association between them could be with potential therapeutic implications. While interactions of lncRNAs are more complex than previously thought, it still caused considerable concern and interest. It has been proven that lncRNAs plays an imperative role in tumors and participates in the generation and development of many diseases. In the meantime, a large amount of recent evidence shows that lncRNAs is also essential in neuropathic pain.³⁴

The Profiles of Differentially Expressed LncRNAs in Neuropathic Pain

In recent years, with the rapid improvement of sequencing technology, many studies have conducted more in-depth research on neuropathic pain from the perspective of genes. Stacks of differentially expressed genes were found in the dorsal horn of the spinal cord, DRG and other regions in neuropathic pain models.³⁵ Among them, it would have indicated that long-chain non-coding RNAs play a fundamental role in disease regulation. Therefore, the lncRNAs will become crucial targets for the treatment of neuropathic pain.^36,37

Gene sequencing technology was being used in the rat neuropathic pain model to find many differentially expressed genes in the spinal cord and DRG. Therefore, bioinformatics analysis technology can be an ideal method to perform enrichment analysis and construct interactive network, revealing the molecular mechanism of lncRNAs in nerve damage and repair process.³⁶ LncRNAs still, in turn, regulate neurite outgrowth of DRG neuron.³⁶ Hu et al identified the differentially expressed lncRNAs in the spared nerve injury (SNL) model and found 1259 down-regulated and 2473 up-regulated lncRNAs in SNL rats. Among them, lncRNAs PKIA-AS1, small nucleolar RNA host gene 4(SNHG4), SH3 and cysteine-rich containing protein 3(STAC3) and CIRBP-AS1 were significantly up-regulated.³⁸ Besides, more than 800 differentially expressed genes have been discovered in the chronic constriction injury model.³⁹ A variety of differentially expressed genes were also detected in the neuropathic pain model of mice^42–44 (Table 1). These studies provide the premise for studying the genetic level of neuropathic pain.

Table 1 The Number of Differentially Expressed lncRNAs by Using Sequencing Technology in Multiple Models

Major Neuropathic Pain Models

Promising new models for chronic pain provided a good base for further in-depth study of mechanisms. Photochemical and excitotoxic spinal cord injury are main central pain models. But the peripheral models seem to be more popular, which include chronic constriction injury (CCI), SNL, diabetic neuropathic pain (DNP), sciatic nerve injury (SCI) models and so on. In those studies, mechanical and thermal thresholds are important indicators to evaluate the hyperalgesia; and the content of inflammatory mediators usually reflects the degree of neuroinflammation. Nowadays, mature model of peripheral neuropathic pain has been established and major animal models are shown in Figure 1.⁴⁸

Figure 1 The major animal models of neuropathic pain. (A) The CCI model loosely ties the unilateral sciatic nerve below the mid-femur region with four chromic gut ligatures. It shows behavioral signs of spontaneous pain and hyperalgesia (thermal and tactile allodynia) and duration of pain signs persist over 2 months. (B) L5 and L6 spinal nerves of SNL model are tied unilaterally and closely at the distal side of dorsal root ganglia. It could indicate allodynia and hyperalgesia and last for at least 4 months. (C) DNP model was established by a single i.p. injection of streptozocin (STZ) and it induces thermal and mechanical hyperalgesia for 2–3 weeks.49 (D) In SNI model, the common peroneal and tibial nerves was ligated and axotomized, but sural nerve remained intact. Paw withdrawal threshold after SNI is significantly lower than sham group and hyperpathia lasts for at least 1 month.50

Latest Research Advances of LncRNAs in Neuropathic Pain Models

Based on the numerous researches on neuropathic pain-related lncRNAs, a large number of studies have found that lncRNAs are highly correlated with neuropathic pain, which provides a new target for its treatment.

SNL Model

There seems many lncRNAs expressed differentially in the SNL model (Table 2). Interaction between the lncRNAs and proteins is important. Firstly, Zhao et al discovered that the voltage-dependent potassium channel-related lncRNAs, KCNA2 antisense RNA, could regulate the excitability of DRG neurons by acting on zinc finger protein in rat, affecting the development and maintenance of neuropathic pain.⁵¹ The expression of lncRNA H19 in the DRG increased after SNL, and it is mainly up-regulated in Schwann cells—a non-primary sensory neuron, which likely be closely related to neuropathic pain.⁵² Recent studies have found that lncRNA embryonic stem cell expression 1 (lncenc1) is significantly upregulated in the DRG. Lncenc1 could bind to the RNA binding protein (RBP) enhancer of Zeste homologous protein 2 (EZH2), affecting the activation of microglia and the occurrence of neuropathic pain.⁵³ Hu et al performed array analysis on the spinal cord and found that lncRNA PKIA-AS1 is highly expressed in SNL rat model. It has proved that PKIA-AS1 can mediate neuropathic pain by regulating the expression and function of CDK6 in experiments. It seems that most of lncRNAs may interact with receptor indirectly in SNL model.

Table 2 Differential Expression of lncRNAs in SNL Model

Besides, lncRNAs directly affects neuropathic pain as well as interacts with miRNAs’ downstream. Linc00052 is significantly expressing in the spinal cord of SNL rats. The knockout of Linc00052 can inhibit the neuroinflammatory process through the miR-448-JAK1 axis, alleviating the development of neuropathic pain.⁵⁴ Studies have found that lncRNA p21 has been confirmed in vivo and in vitro experiments that it may act on miR-181b/Tnfaip1 to activate the downstream AKT/CREB axis, regulating cell apoptosis and inflammatory factor secretion, then affecting neuropathic pain.⁵⁵ The small nucleolar RNA host gene (SNHG) family not only involves in the proliferation, metastasis and prognosis of tumors cells, but also affects the progression of neuropathic pain. LncRNA SNHG1 mainly induces pain by directly regulating the level of CDK4 in the spinal cord of SNL rats.⁵⁶ SNHG4 regulates miR-423-5p negatively, which promotes neuro-inflammation and affects the progression of neuropathic pain.⁵⁷ In addition, a significant up-regulation of SNHG5 has been revealed in the mouse SNL model. After knocking out the SNHG5 gene, the mechanical threshold and thermal threshold were significantly increasing. At the same time, the studies suggested that the activation of microglia could be inhibited by regulating the miR-154-5p/CXCL13 axis, then neuropathic pain is reduced as well.⁵⁸ Such upregulation or downregulation of miRNA was highly correlated with the expression level of lncRNAs in the SNL model. As aforementioned, lncRNAs usually act as a “molecular sponge” to regulate neuroinflammation by lncRNAs–miRNAs axis. The result proven herein, affirms the indispensable role played by miRNA in development of neuropathic pain. As a result, lncRNAs is a new target for the treatment of neuroinflammatory neuropathic pain.³⁸

CCI Model

A large number of differentially expressed lncRNAs genes related to neuropathic pain were found in the spinal cord and DRG in CCI models (Table 3). Many studies have suggested that lncRNA X–inactive specific transcript (XIST) is significantly up-regulated in the spinal cord tissue and microglia of the CCI model, and it plays a significant role by regulating downstream molecules. For one thing, the development of neuropathic pain can be affected by acting on downstream miR-154-5p, which directs the expression of TLR5.⁵⁹ For another, lncRNA XIST was also reported that it could increase the expression of STAT3 (Signal transducer and activator of transcription 3) by regulating downstream miR-544, which then causes pain aggravation.⁶⁰ Besides, lncRNA XIST can not only negatively regulate miR-150 and zinc-finger E-box-binding (ZEB),⁶¹ but also act on miR-137 to enhance the expression of TNFAIP1, which can promote inflammation and aggravate the development of neuropathic pain.⁶² It demonstrates that lncRNA XIST plays an essential role in neuropathic pain. The targeted miRNAs of lncRNAs could be predicted by bioinformatics analysis technology, and the interrelations were followed up with animal experiments, which is helpful for the prediction of therapeutic targets. lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is one of the diagnostic markers in many diseases.^63,64 Research indicated that the expression of lncRNA MALAT1 was significantly up-regulated in the spinal cord and microglia of the CCI model, and it is considered to be a prominent target for pain regulation. Neuropathic pain could be regulated by changing the expression of miR-154-5p and AQP9 by lncRNA MALAT1.⁶⁵ LncRNA MALAT1 also interacts with downstream miR-129-5p to regulate the expression of HMGB1 (High-mobility group box 1), which can promote hyperalgesia.⁶⁶ In addition, deletion of MALAT1 gene can alleviate the development of neuroinflammation by inhibiting expression of COX-2, IL-1β and IL-6, while promoting up-regulation of miR-206 and inhibiting overexpression of ZEB2, which can improve the progression of neuropathic pain.⁶⁷ LncRNA XIST exists in a variety of biological processes as a ceRNA regulator network of miRNA-mRNA,⁶⁸ and it could potentially be involved in the therapy of neuropathic pain. As can be observed, the pain networks are highly complex and interconnected. For example, the expression of HMGB1 gene can be affected by lncRNA FIRRE and NEAT1 at the same time. Besides, other significantly differentially expressed lncRNAs and the downstream pathway in the CCI model provide new ideas and targets for its treatment.

Table 3 Differential Expression of lncRNAs in CCI Model

DNP Model

The dominating manifestation of DNP model, common complications of type 2 diabetes, is accompanied by a burning sensation, acupuncture sensation and other abnormal sensations.^81,82 The pathogenesis of diabetic neuropathic pain is complicated.⁸² Most recently, lncRNAs have attracted attention due to the continuous research on the mechanism of diabetic peripheral nerve pain. Related differentially expressed lncRNAs genes were parading in the DNP rat model (Table 4). Liu et al showed that inhibition of lncRNA BC168687 could reverse the threshold of mechanical pain and thermal pain in DNP rats. It mainly interferes with the expression of TRPV1 in DRG and inhibits phosphorylation (P)-ERK and p-p38 signaling pathways at the same time;⁸³ They also found that lncRNA BC168687 acts on the P2X7 receptor in DRG, which relieves neuropathic pain by inhibiting its expression.⁸⁴ In addition, lncRNA NONRATT021972 is a gene that plays a pivotal role in DNP. After its expression was inhibited, in the DNP model, results showed that it could interact with the P2X3 and P2X7 receptors in DRG to alleviate the development of peripheral pain complications in diabetes.^85,86 Increased expression of lncRNA uc.48+ was also demonstrated in DNP rat DRG and serum of diabetic patients, which can up-regulate the P2X3 receptor of DRG to regulate the excitability of neurons to affect the development of pain.⁸⁷ Besides, Xiong et al found that lncRNA uc.48+ could promote the release of calcitonin gene-related peptide (CGRP) in the spinal cord. When the expression of lncRNA uc.48+ is down-regulated, it can inhibit the phosphorylation of downstream p38 and ERK1/2, which will reduce the release of IL-1β and TNF-α as well as alleviate hyperalgesia in DNP rats.⁸⁸ It is known that diabetic neuropathy is one of the diabetic complications and the patients are concomitant with maintaining a low quality of life. Now this illustrates neuroinflammation and microglia activation are critical in origination and development of DNP. Therefore, these related lncRNAs may become noteworthy targets for the treatment of DNP in the future.

Table 4 Differential Expression of lncRNAs in CCI Model. GENE

Other Neuropathic Pain Models

Moreover, lncRNAs are found to be actively involved in other models (Table 5). Trigeminal neuralgia (TN) is also a prevalent neuropathic pain in clinical practice. Cai et al found that lncRNA Gm14461 is related to the activation and autophagy of astrocytes in a mouse model of trigeminal neuralgia. When the lncRNA was silencing, loss of Gm14461 increased autophagy in astrocytes and played an analgesic effect by regulating AMPK and Akt/mTOR signaling pathways.⁸⁹ LncRNA Gm14461 is also a factor to regulate CGRP (calcitonin gene-related peptide) and P2X3/7 receptors, which affect the release of inflammatory mediators and pain.⁹⁰ Similar to the role of lncRNA uc.48+ in the DNP model, in the TN model, lncRNA uc.48+ will up-regulate the expression of P2X7 receptors and enhance the phosphorylation of ERK1/2 in the trigeminal nerve meridian, which will heighten pain sensitization.⁹¹ In the rat brachial plexus injury (BPI) model, the expression of lncRNA Malat1 decreases in the spinal cord, which may affect the excitability of neurons by regulating the level of calcium ions.⁹² In addition, up-regulation of JHDM1D-AS1 can reduce the inflammation and neuronal damage of the spinal cord after brachial plexus injury by targeting miR-101-3p-DUSP.⁹³ In the rat model of cancer pain, the expression of lncRNA-NONRATT021203.2 was increased, which can participate in the production of pain by targeting CXCL9 (C-X-C motif chemokine ligand 9).⁹⁴ Postherpetic neuralgia (PHN) is also a considerable neuropathic pain. It confirmed that KCNA2-AS is highly expressing in the spinal cord of the PHN (postherpetic neuralgia) rat model. The pain is relieved when KCNA2-AS was knocking out. The possible reason is that it reduces the translocation of pSTAT3 to the nucleus and inhibits the activation of astrocytes.⁹⁵ However, lncRNA NEAT1 can target the expression of AQP4 by inhibiting miR-128-3p in the rat SCI model, which affects the development of neuropathic pain.⁹⁶ SNI (spare nerve injury) model as a common model in neuropathic pain research. But there are few reliable studies of lncRNAs in this model. Silencing of LINC01119 can alleviate the hypersensitivity and reduced the increase in IL−6, IL−1β, and TNF−α via binding with ELAVL1 and increasing BDNF mRNA stability and expression level. Here, we briefly reviewed recent studies of lncRNAs in neuropathic pain, focusing on different rodent neuropathic pain models. Many expressions of lncRNAs are altered in rodent rats after the establishment of chronic pain. Therefore, lncRNAs might be a good potential therapeutic target for neuropathic pain.

Table 5 Differential Expression of lncRNAs in Other Models

Discussion and Future Perspectives

Chronic neuropathic pain is a severe situation, accompanying by injury and dysfunction of the somatosensory nervous system.⁶ However, at present, a lack of efficient curative treatments remains a severe challenge for neuropathic pain. It proved that the rodent models of chronic neuropathic pain play an essential role in parent studies. And detailed inter-molecular interactions such as lncRNAs and miRNAs were revealed, which could provide therapeutic promise for the treatment of chronic neuropathic pain.

With the advance in sequencing technology, the studies of lncRNAs in have become a hotspot of the filed. It has been demonstrated that played an essential role in neuropathic pain. Therefore, lncRNAs are likely expected to become a potential target for neuropathic pain. We wonder if these sequencing results of lncRNAs are reliable. For instance, even within the same model there are major differences among sequencing studies published in the literature. The sequencing technology should exhibit excellent sensitivity, repeatability, and stability. In other words, the current study confirms that lncRNAs lack specificity in different models. Perhaps due to complex influencing factors in the system, a few discrepancies are evident in the sequencing results.

As is well known, lncRNAs do not participate in protein-coding directly. However, it plays an imperative part in co-transcriptional regulation, post-transcriptional and Post-translational protein modifications and so on.²⁰ Related research found that the potential regulatory effect of lncRNAs is very distinguished in various diseases. In the above experiments of different neuropathic pain animal models, but also present a significant role in regulating the generation as well as the progression of neuropathic pain. The previous focus was mainly on the changes in the excitability of central and peripheral sensory neurons and the interaction of surrounding microglia, macrophages and other immune cells in the research of neuropathic pain, which led to the persistence of pain signals. What’s more, we have shown in this review that the interactions of the lncRNAs, miRNA and proteins are very complicated. As such, the studies of those non-coding RNAs can be used to understand better the mechanisms of molecular interaction. Current works of lncRNAs have been shown in Figure 2. However, there is limited research focused on clinical prospective studies of lncRNAs in neuropathic pain. Because of neurocytes are nonrenewable, human specimens are generally available. Currently, several researches are focusing attention on the animal and cell studies.

Figure 2 Differentially expressed lncRNA in differential neuropathic pain models. ↑: up-regulation; ↓: down-regulation. Differential shapes of lncRNAs denote gene symbol without biological meaning. Center works represent different models of neuropathic pain.

Gender differences of immune cells were found among the neuropathic pain. The magnitude of the injury-induced macrophage expansion was different between the female and male mice.¹⁶ Microglia also showed sex differences. It is critical to the development of the neuropathic pain in male mice but not in female.⁹⁹ No previous studies have, however, investigated the differentially expressed lncRNAs in neuropathic pain models of different sex. The exact role of lncRNAs in mediating hypersensitivity in sexually dimorphic is still to be defined in the in the subsequent studies.

In conclusion, current results of lncRNAs in neuropathic pain indicate the potential of using sequencing technology. Despite the current researches lacking clinical implications and specificity of lncRNAs in different models, sufficient evidence has been presented to prove that lncRNAs have critical roles in the development of neuropathic pain. Therefore, additional studies are required to look for potential specific biomarkers and treatment in neuropathic pain from basic research to clinical applications.