Newly discovered functions of miRNAs in neuropathic pain: Transitioning from recent discoveries to innovative underlying mechanisms

Abstract

Neuropathic pain is a widespread clinical issue caused by somatosensory nervous system damage, affecting numerous individuals. It poses considerable economic and public health challenges, and managing it can be challenging due to unclear underlying mechanisms. Nevertheless, emerging evidence suggests that neurogenic inflammation and neuroinflammation play a role in developing pain patterns. Emerging evidence suggests that neurogenic inflammation and neuroinflammation play significant roles in developing neuropathic pain within the nervous system. Increased/decreased miRNA expression patterns could affect the progression of neuropathic and inflammatory pain by controlling nerve regeneration, neuroinflammation, and the expression of abnormal ion channels. However, our limited knowledge of miRNA targets hinders a complete grasp of miRNA’s functions. Meanwhile, exploring exosomal miRNA, a recently uncovered role, has significantly advanced our comprehension of neuropathic pain’s pathophysiology in recent times. In this review, we present a comprehensive overview of the latest miRNA studies and explore the possible ways miRNAs might play a role in the development of neuropathic pain.

Introduction

Neuropathic pain, a condition arising from somatosensory nervous system damage, is a prevalent global clinical issue impacting numerous individuals worldwide . ¹ It poses substantial public health and economic challenges and proves challenging to manage due to unclear underlying mechanisms. However, emerging evidence points to the involvement of neuroinflammation and neurogenic inflammation in the development of pain. ² Furthermore, a growing body of evidence indicates that the stimulation of neuroinflammation and neurogenic inflammation, in the peripheral and central nervous systems, plays a critical role in the prolonged existence of pain.^3,4 Previous studies have investigated the possibility that chronic neuropathic pain could stem from an inflammatory response within the nervous system triggered by nerve damage. ⁵ Recent research findings suggest that microRNAs (miRNAs) exhibit important anti-inflammatory effects, influencing pathogenesis in various contexts, including tumors, inflammation, cardiovascular and respiratory diseases.^6–9 However, the exact pathogenesis of neuropathic pain remains unclear. Newer investigations employing microarray technologies have uncovered the significant involvement of miRNAs in the emergence and advancement of neuropathic pain. Their regulatory impact extends to various aspects, including neuronal plasticity, excitability, ion channels, synaptic plasticity, and neuroinflammation. The gathered evidence indicates that changes in miRNA expression patterns may be critical players in the development of neuropathic pain. Nevertheless, the lack of understanding regarding miRNA targets hinders a comprehensive grasp of miRNAs’ biological functions in this context. This review aims to shed light on miRNA expression profiling studies related to neuropathic pain and explores specific miRNAs' molecular mechanisms that significantly contribute to neuropathic pain pathogenesis. Furthermore, we discuss the potential prognostic, diagnostic, and therapeutic implications of these miRNAs in the clinical management of neuropathic and neurogenic pain.

The roles of miRNAs

MicroRNAs (miRNAs) are prevalent in mammalian cells and are crucial in regulating approximately 30% of human genes. In the human genome, there are over 1800 miRNAs that target around 60% of human mRNAs. ¹⁰ Mutations in miRNA and related pathway genes have been linked to various human disorders, including neuropathic pain. MicroRNAs are abundantly distributed in both the central and peripheral nervous systems, which contain components related to pain processing. Although miRNAs do not directly engage in synthesizing peptides, they belong to the group of noncoding RNAs that hold substantial sway over miRNA stability and the translation of proteins. ¹¹ miRNAs exert their influence on gene expression after transcription by binding to the 3′UTR of target mRNAs, resulting in a notable decrease (84%) in the production of proteins. ¹² It is noteworthy that a single miRNA can target multiple mRNA transcripts, and multiple miRNAs can act on a single mRNA molecule simultaneously.^12,13 There is a widely accepted consensus regarding the critical roles miRNAs play in regulating various essential biological processes, such as early development, cell proliferation, apoptosis, lipid metabolism, cell differentiation, and disease progression. ¹⁴ Research conducted in experimental models has indicated that changes in miRNA expression patterns could impact the development of a wide range of conditions, such as cardiovascular diseases, cerebrovascular diseases, and neurodegenerative disorders.^15,16

Regulatory mechanisms of miRNAs in neuropathic pain

Several research investigations showed notable irregularities in miRNA expression in animals after experiencing peripheral nerve injury. Some of the miRNAs that have been discovered are linked to neuroinflammation, abnormal ion channel expression, and nerve regeneration, suggesting that altered miRNA expression could play a role in the development of neuropathic pain and might have the potential as useful diagnostic markers, enhancing the categorization of neuropathic pain. Nevertheless, the lack of information regarding miRNA targets poses a challenge to fully understanding the functions of miRNAs. In this section, we present an extensive summary of the existing understanding of miRNAs and investigate how they may play a role in neuropathic pain (Table 1).

Table 1.

The role of Micro RNAs in neuropathic pain.

Mechanism	Micro RNA	Target	Effect	Reference
Neuropathic pain	miR-128-3p ↑	ZEB1 ↓	Eased the development of neuropathic pain.	17
	miR-23a ↑	CXCR4/TXNIP/NLRP3 ↓	Suppressed the formation of neuropathic pain.	18
	miR-221 ↓	SOCS1 ↑	Mitigated neuropathic pain and neuroinflammation.	19
	miR-22 ↑	Mtf1 ↑	Facilitated the growth and sustenance of pain associated with inflammation.	20
	miR-381 ↑	HMGB1 and CXCR4 ↓	Hindered the progression of neuropathic pain.	18
	miR-216a-5p ↑	KDM3A ↓	Relieved rats of neuropathic pain.	21
	miR-150 ↑	ZEB1 ↓	Suppressed neuropathic pain in vivo.	22,23
	miR-155 ↓	SOCS1 ↑	Reduced the intensity of neuropathic pain.	24
	miR-144 ↑	RASA1 ↓	Aided in preventing the progression of neuropathic pain.	17
	miR-362-3p ↑	BAMBI ↓	The progression of neuropathic pain was repressed.	25
	miR-221 ↓	SOCS3 ↑	Diminished discomfort and decreased inflammatory marker expression.	26
	miR-93 ↑	STAT3 ↓	Suppressed the formation of neuropathic pain in rats with chronic constriction injury.	27
	miR-155 ↓	TRPA1 ↓	Dampened the neuropathic pain triggered by OXL.	28
	miR-183 ↑	TGF-α/CCL2/CCR2 ↓	The pain associated with osteoarthritis was improved.	29
	miR-140 ↑	S1PR1 ↓	Inhibited neuropathic pain induced by chronic constriction injury.	21
	miR-28-5p ↑	ZEB1 ↓	Diminished sensations of neuropathic pain.	30
	miR-429 ↑	ZEB1 ↓	Diminished the progression of neuropathic pain in vivo.	22,23
	miR-136 ↑	ZEB1 ↓	Prevented the advancement of neuropathic pain.	31
Infiltration of immune cells	miR-214-3p ↑	CSF1 ↓	Diminished the neuroinflammatory response and pain-related behavior.	32
	miR-146a-5p ↑	IRAK1/TRAF6 ↓	Prevented the formation of neuropathic pain caused by chronic constriction injury.	33
	miR-590-3p ↑	RAP1A ↓	A decrease in T cell infiltration resulted in the obstruction of diabetic peripheral neuropathy pain (DPNP).	34
	miR-135s ↑	KLF4 ↓	Promoted the regrowth of retinal ganglion cell axons following optic nerve damage in mature mice.	35
	miR-155 ↓	SPRR1A ↑	Improved the viability of neurons and facilitated the extension of axons.	36
	miR-192-5p ↑	XIAP ↑	Reduced the programmed cell death of nerve cells and supported the recovery and regrowth of peripheral nerves following injury.	37
	miR-125b ↑	JAK/STAT ↓	Facilitated the restoration and growth process after damage to the spinal cord.	38
	miR-210 ↑	EFNA3 ↓	Stimulated the regrowth of sensory axons while impeding programmed cell death.	39
	miR-138 ↓	SIRT1 ↑	Facilitated the regrowth of axons in mammals.	40
	miR-21 ↑	EGFR ↑	Improved the regrowth of axons following optic nerve crush.	41
	miR-21 ↑	PTEN ↓	Stimulated neurite growth.	42
	miR-21 ↑	TGFβI/TIMP3/EPHA4 ↓	Facilitated the multiplication of schwann cells and the regrowth of axons.	43
	miR let-7 ↑	Ntn1 ↓	Diminished the extension of axons.	44
	miR-9 ↑	Dcc ↓	Diminished the extension of axons.	44
	miR-199a-3p ↑	mTOR ↓	Weakened neurite growth.	42
	miR-26a ↑	GSK3β/Smad1 ↓	Aided the regrowth of axons in vivo.	45
	miR-9 ↑	FoxP1 ↓	Inhibited axon regeneration in vitro and in vivo.	46
	miR-455-5p ↓	GSK3β/Tau ↑	Facilitated the expansion and renewal of axons.	47
	miR-19a ↑	PTEN ↑	Encouraged the regrowth of axons following optic nerve injury in mature mice.	48
Voltage-gated potassium channels	miR-137 ↓	Kv1.2 ↑	Relieved sensitivity to touch and extreme sensitivity to heat.	49
	miR-183-5P ↑	TREK-1 ↓	Effectively improved the condition of neuropathic pain.	50
	miR-17-92 ↓	Multiple voltage-gated potassium channels ↑	Reduced the hypersensitivity to touch caused by nerve damage.	51
Voltage-gated calcium channels	miR-103 ↑	Cav1.2-LTC ↓	Effectively alleviate discomfort.	52
Voltage-gated sodium channel	miR-219 ↑	CaMKIIγ ↓	Blocked and reverted neuropathic pain and increased sensitivity of spinal neurons triggered by complete Freund’s adjuvant.	53
	miR-183 ↑	α2δ-1 and α2δ-2	Halted the increase in inherent mechanical sensitivity in pain receptors.	54
	miR-32-5p ↑	Cav3.2 ↓	Reversed mechanical allodynia.	55
	miR-124a ↑	MeCP2 ↓	Reduced the perception of pain caused by inflammation.	56
	miR-30b ↑	Nav1.7 ↓	Alleviated neuropathic pain.	57
	miR-7a ↑	β2 subunit ↓	Inhibited the sensation of neuropathic pain.	51
	miR-30b ↑	Nav1.3 ↓	Inhibited the sensation of neuropathic pain.	58
	miR-183 ↑	Nav1.3/Nav1.7/Nav1.8 ↓	Blocked the presence of pain-associated elements and improved the pain caused by osteoarthritis.	29
	miR-384-5p ↑	SCN3A ↓	Markedly reduced heightened sensitivity to touch and extreme sensitivity to heat in rats with chronic constriction injury.	59
	miR-182 ↑	Nav1.7 ↓	Relieved neuropathic pain caused by spared nerve injury.	60
	miR-96 ↑	Nav1.3 ↓	Eased the discomfort of neuropathic pain.	61
	miR-30b-5p ↑	Nav1.6 ↓	Attenuated pain.	62

PTEN: phosphatase and tensin homolog; DPNP: diabetic peripheral neuropathic pain; KDM3A: lysine-specific demethylase 3A; CSF1: Colony-stimulating factor 1; TGFβI: transforming growth factor-beta-induced protein; TRAF6: Tumor necrosis factor receptor-associated factor 6; TIMP3: tissue inhibitor of metalloproteinases 3; HMGB1: high mobility group box 1; FoxP1: forkhead box protein P1; CXCR4: Chemokine CXC receptor 4; GSK3β: glycogen synthase kinase 3β; TGF-α: Tumor necrosis factor α; KLF4: kruppel-like factor 4; CCL2: CC chemokine ligand 2; IRAK: IL-1R-associated kinase; CCR2: CC chemokine receptor type-2; MeCP2: Methyl-CpG binding protein 2; ZEB1: Zinc finger E-box-binding homeobox 1; TXNIP: Thioredoxin interacting protein; NLRP3: NOD-like receptor protein 3; Dcc: deleted in colorectal cancer; S1PR1: Sphingosine-1-phosphate receptor 1; Ntn1: netrin-1; RASA1: RAS P21 protein activator 1; RAP1A: Ras-related protein 1A; STAT: signal transducer and activator of transcription; Mtf1: Mitochondrial Transcription Factor 1; BAMBI: bone morphogenetic protein and activin membrane-bound inhibitor; OXL: Oxaliplatin; XIAP: X-linked inhibitor of apoptosis protein; EFNA3: ephrin-A3; SPRR1A: small proline-rich repeat protein 1A; EPHA4: erythropoietin-producing human hepatocellular receptor A4; SOCS: Suppressor of cytokine signaling; mTOR: mechanistic target of rapamycin; TRPA1: transient receptor potential cation channel subfamily A member 1; SIRT1: Sirtuin 1; Nav: voltage-gated sodium channels; JAK: Janus kinase gene; Kv: voltage-gated potassium channels; TREK-1: TWIK-related K channel 1; EGFR: epidermal growth factor receptor; Cav1.2-LTC: Cav1.2-comprising L-type calcium channel; CaMKII: calmodulin (CaM)-activated kinase II.

Regulation of neuroinflammation in neuropathic pain enhancement

MiRNAs have shown their involvement in nearly all known biological processes and a range of pathological situations, which includes neuropathic pain. In chronic inflammatory pain scenarios, the involvement of sensory neurons, particularly thermoreceptors, and mechanoreceptors, plays a significant role in the development of neuropathic pain. This encompasses various conditions such as complex regional pain syndrome (CRPS), polyneuropathies, postherpetic neuralgia (PHN), and fibromyalgia syndrome. ⁶³ Many miRNAs are now recognized as crucial regulators in the pathophysiology of this condition, controlling various aspects of neuroinflammation and neuronal gene expression. Researchers have directly observed distinct miRNA expression in the dorsal root ganglion (DRG) following the onset of neuropathic and inflammatory pain. ⁶⁴ Hence, it is crucial to investigate the role of miRNAs in regulating the migration of immune cells and the process of neuroinflammation following nerve injury (Figures 1–3).

Figure 2.

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Figure 1.

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Figure 3.

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Immune cell infiltration

Neuropathic pain is commonly linked to an immediate infiltration of immune cells following damage to peripheral nerves. Recent research indicates that miRNAs released by immune and nonimmune cells play a crucial role in immune regulation. In diabetic peripheral neuropathic pain, miR-590-3p has been found to control the entry of immune cells into neural tissues. ³⁴ Similarly, after spinal nerve ligation (SNL), rats exhibited reduced levels of miR-214-3p in spinal astrocytes, leading to their hyperactivity due to the upregulation of CSF1. ³² Additionally, alterations in miRNA patterns in microglial cells suggest that these cells exhibit unique functions depending on particular tissue or level of the disease process.

Neuroinflammation

Preliminary studies have unveiled the dual capacity of miRNAs to activate or inhibit the immune system, suggesting their potential involvement in the progression and development of autoimmune and inflammatory conditions, including neuropathic pain. Throughout the various stages of these conditions, specific miRNAs have been observed to undergo alterations in expression. To exert their influence in neuropathic pain, miRNAs signal through multiple pathways, including TXNIP/NLRP3 inflammasome, MAP kinase, IRAK/TRAF6, TLR4/NF-κB, and TLR5 and TNF-α signaling. ⁶⁵ Among the key receptors in the innate immune system, TLR plays a critical role by inducing the release of pro-inflammatory cytokines, initiating the production of inflammatory mediators responsible for pain, fever, and other inflammatory reactions, and exerting regulatory influence over the inflammatory cascade. ⁶⁶ In cultured astrocytes from mice after spinal nerve ligation (SNL), exposure to IL-1β or TNF-α increased by Tumor necrosis factor receptor-associated factor 6 (TRAF6) levels. TRAF6 is a crucial mediator involved in TLR signaling, NF-κB activation, and the expression of proinflammatory cytokines and interferons. IL-1 receptor-associated kinase (IRAK) plays a role in MyD88 signaling. The interaction between TRAF6 and IRAK leads to further activation of Janus kinase N-terminal region (JAK) and nuclear factor κ-activated B-cell light chain enhancer (NF-κB). ⁶⁷ By targeting miRNA-146a-5p, which inhibits specific sections of TRAF6 and IRAK1 mRNA 3′UTR in TLR signaling pathways and reduces their NF-κB activation, protein production, the NLRP3 inflammasome signaling pathway, and neuropathic pain levels are adversely affected. ⁶⁸ By upregulating miR-381, neuropathic pain behaviors were relieved in rats with chronic constriction nerve injury (CCI) due to the inhibition of HMGB1 expression. Conversely, this positive effect was reversed when miR-381 inhibitors were utilized. ⁶⁹ Similarly, overexpressing miR-362-3p led to significantly suppressing of proinflammatory cytokine levels by regulating BAMBI expression, thereby impeding the neuroinflammatory process and alleviating neuropathic pain in CCI mice. ⁷⁰ The TGF-β family includes a group of cytokine secreted and have a crucial impact on multiple biological processes, including cell division, survival, differentiation, and migration. ⁷¹ Among the members of this family is transforming growth factor α (TNF-α), which is highly associated with neuropathic pain. Furthermore, a member of the C-C chemokine family, C-C motif chemokine ligand 2 (CCL2), exhibits a high affinity for C-C chemokine receptor 2 (CCR2). Recent research indicates that miR-183 overexpression decreases the CCLR2/TGF-α/CCL2 signaling axis, consequently suppressing the expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) as well as pain-related markers (Nav1.7, TRPV1, Nav1.8, Nav1.3) in the dorsal root ganglion (DRG). ²⁹ In addition, a transcription factor known as zinc finger E-box-binding homeobox 1 (ZEB1) participates in diverse disease processes by repressing ZEB1 expression. A group of miRNAs, namely miR-136, miR-128-3p, miR-28-5p, miR-200b, miR-150, and miR-429, collaboratively regulate the advancement of neuropathic pain and neuroinflammation through the inhibition of ZEB1 expression.^30,31 The G-protein-coupled receptor group, which includes CXCR4, plays a significant role in regulating the maturation of glial cells and neurons in the central nervous system. ⁷² The role of CXCR4 in various processes involved in sensing pain stimuli is becoming more apparent. Additionally, a versatile protein called thioredoxin-interacting protein (TXNIP) is critical in numerous cellular activities, encompassing metabolism, growth, division, and apoptosis. ⁷³ The process of inflammation is initiated by the activation of a complex known as the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3). This complex leads to the generation and release of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and IL-18, playing a crucial role in the onset of inflammation. ⁷⁴ MiR-23a exerts a significant impact on neuropathic pain (NP) by regulating spinal glial cells through the specific targeting of TXNIP/NLRP3 and CXCR4 on the inflammasome axis. Intrathecal administration of miR-23a effectively reduced hyperalgesia by mimicking the suppression of spinal CXCR4 through lentiviral delivery and inhibiting the overexpression of NLRP3 or TXNIP. ¹⁸ In a rat model of NP, the expression of miR-144 and miR-140 was found to be decreased in the dorsal root ganglion (DRG). Additionally, intrathecal injection of miR-144 and miR-140 agomir, which target RASP21 protein activator 1 (RASA1) and sphingosine-1-phosphate receptor 1 (S1PR1), respectively, led to reduced secretion of inflammatory factors and amelioration of hyperalgesia. ²¹ Moreover, in rats experiencing neuropathic pain following chronic nerve injury (CCI), miR-216a-5p alleviated the condition by targeting KDM3A and deactivating the Wnt/β-catenin signaling pathway. ⁷⁵ In the context of CCI rats, miR-93 directly targets the 3′UTR of the Signal transducer and activator of transcription 3 (STAT3), thus preventing the progression of the disease. ²⁷ NF-κB plays a vital role as a mediator in the inflammatory cascade. When activated, the NF-κB pathway triggers both M1 and M2 macrophages to release pro-inflammatory cytokines, thereby intensifying the overall inflammatory response. ⁷⁶ On the other hand, MAPK, a mitogen-activated kinase, plays a significant role in various physiological and pathological processes, including neuropathic pain, through the phosphorylation of serine/threonine and tyrosine residues. ⁷⁷ At the same time, a distinct set of proteins called cytokine signaling inhibitors (SOCS) maintains the equilibrium of Th2-Th1 cells, moderates the negative feedback of cytokine signaling, and lessens Th2-induced inflammation. SOCS1 and SOCS3 activation by various inflammatory and anti-inflammatory cytokines inhibits cytokine actions. ⁷⁸ Several researchers have observed that inhibiting miR-221 or miR-155 alleviates neuroinflammation and neuropathic pain by increasing the expression of suppressor of cytokine signaling 1 (SOCS1) through inhibition of p38-MAPK and NF-κB. ⁷⁹ Furthermore, in diabetic peripheral neuropathy (DPN), the inhibition of miR-221 led to pain reduction and a decrease in the expression of inflammatory factors (IL-1β, IL-6, PEG2, TNF-α, and BK) by specifically targeting SOCS3. ²⁶ Similarly, in the context of oxaliplatin-induced peripheral neuropathic pain, miR-155 levels in the spinal cord rose. Interestingly, administering a miR-155 inhibitor through intrathecal injection led to a reduction in hyperalgesia in rats, potentially achieved by obstructing the oxidative stress-TRPA1 pathways. ²⁸ Suppression or inhibition of miRNA-22 led to diminished mechanical allodynia and heat hyperalgesia triggered by Complete Freund’s adjuvant (CFA). On the contrary, the upregulation of miRNA-22 induced pain-related behaviors. The increased miRNA-22 directly interacted with the Mtf1 promoter, resulting in the activation of RNA polymerase II and an elevation in Mtf1 expression. The heightened expression of Mtf1 contributed to spinal central sensitization, as indicated by elevated levels of p-ERK1/2, GFAP, and c-Fos in the dorsal horn. ²⁰ The results indicate that utilizing epigenetic approaches to modulate miRNAs for reducing neuroinflammation could present a hopeful and innovative therapeutic strategy for managing nociceptive hypersensitivity and neuropathic pain arising from peripheral nerve injuries.

Regulation of nerve regeneration

Following nerve injury, the preservation of neurons is a crucial prerequisite for the regeneration of nerves and the restoration of function. Prior studies have indicated that prolonged pain can result in damage or potential cell death of the spinal cord and peripheral nerve cell bodies. ⁸⁰ Recent research has revealed that injured peripheral neurons can trigger the release of intrinsic neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF), which facilitate neuronal survival and axon regeneration. ⁸¹ In this context, miRNAs also have a substantial impact on the process of nerve regeneration. Intriguingly, abnormal expression of several miRNAs that target genes crucial for nerve regeneration has been noted in the dorsal root ganglion (DRG). ⁸² Furthermore, it has been proposed that reducing miR-192-5p expression can increase the levels of the X-linked inhibitor of apoptosis protein (XIAP), thereby reducing nerve cell apoptosis and promoting regeneration after sciatic nerve injury (SNI). ³⁷ Furthermore, the upregulation of miR-210 promotes the survival of neurons by inhibiting apoptosis through its interaction with ephrin-A3 (EFNA3), thus facilitating the process of axon regeneration. ³⁹ MiR-135a and miR-135b effectively downregulate Kruppel-like factor 4 (KLF4), a potent inhibitor of axonal regeneration and outgrowth, thus promoting axonal outgrowth and cortical neuron migration. ³⁵ In rats experiencing spinal cord injury (SCI), the simultaneous use of 3D fiber hydrogel scaffolds and continuous administration of a blend of axon miRNAs (miR-222, miR-431, and miR-132) led to notable enhancement in axon regeneration. ⁴⁹ Prior research has demonstrated that elevated levels of miR-125b facilitate axon regeneration after spinal cord injury through regulation of the JAK/STAT pathway. Furthermore, miR-125b offers neuroprotection by diminishing neuronal apoptosis and dampening inflammatory responses. ³⁸ In vivo, the deletion of miR-155 enhances injury-induced expression of SPRR1A, a gene related to regeneration, in neurons, and also reduces inflammatory signaling in macrophages, leading to improved axon regeneration. ³⁶ Mice subjected to optic nerve compression exhibited notable augmentation of miR-19a levels within retinal ganglion cells, leading to a substantial enhancement of axon regeneration in vivo. ⁴⁸ Furthermore, alterations in the expression of miR-199a-3p and miR-21, induced by injury, impact axon development by regulating protein synthesis systemically and within axons through the modulation of the mTOR/PTEN pathway. ⁴² The mTOR/PTEN pathway is a critical regulator of axonal regeneration. PTEN, a tumor suppressor phosphatase, functions as a PIP3 phosphatase, effectively restraining mTOR signaling. Meanwhile, mTOR, a serine/threonine protein kinase, facilitates protein synthesis and mRNA translation, leading to improved axon growth. ⁸³ The combined action of these two elements collaboratively governs the growth of axons. MiRNA-21 regulates the expression of target genes, including EPHA4, TGF-I, and TIMP3, critical for promoting Schwann cell (SC) proliferation and facilitating axon regeneration in repairing injured peripheral nerves. ⁴³ Moreover, the naturally occurring miR-26a within sensory neurons plays a role in aiding the regeneration of sensory axons following spinal cord injury (SNI) by facilitating the activation of Smad1 while inhibiting the expression of glycogen synthase kinase 3β (GSK3β). ⁴⁵ On the other hand, numerous miRNAs act as negative regulators of neuronal regeneration, thus supporting the progression of neuropathic conditions. As an example, miR-21 regulates the epidermal growth factor receptor (EGFR) pathway, resulting in the excessive activation of astrocytes and the formation of glial scar tissue, which acts for obstacle to axon regeneration. ⁴¹ Blocking miR-455-5p leads to the inhibition of axonal growth and regeneration in sensory neurons of mice, and at the same time, reduces the activation of the GSK3β/Tau protein pathway. ⁴⁷ miR-9 and miRlet-7 also hinder axonal regeneration by targeting the protein levels of Dcc and Ntn1, respectively. ⁴⁴ Additionally, miR-9 is involved in regulating FoxP1 triggered by injury and has a role in axon regeneration. However, sensory neurons with high levels of endogenous miR-9 were found unable to regenerate their axons. ⁴⁶ Additionally, a newly discovered mechanism of controlling the inherent potential for axon regeneration includes a reciprocal negative regulatory loop between SIRT1 and miR-138. ⁴⁰ These discoveries introduce a new perspective for future investigations into axon regeneration in the context of neuropathic pain. Despite substantial advancements in comprehending the basic processes of peripheral nerve regeneration and leveraging these pathways for promoting regeneration following peripheral nerve injury (PNI), various challenges remain in devising therapies that facilitate comprehensive regeneration and functional restoration of neurons.

Regulation of neuronal ion channels

Following nerve fiber injury, alterations in the nerve endings and dorsal root ganglion (DRG) of the spinal cord may occur, affecting the structure and function of ion channels. These changes can lead to the abnormal discharge of neurons and subsequently result in pain. ⁸⁴ Consequently, ion channels play a pivotal role in determining neuronal excitability, and they might be subject to miRNA regulation under painful conditions. It is worth noting that voltage-gated channels, implicated in the pain pathway, have become the primary focus of interventions for treating neuropathic pain.

Calcium channels

In addition to voltage-gated potassium and sodium channels, calcium channels are equally essential in the pain sensitization process after nerve injury, as they participate in neurotransmitter release, regulate neuronal excitability, and influence intracellular alterations, including gene activation. ⁸⁵ In murine spinal neurons, the expression of miR-124a and miR-219, which negatively regulate spinal CaMKIIγ and the proinflammatory marker MeCP2, was significantly reduced after the development of inflammatory pain induced by either formalin or CFA injection. ⁵³ Furthermore, a decrease in miR-103 expression was observed in the spinal neurons of SNL rats. This concomitantly impacted the translational levels of the three constituents forming the Cav1.2-L-type calcium channel, an ion channel linked to pain sensitization. ⁸⁶ Moreover, miR-32-5p, through histone methylation-mediated targeting of Cav3.2 channels, downregulates its expression in trigeminal ganglion (TG) neurons, thereby controlling trigeminal neuropathic pain. ⁵⁵ In mice, the miR-183 cluster exerts control over more than 80% of genes associated with neuropathic pain by modulating the auxiliary voltage-gated calcium channel subunits α2δ-1 and α2δ-2. This regulation affects basal mechanical sensitivity and mechanical allodynia. ⁵⁴ These discoveries underscore the crucial involvement of miRNA-related channel abnormalities in developing nerve injury-induced neuropathic pain. They emphasize the significance of abnormal sensory input in maintaining neuropathic pain and suggest the potential efficacy of targeted chemo genetic silencing as a therapeutic approach for neuropathic pain treatment.

Voltage-gated potassium channels

Neurons’ excitability is significantly controlled by voltage-gated potassium channels, which govern action potential generation, neuronal firing frequency, and the release of neurotransmitters. ⁸⁷ The miRNA cluster miR-17-92, comprising six distinct members, downregulates the expression of potassium channels, particularly type A currents, leading to decreased outward potassium currents and the development of mechanical allodynia. ⁸⁸ Likewise, suppressing miR-137 leads to a decrease in mechanical allodynia and thermal hyperalgesia, normalizes Kv currents, reduces excessive neuronal activity in the dorsal root ganglion (DRG), and restores the expression of the potassium channel Kv1.2. ⁸⁹ Furthermore, miR-183-5p plays a role in controlling CCI-induced neuropathic pain by suppressing the expression of TREK-1, a Kv channel. ⁵⁰

Voltage-gated sodium channels

The Nav1.3 voltage-gated sodium channel, a variant susceptible to tetrodotoxin encoded by SCN3A, produces fast-repriming sodium ion currents. These currents can promote repetitive firing patterns and abnormal discharges in injured neurons, leading to heightened neuronal excitability and closely linked to neuropathic pain. ⁹⁰ Similar sodium-ion channels include Nav1.6, encoded by SCN8A, and Nav1.7, encoded by SCN9A. ⁹¹ MiR-96 administration via intrathecal delivery suppresses the upregulation of Nav1.3 caused by CCI. Additionally, miR-96 has been found to decrease the expression of Nav1.3 mRNA in embryonic DRG neurons in in vitro studies. ⁶¹ Additionally, miR-384-5p regulates the emergence of neuropathic pain by controlling SCN3A. ⁵⁹ In rats, miR-182 effectively reduced the neuropathic pain induced by SNI by regulating Nav1.7. ⁶⁰ Remarkably, the heightened presence of miR-30b resulted in a marked decline in the levels of Nav1.7, Nav1.6, and Nav1.3 in both dorsal root ganglion (DRG) neurons and the spinal cord, thereby leading to a considerable alleviation of neuropathic pain induced by spinal nerve ligation (SNL) or oxaliplatin. ⁵⁷ Similarly, the heightened presence of miR-183 reduced osteoarthritic pain by suppressing the synthesis of Nav1.8, Nav1.3, and Nav1.7. ²⁹ Moreover, elevated levels of miR-7a in primary sensory neurons of damaged DRGs hindered the increase in the β2 subunit of voltage-gated sodium channels. They reverted the persistent hyperexcitability of pain-sensing neurons to their regular state. ⁵¹

Role of exosomal miRNAs in neuropathic pain

Exosomes (Exos) are a group of nanosized extracellular vesicles (EVs) ranging in size from 40 to 200 nm, which are released by various cell types and participate in paracrine interactions among different cell types, such as glial cells, mesenchymal stem cells, neurons, leukocytes and endothelial cells. ⁹² Functioning as unique secreted agents, these biological nanoparticles are abundant in various genetic components like miRNAs, long noncoding RNA, lipids, and proteins. They can easily spread throughout biofluids, impacting biochemical reactions and cell survival in normal and disease-related circumstances, particularly in neurodegenerative and inflammatory conditions. ⁹³ Notably, exosomal miRNAs can be found in significant quantities in various bodily fluids like blood, saliva, breast milk, and urine. ⁹⁴ Pain often arises from inflammation, with various cytokines, chemokines, and other factors contributing to the onset of acute inflammatory pain. In cases of chronic inflammation, the processes leading to peripheral and cerebral sensitization can be initiated. ⁹⁵ Exosomal miRNAs, once produced, can be transported to different sites and act on neurons, microglia, macrophages, or other tissue cells. These entities control neuropathic pain through their impact on the release of inflammatory substances and oxidative stress, as well as their ability to regulate neural remodeling and nerve regeneration processes. Exosomes can modulate the release of nociceptive mediators from cells, which are known to be involved in neuroinflammation and are recognized for their role in sensitizing sensory terminals. ⁹⁶ Immunological cells, such as T lymphocytes and antigen-presenting dendritic cells (DCs), are capable of releasing and absorbing exosomal miRNAs, suggesting that exosomal miRNA transfer could serve as a novel mode of intercellular communication. ⁹⁷ Consequently, exosomal miRNA transmission holds significant implications for various systems and processes, including immune responses and neuron-glia communication. ⁹⁸ Fascinatingly, these exosomal miRNAs possess the ability to elicit both anti-inflammatory and proinflammatory responses. As previously discussed, specific exosomal miRNAs can release cytokines or other proinflammatory agents, influencing target organs directly. ⁹⁹ For instance, exosomes derived from neutrophils and synovial fibroblasts, and chondrocytes have been observed to stimulate macrophages to produce IL-1 and metalloproteinases. Additionally, the production of these inflammatory substances has been linked to the transfer of miR-449a-5p and miR-206 within exosomes’ contents. ¹⁰⁰ MiR-449a-5p plays a crucial role in inhibiting ATG4B, which regulates macrophage autophagy, triggers inflammasome activation, and exacerbates the inflammatory response. Studies have indicated that serum exosomes from neuropathic mice exhibit increased levels of miR-21. Further investigations revealed macrophage-derived exosomes containing miR-21-5p promote pyroptosis through A20, leading to a proinflammatory phenotype and aggravating podocyte damage in diabetic nephropathy mice. ¹⁰¹ Notably, intrathecal addition of miR-21-5p antagomir or conditional deletion of miR-21 in sensory neurons reduced hyperalgesia and macrophage recruitment in the DRG. A20 acts as an inhibitor of the NF-κB signaling pathway. Similarly, research showed that DRG sensory neurons release miR-23a-enriched extracellular vesicles (EVs) upon nerve damage, which are then taken up by macrophages to promote M1 polarization in vitro. Moreover, intrathecal delivery of an EV-miR-23a antagomir, which blocks A20 to enhance NF-κB signaling, reduced M1 macrophages, and improved neuropathic hyperalgesia. ¹⁰² Conversely, specific exosomal miRNAs exhibit anti-inflammatory and pain-relieving properties in chronic pain models by transporting healing substances to damaged neurons in both the central nervous system (CNS) and peripheral nervous system (PNS). ¹⁰³ The beginning of inflammation is thought to be the main cause of pain. Furthermore, exosomal miRNAs can suppress the generation of proinflammatory cytokines like PGE2, IL-1β, TNF-α and IL-6 at the injury site, while stimulating the release of IL-10, leading to pain relief effects. ⁹³ These miRNAs possess the ability to modulate nociception, and when administered intrathecally, miR-25, miR-103, miR-544, miR-124, and miR-23b have been shown to reduce inflammatory and neuropathic pain by modifying the intracellular activities of neurons, astrocytes, and microglia.^104,105 MiR-124 suppresses GRK2 expression, thus controlling the phenotypic balance between M1 and M2 cells in the spinal cord. Similarly, akin to certain neurotrophic factors like BDNF, GDNF, NGF, FGF-1, and IGF-1, exosomal miRNAs possess the capacity to enhance axonal growth and neuronal viability, intensifying therapeutic outcomes. ¹⁰⁶ Until now, the targeted and modular EV loading (TAMEL) approach has not been utilized in experimental pain research. Despite numerous studies attempting to shed light on the function of exosomal miRNAs in neuropathic pain, much remains to be unraveled about their cellular and molecular roles, as well as their downstream targets.

The emerging role of exosomal RNAs in neuropathic pain

Common pain relievers like acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), local anesthetics, and opioids can effectively manage acute pain. However, when it comes to neuropathic pain, these options often fall short due to limited efficacy or unwanted side effects. ¹⁰⁷ Therefore, current research endeavors should prioritize the discovery and development of mechanism-based therapies aimed at alleviating neuropathic pain. Laboratory investigations reveal that exosomes can regulate neuropathic pain and can be released by various cell types, including stem cells. ¹⁰³ Moreover, evidence indicates that exosomes derived from stem cells can closely replicate the functional effects of their parent cells, making them vital players in tissue repair processes. ¹⁰⁸ Exosomes derived from stem cells offer a novel therapeutic approach and research focus for neuropathic pain, as they not only aid in nerve repair but also eliminate the risks associated with stem cell transplantation, such as immunosuppression, genetic alterations, and malignant transformation, thanks to their paracrine effects. ¹⁰⁹ These stem cell-derived exosomes are believed to transfer beneficial neurotrophic factors like IGF-1, FGF-1, NGF, BDNF, and GDNF to injured neurons. Intrathecal administration of mesenchymal stem cell exosomes in rats with spinal cord injuries has demonstrated a reduction in neuropathic pain by shifting microglia from the M1 to the M2 phenotype and inhibiting the release of inflammatory cytokines like NF-κB, IL-6, IL-1, and TNF-α. ¹¹⁰ The TLR4/miR-216a-5p axis has been shown to have a regulatory impact on microglial polarization. ³⁷ Mesenchymal stem cells (MSCs) are an up-and-coming stem cell type for treating various ischemic conditions and tissue damage due to their versatile differentiation potential and strong immune regulatory functions. ¹¹¹ Recent research in the laboratory revealed that MSCs can migrate to injured nerve tissues and promote the regeneration of damaged neurons. ¹¹² Exosomes derived from MSCs regulate the growth of neurites by affecting both the quantity and overall length of neurites through the transmission of miR-133b to nerve cells.^113,114 Moreover, exosomes containing an abundance of miR-17-92 clusters have the potential to boost neuroplasticity and improve functional recovery by targeting PTEN, thereby activating the GSK-3β/Akt/PI3K/mTOR signaling pathway. ¹¹⁵ Likewise, small exosomes derived from Schwann cells (SCs) that contain miR-21-5p exert a negative regulatory effect on PTEN, leading to improved growth and survival of sensory neurons. ¹¹⁶ In order to promote neurite outgrowth in laboratory settings and support nerve regeneration in living organisms, secreted extracellular vehicles (EVs) from SCs carry miR-23b-3p from mechanically stimulated SCs to neurons, leading to a decrease in the levels of neuronal neuropilin 1 (Nrp1). ¹¹⁷ Exosomes derived from umbilical cord mesenchymal stem cells enhance axon regrowth and functional improvement in the spinal cord of rats by targeting Cblb/Cbl-mediated NGF/TrkA signaling through miR-199a-3p/145-5p. ¹¹⁸ After spinal cord injury, neural stem cell-released exosomes, stimulated by IGF-1, reduce apoptosis and promote nerve regeneration, partially through a YY1/miR-219a-2-3p mechanism. ¹¹⁹ Likewise, miR-181c-5p acts as a negative regulator of Bcl-2-interacting cell death mediators (BIM), thereby preventing neuronal apoptosis and maintaining cortical neuron health, which ultimately facilitating axon regeneration. ¹²⁰ A recent investigation revealed that stem cell-derived exosomes enriched with miR-29 could alleviate proinflammatory responses in a rat model of osteoarthritis. ¹²¹ Similarly, miR-199-3p overexpression in mice attenuated postherpetic neuralgia induced by TRX by targeting MECP2. ¹²² Microglial cells incorporated exomiR-181c-5p, which prevented the release of inflammatory substances. Intrathecal treatment with exomiR-181c-5p in CCI rats reduced neuroinflammatory symptoms and neuropathic pain. ⁷⁰ To counter neuroinflammation, exosomes originating from human umbilical cord MSCs increased the expression of autophagy-related proteins (LC3-II and beclin1) and hindered NLRP3 inflammasome activation via the TRAF6/miR-146a-5p pathway in the spinal cord dorsal horn. ¹²³ Additionally, xenogenic injection of human MSC exosomes enriched with miR-26a-5p led to the downregulation of cyclooxygenase-2 (PTGS2) in rat synovial fibroblasts, resulting in reduced pathogenic alterations. ¹²⁴ Exosomes derived from MSCs exhibit pain-relieving properties in chronic pain models by delivering particular miRNAs to both the central nervous system (CNS) and peripheral nervous system (PNS). ¹²⁵ Exosomal miRNAs offer distinct advantages as biomarkers compared to freely circulating miRNAs: firstly, exosomes harbor a wide variety of miRNAs, making them reliable vehicles for miRNA research ¹ ; secondly, the bilayer membrane structure of exosomes enhances miRNA stability, susceptibility to amplification, and reduces unfavorable outcomes ² ; and lastly, exosomes can efficiently traverse the blood-brain or blood-spinal cord barriers. ⁴ Despite the potential benefits of MSC-derived exosomes, the primary challenge in stem cell therapy remains the limited therapeutic effectiveness due to the low survival rate of transplanted cells in injured tissues. The results indicate that stem cell-derived exosomal miRNAs can control pain by diminishing proinflammatory cytokines and fostering neuronal regeneration and differentiation, presenting a fresh and innovative therapeutic strategy for the treatment of nerve injuries. In conclusion, neuropathic pain poses a major risk to patients’ physical and emotional well-being, and the absence of reliable and efficient treatment methods adds to the difficulty of addressing this therapeutic challenge. The extensive investigation of miRNA in the development of neuropathic pain has opened promising avenues for clinical translation, particularly due to its remarkable impact on neuroinflammation, nerve regeneration, and other aspects, offering hope for improved clinical management of neuropathic pain. However, challenges such as miRNA’s bioactivity, stability, safety, and tissue specificity must be addressed before its widespread therapeutic application. Efficient transmembrane delivery and careful consideration of enzymatic reactions are additional critical factors during miRNA targeting. As a result, extensive focus is currently directed toward investigating delivery agents, such as exosomes, liposomes, viral vectors, miRNA mimics, and inhibitors. Patient safety remains a top priority during clinical applications, necessitating a thorough assessment of the potential immunological response triggered by exogenous miRNA treatment. Additional preclinical investigations and clinical trials are crucial to support the successful application of these findings in clinical settings, as the current research is primarily confined to studies conducted in cell cultures and animal models. With ongoing research into miRNA mechanisms and the utilization of advanced technologies like miRNA gene chips and high-throughput methods, miRNAs can become novel biological markers for disease diagnosis and offer new targets and strategies for understanding and treating neuropathic pain in the future.

ORCID iD

Amir Azimian https://orcid.org/0000-0002-2532-8198