Nerve injury‐induced change in gene expression in primary sensory neurons of dorsal root ganglion (DRG) is critical for neuropathic pain genesis. N 6 ‐methyladenosine (m 6 A) modification of RNA represents an additional layer of gene regulation. Here, it is reported that peripheral nerve injury increases the expression of the m 6 A demethylase fat‐mass and obesity‐associated proteins (FTO) in the injured DRG via the activation of Runx1, a transcription factor that binds to the Fto gene promoter. Mimicking this increase erases m 6 A in euchromatic histone lysine methyltransferase 2 ( Ehmt2 ) mRNA (encoding the histone methyltransferase G9a) and elevates the level of G9a in DRG and leads to neuropathic pain symptoms. Conversely, blocking this increase reverses a loss of m 6 A sites in Ehmt2 mRNA and destabilizes the nerve injury‐induced G9a upregulation in the injured DRG and alleviates nerve injury‐associated pain hypersensitivities. FTO contributes to neuropathic pain likely through stabilizing nerve injury‐induced upregulation of G9a, a neuropathic pain initiator, in primary sensory neurons.

Nav1.3 is a tetrodotoxin-sensitive isoform among voltage-gated sodium channels that are closely associated with neuropathic pain. It can be up-regulated following nerve injury, but its biological function remains uncertain. MicroRNAs (miRNAs) are endogenous non-coding RNAs that can regulate post-transcriptional gene expression by binding with their target mRNAs. Using Target Scan software, we discovered that SCN3A is the major target of miR-30b, and we then determined whether miR-30b regulated the expression of Nav1.3 by transfecting miR-30b agomir through the stimulation of TNF-α or by transfecting miR-30b antagomir in primary dorsal root ganglion (DRG) neurons. The spinal nerve ligation (SNL) model was used to determine the contribution of miR-30b to neuropathic pain, to evaluate changes in Nav1.3 mRNA and protein expression, and to understand the sensitivity of rats to mechanical and thermal stimuli. Our results showed that miR-30b agomir transfection down-regulated Nav1.3 mRNA stimulated with TNF-α in primary DRG neurons. Moreover, miR-30b overexpression significantly attenuated neuropathic pain induced by SNL, with decreases in the expression of Nav1.3 mRNA and protein both in DRG neurons and spinal cord. Activation of Nav1.3 caused by miR-30b antagomir was identified. These data suggest that miR-30b is involved in the development of neuropathic pain, probably by regulating the expression of Nav1.3, and might be a novel therapeutic target for neuropathic pain.Perspective: This study is the first to explore the important role of miR-30b and Nav1.3 in spinal nerve ligation-induced neuropathic pain, and our evidence may provide new insight for improving therapeutic approaches to pain.

Voltage-gated sodium channels, which are involved in pain pathways, have emerged as major targets for therapeutic intervention in pain disorders. Nav1.7, the tetrodotoxin-sensitive voltage-gated sodium channel isoform encoded by SCN9A and predominantly expressed in pain-sensing neurons in the dorsal root ganglion, plays a crucial role in nociception. MicroRNAs are highly conserved, small non-coding RNAs. Through binding to the 3′ untranslated region of their target mRNAs, microRNAs induce the cleavage and/or inhibition of protein translation. Based on bioinformatics analysis using TargetScan software, we determined that miR-30b directly targets SCN9A. To investigate the roles of Nav1.7 and miR-30b in neuropathic pain, we examined changes in the expression of Nav1.7 in the dorsal root ganglion by miR-30b over-expression or knockdown in rats with spared nerve injury. Our results demonstrated that the expression of miR-30b and Nav1.7 was down-regulated and up-regulated, respectively, in the dorsal root ganglion of spared nerve injury rats. MiR-30b over-expression in spared nerve injury rats inhibited SCN9A transcription, resulting in pain relief. In addition, miR-30b knockdown significantly increased hypersensitivity to pain in naive rats. We also observed that miR-30b decreased Nav1.7 expression in PC12 cells. Taken together, our results suggest that miR-30b plays an important role in neuropathic pain by regulating Nav1.7 expression. Therefore, miR-30b may be a promising target for the treatment of chronic neuropathic pain.

The sodium channel 1.7 (Nav1.7), which is encoded by SCN9A gene, is involved in neuropathic pain. As crucial regulators of gene expression, many miRNAs have already gained importance in neuropathic pain, including miR-182, which is predicted to regulate the SCN9A gene. Nav1.7 expression in L4-L6 dorsal root ganglions (DRGs) can be up regulated by spared nerve injury (SNI), while miR-182 expression was down regulated following SNI model. Exploring the connection between Nav1.7 and miR-182 may facilitate the development of a better-targeted therapy. In the current study, direct pairing of miR-182 with the SCN9A gene was verified using a luciferase assay in vitro. Over-expression of miR-182 via microinjection of miR-182 agomir reversed the abnormal increase of Nav1.7 at both mRNA and protein level in L4-6 DRGs of SNI rats, and significantly attenuated the hypersensitivity to mechanical stimulus in the rats. In contrast, administration of miR-182 antagomir enhanced the Nav1.7 expression at both mRNA and protein level in L4-6 DRGs, companied with the generation of mechanical hypersensitivity in naïve rats. Collectively, we concluded that miR-182 can alleviate SNI- induced neuropathic pain through regulating Nav1.7 in rats.

No abstract

Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel in primary sensory neurons of the dorsal root ganglia (DRGs). Pharmacological and genetic studies implicated TRPM3 in various pain modalities, but TRPM3 inhibitors were not validated in TRPM3 2/2 mice. Here we tested two inhibitors of TRPM3 in male and female wild-type and TRPM3 2/2 mice in nerve injury-induced neuropathic pain. We found that intraperitoneal injection of either isosakuranetin or primidone reduced heat hypersensitivity induced by chronic constriction injury (CCI) of the sciatic nerve in wild-type, but not in TRPM3 2/2 mice. Primidone was also effective when injected locally in the hindpaw or intrathecally. Consistently, intrathecal injection of the TRPM3 agonist CIM0216 reduced paw withdrawal latency to radiant heat in wild-type, but not in TRPM3 2/2 mice. Intraperitoneal injection of 2 mg/kg, but not 0.5 mg/kg isosakuranetin, inhibited cold and mechanical hypersensitivity in CCI, both in wild-type and TRPM3 2/2 mice, indicating a dose-dependent off-target effect. Primidone had no effect on cold sensitivity, and only a marginal effect on mechanical hypersensitivity. Genetic deletion or inhibitors of TRPM3 reduced the increase in the levels of the early genes c-Fos and pERK in the spinal cord and DRGs in CCI mice, suggesting spontaneous activity of the channel. Intraperitoneal isosakuranetin also inhibited spontaneous pain related behavior in CCI in the conditioned place preference assay, and this effect was eliminated in TRPM3 2/2 mice. Overall, our data indicate a role of TRPM3 in heat hypersensitivity and in spontaneous pain after nerve injury.

Neuropathic pain genesis is related to gene alterations in the dorsal root ganglion (DRG) following peripheral nerve injury. Transcription factors control gene expression. In this study, we investigated whether octamer transcription factor 1 (OCT1), a transcription factor, contributed to neuropathic pain caused by chronic constriction injury (CCI) of the sciatic nerve. CCI produced a time-dependent increase in the level of OCT1 protein in the ipsilateral L4/5 DRG, but not in the spinal cord. Blocking this increase through microinjection of OCT1 siRNA into the ipsilateral L4/5 DRG attenuated the initiation and maintenance of CCI-induced mechanical allodynia, heat hyperalgesia, and cold allodynia and improved morphine analgesia after CCI, without affecting basal responses to acute mechanical, heat, and cold stimuli as well as locomotor functions. Mimicking this increase through microinjection of recombinant adeno-associated virus 5 harboring full-length OCT1 into the unilateral L4/5 DRG led to marked mechanical allodynia, heat hyperalgesia and cold allodynia in naive rats. Mechanistically, OCT1 participated in CCI-induced increases in Dnmt3a mRNA and its protein and DNMT3a-mediated decreases in Oprm1 and Kcna2 mRNAs and their proteins in the injured DRG. These findings indicate that OCT1 may participate in neuropathic pain at least in part by transcriptionally activating Dnmt3a and

Despite the great increase in human lifespan with improved medical care, the physiological and pathological changes such as memory and cognitive disorders and associated anxiety and depression are major concern with aging. Molecular mechanisms underlying these changes are little known. The present study examined the differentially expressed genes (DEGs) and the genes with differentially expressed isoforms in three brain regions, anterior cingulate cortex (ACC), amygdala and hippocampus, throughout the lifespan of mice. Compared to 2-month old mice, both 12-and 24-month old mice displayed memory and cognitive impairments in the Morris water maze, Y-maze, and novel object recognition tests and depression-and anxiety-like behaviors in the tail suspension, forced swimming, open field, and elevated plus maze tests. RNA sequencing analysis identified 634 and 1078 DEGs in ACC, 453 and 1015 DEGs in the amygdala and 884 and 1054 DEGs in hippocampus in the 12-and 24-month old mice, respectively. Similarly, many genes with differentially expressed isoforms were also identified in these three brain regions in the 12-and 24-month old mice. Further functional analysis revealed that many DEGs and the genes with differentially expressed isoforms in the ACC and amygdala were mapped to depression-and anxiety-related genes, respectively and that a lot of DEGs and the genes with differentially expressed isoforms in hippocampus were mapped to cognitive dysfunction-related genes from both 12-and 24-month old mice. All of these mapped DEGs and the genes with differentially expressed isoforms were closely related to neuroinflammation. Our findings indicate that these neuroinflammation-related DEGs and the genes with differentially expressed isoforms are likely new targets in the management of memory/cognitive impairment and emotional disorders during the aging.