Research paperAbnormal DNA methylation in the lumbar spinal cord following chronic constriction injury in rats
Introduction
Neuropathic pain is a chronic pain, which is caused by primary or secondary damage or dysfunction of the nervous system, and is characterized by hyperalgesia, allodynia and spontaneous pain [18]. The pathogenesis of neuropathic pain is complex and not clearly understood. The transcription and expression level of pain-associated genes are highly involved in the generation and maintenance of neuropathic pain [25]. Decreased gamma-aminobutyric acid (GABA)-ergic inhibition in the spinal dorsal horn plays an important role in neuropathic pain [4], [28]. Glutamate decarboxylase 67 (GAD 67) is a key synthetic enzyme for the main inhibitory transmitter GABA, and diminishes in the spinal dorsal horn in rats following chronic constriction injury (CCI) [19], [29].
Epigenetic mechanisms that induce heritable changes in gene expression without causing alterations in the DNA sequence can regulate the transcription and expression of pro- or antinociceptive genes [5], [8], and may play an important role in neuropathic pain [3], [27]. DNA methylation is one of the earliest and most characteristic epigenetic mechanisms in mammals, and is a major contributor to the stability of gene expression [12]. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), which transfer a methyl group from S-adenosyl-l-methionine to cytosine bases of cytosine–phosphate–guanine (CpG) in DNA [9]. The CpG dinucleotides tend to cluster in regions called CpG islands, defined as regions of more than 200 bases with a G + C content of at least 50% and a ratio of observed to statistically expected CpG frequencies of at least 0.6 [23]. The methylation of CpG islands in the gene promoter plays an important role in regulating gene expression. Increased methylation of CpG islands in the gene promoter can downregulate gene expression, and decreased methylation of CpG islands in the gene promoter can upregulate gene expression [26]. There are two functionally different classes of DNMTs—maintenance methyltransferases DNMT1 and de novo methyltransferases DNMT3a and DNMT3b [16]. Methylated DNA relies on interfering with transcription factors directly and mainly via methyl-CpG-binding proteins (MBDs) indirectly to inhibit gene transcription [2], [20]. Methyl-CpG binding protein 2 (MeCP2) and MBD 1–3 are the most studied MBDs in the central nervous system (CNS) [16].
GAD 67 is encoded by gene GAD 1. Recent evidences indicate that DNA methylation can regulate the expression of GAD 67 by regulating the methylation of GAD 1 promoter in the psychotic brain [6], [10]. And there is a CpG island in rat GAD 1 promoter by scanning the rat GAD 1 promoter region (GenBank accession number AF110132) and recent rat GAD 1 sequence from UCSC Genome Browser on Rat Nov. 2004 (Baylor 3.4/rn4) Assembly. In this study, in order to discover whether DNA methylation regulates GAD 67 expression in the spinal cord in CCI rats and is involved in neuropathic pain, we examined mRNA levels of DNMTs, MBDs and GAD 67, and methylation of GAD 1 promoter in the lumbar spinal cord in CCI rats on day 14 after surgery.
Section snippets
Animal surgeries and neuropathic pain measurements
This study was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Animal Care Committee of the Fujian Provincial Cancer Hospital in China approved all the procedures. Sixteen male Sprague-Dawley (SD) rats, weighing 220–250 g, provided by the animal experiment center of Fujian Medical University were divided into CCI rats (n = 8) and sham operation rats (n = 8). Under deep anesthesia induced by intraperitoneal injection of 10% chloral
Changes in behavior and neuropathic pain measurements
CCI rats suffered from neuropathic pain showing various signs of protecting postures and moving disorders such like foot and toe closing together, dorsiflexion, eversion and marked limp. Sham operation rats showed no such signs. MWT and TWL had similar changes (Fig. 1). There was no significant difference in MWT and TWL among all the rats before surgery (P > 0.05). MWT and TWL of sham operation rats showed no significant change compared with that of before operation (P > 0.05). The MWT and TWL of
Discussion
Pain-associated gene' transcription and expression, on which DNA methylation may exert a major effect, are highly relevant to the genesis of neuropathic pain. DNA methylation is mediated by DNMTs and MBDs. DNMT1 is the primary maintenance methyltransferase, which associates with hemi-methylated CpG during DNA replication and preserves methylation patterns in daughter cells [24]. DNMT3a and DNMT3b are the primary de novo methyltransferases, which target unmethylated CpG and establish new
Conclusions
In this study, GAD 67 expression decreased, GAD 1 promoter methylation increased, DNMT3a, DNMT3b and MeCP2 expression increased, and MBD2 expression decreased in the lumbar spinal cord in CCI rats on day 14 after surgery. These results indicate that decreased GAD 67 expression may be associated with increased GAD 1 promoter methylation, which may be mediated by DNMT3a, DNMT3b, MeCP2 and MBD2 in CCI rats. These indicate that abnormal DNA methylation may be highly involved in CCI-induced
Acknowledgements
This study was supported by National Natural Science Foundation of China (81400921) and Natural Science Foundation of Fujian Province of China (2015J05144).
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