Spinal Cord DNMT1 Contributes to Diabetic Neuropathic Pain Mediated by miR-152-3p Downregulation

Abstract

Diabetic neuropathic pain (DNP) is a common complication of diabetes, yet there are no safe and effective therapeutic options. Emerging evidence has indicated that DNA methylation mediated by DNA methyltransferases (DNMTs) is associated with neuropathic pain. However, how DNMTs respond to DNP and the underlying mechanism has not been established. In this study a DNP model was created and DNMT1, but not DNMT3a or DNMT3b, was shown to be upregulated in the spinal cords of mice with DNP. Moreover, DNMT1 was predominantly expressed in spinal cord neurons. Knockdown of DNMT1 in neurons improved nociceptive hypersensitivity in mice with DNP. Furthermore, bioinformatics analysis and real-time quantitative PCR results suggested that downregulated miR-152-3p in the spinal cord of mice with DNP may be an upstream DNMT1 molecule. Overexpression of miR-152-3p reduced DNMT1 expression in the spinal cord and alleviated nociceptive hypersensitivity in mice with DNP. Knockdown of miR-152-3p caused an increase in DNMT1 expression in the spinal cord and induced nociceptive hypersensitivity in naive mice. Moreover, knockdown of DNMT1 reversed miR-152-3p knockdown-induced nociceptive hypersensitivity in naive mice. These results suggest that downregulation of miR-152-3p in the spinal cord is involved in the development of DNP by upregulating DNMT1. These data demonstrate a new mechanism underlying the development of DNP and provide a new therapeutic target for DNP.

Main article text

1. INTRODUCTION

The prevalence of diabetes is increasing and diabetes is now the ninth leading cause of death worldwide [1], reaching 537 million in 2021 [2] for adults 20–79 years of age. Among the most serious complications of type 2 diabetes mellitus (T2DM) is diabetic neuropathic pain (DNP), the prevalence of which correlates positively with the duration of the disease [3]. Despite the severity of DNP, there is currently no definitive treatment, which significantly increases the mortality rate [4]. Therefore, understanding the pathogenesis of DNP is essential for identifying precise drug targets and developing new treatment strategies.

DNA methyltransferases (DNMTs), including DNMT1, DNMT3a, and DNMT3b, are essential for establishing and maintaining DNA methylation patterns and have an important role in the formation of DNP [5,6]. The expression of DNMT3a, but not DNMT3b, is upregulated in the spinal cord and injured dorsal root ganglion (DRG) in neuropathic pain models induced by chronic constriction injury (CCI) or spinal nerve ligation (SNL), while a DNMT inhibitor or DNMT3a knockdown significantly attenuates pain hypersensitivities [7,8]. However, another study showed that DNMT1 is upregulated in the injured DRG after peripheral nerve injury [9]. These data suggest that in neuropathic pain with different causes, the DNMT subtypes that are involved also differ. Therefore, it is important to determine which DNMTs have a role in the formation of DNP.

Micro (mi)RNAs are a class of small RNAs (approximately 20–24 nucleotides) that can inhibit the expression of many genes at the post-transcriptional level [10]. Emerging evidence highlights the significant roles of miRNAs in the development of neuropathic pain [11] and the expression of DNMTs. MiR-23a directly targets CXCR4 to regulate pain via the TXNIP/NLRP3 inflammasome axis in spinal glial cells in mice with partial sciatic nerve ligation (pSNL)-induced neuropathic pain. Additionally, small RNA sequencing has identified 22 differentially expressed miRNAs in the DRGs of chronic compression DRG rats [12]. Moreover, miR-547-3p has been identified as a key mediator of neuroinflammation and neuropathic pain [12]. Clinical studies have shown a strong negative correlation between hsa-miR-29a and hsa-miR-500a expression and the intensity of neuropathic pain [13]. In addition, miRNAs have been shown to negatively regulate DNMT expression, further affecting the level of relevant gene methylation [14–16]. However, it is unknown if miRNA and DNMT expression affect the occurrence and progression of DNP.

In this study the levels of DNMT1, DNMT3a, and DNMT3b expression in the spinal cord of DNP mice were determined. In addition, miR-152-3p was shown to be an upstream regulator of DNMT1 and the role of miR-152-3p in the progression of DNP was clarified.

2. RESULTS

2.1. Increased expression of DNMT1 in the spinal cords of mice with DNP

High-fat fed/low-dose streptozotocin (STZ)–induced T2DM mice were used as animal models of DNP. The fasting blood glucose (FBG) level in the diabetic group mice was significantly increased on days 5 (P<0.01) and 21 after STZ injection compared to control mice (P<0.01; Fig 1A). Moreover, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of diabetic mice were significantly decreased on day 21 after STZ injection compared to control mice (P<0.01 and P<0.01, respectively; Fig 1B, C), which indicated that diabetic mice developed mechanical allodynia and thermal hyperalgesia.

Figure1 |

Expression of DNMTs in the spinal cords of mice with diabetic neuropathic pain (DNP). (A) The fasting blood glucose (FBG) concentration on day 5 after STZ injection and at the end of the experiment on day 21 after STZ injection in mice. (B) Mechanical allodynia is shown as a mechanical withdrawal threshold (MWT). (C) Thermal hyperalgesia was demonstrated as thermal withdrawal latency (TWL). (D-F) The levels of DNMT1, DNMT3a, and DNMT3b mRNA expression in the spinal cords of mice with DNP were determined by RT-qPCR. (G-I) The levels of DNMT1, DNMT3a, and DNMT3b mRNA expression in the spinal cords of mice with DNP were determined by western blot analysis. All data are expressed as the mean±SEM. n=6; **P<0.01; ns, not significant.

The levels of DNMT1, DNMT3a, and DNMT3b expression in the spinal cords of mice with DNP were determined to elucidate the molecular mechanisms underlying DNP. As shown in Fig 1D–I, the mRNA and protein levels of DNMT1 mRNA and protein (P<0.01 and P<0.01, respectively), but not DNMT3a or DNMT3b mRNA and protein, in the spinal cords of mice with DNP were significantly increased compared to control mice. These data indicate that DNMT1 may serve as an important epigenetic regulator in the development of DNP.

2.2. DNMT1 is mainly localized in spinal neurons

Double immunofluorescent staining was used to determine the co-localization of DNMT1 cell types with specific markers for neurons, astrocytes, and microglia. DNMT1 was mainly co-localized with a neuronal marker (NeuN) in the spinal dorsal horn, but rarely co-localized with an astrocyte marker (GFAP) or a microglial marker (Iba-1; Fig 2). These data suggest that DNMT1 is predominantly expressed in spinal neurons.

Figure2 |

Cellular localization of DNMT1 in the spinal cords of mice. Representative images show the immunohistochemical staining for DNMT1 (red), neuronal nuclei [NeuN] (green), glial fibrillary acidic protein [GFAP] (green), and Iba-1 (green). Scale bar: 200 μm.

2.3. DNMT1 knockdown in spinal neurons improves pain hypersensitivity in mice with DNP

An adeno-associated vector (AAV) expressing DNMT1 shRNA and mCherry under the control of hSyn promoter (AAV-DNMT1 shRNA) was injected intrathecally into DNP mice to further confirm the role of spinal neuron DNMT1. Control mice were injected with an AAV expressing mCherry alone. Pain behaviors were evaluated 14 d after AAV injection (i.e., 21 d after STZ injection). Figure 3A shows the mCherry fluorescence expressed in the spinal dorsal horn, indicating that AAV was successfully transfected into the spinal neurons. Moreover, intrathecal injection of AAV-DNMT1 shRNA was validated to downregulate DNMT1 mRNA (P<0.01) and protein expression (P<0.05) in mice with DNP (Fig 3B, C). In addition, DNMT1 knockdown in spinal neurons was shown to reverse mechanical allodynia and thermal hyperalgesia in mice with DNP, manifested by increased MWT and TWL after intrathecal injection of AAV-DNMT1 shRNA compared to injection of AAV-mCherry in mice with DNP (Fig 3D, E). However, knockdown of DNMT1 in spinal neurons did not alter the FBG concentration in mice with DNP (Fig 3F). These results suggested that knockdown of DNMT1 in spinal neurons improves pain hypersensitivity in mice with DNP.

Figure3 |

Effect of DNMT1 knockdown in spinal neurons on pain behaviors in mice with DNP. (A) Typical images of AAV-mCherry viral expression in the spinal cord after intrathecal injection of AAV-DNMT1 shRNA. Scale bar: 200 μm (B) DNMT1 mRNA expression in the spinal cord after intrathecal injection of AAV-DNMT1 shRNA. (C) DNMT1 protein expression in the spinal cord after intrathecal injection of AAV-DNMT1 shRNA. (D) The MWT of mice after intrathecal injection of AAV-DNMT1 shRNA. (E) The TWL of mice after intrathecal injection of AAV-DNMT1 shRNA. (F) The FBG concentration of mice after intrathecal injection of AAV-DNMT1 shRNA. All data are presented as the mean±SEM. n=6; ^* P<0.05; ^** P<0.01; ns, not significant.

2.4. Bioinformatic prediction of miRNAs targeting DNMT1 and the effect of miR-152-3p on DNP

Potential miRNAs interacting with DNMT1 were screened using Targetscan, miRDB, and miRWalk databases to elucidate the upstream mechanism underlying DNMT1 regulation of DNP. The top 3 miRNAs targeting DNMT1 were miR-152-3p, miR-148a-3p, and miR-148b-3p. Figure 4A shows the seed region of the top three microRNAs binding to the 3′UTR of DNMT1 in the Targetscan database. The levels of miR-152-3p, miR-148a-3p, and miR-148b-3p expression in the spinal cord of mice with DNP were further determined using RT-qPCR. The expression of miR-152-3p in the spinal cord of mice with DNP mice was significantly decreased compared to the control mice, while the levels of miR-148a-3p and miR-148b-3p were not significantly different between the two groups (Fig 4B), suggesting that miR-152-3p may serve as an important regulator in the development of DNP.

Figure4 |

Identification of miRNAs targeting DNMT1 and observing the effects of miR-152-3p on DNP. (A) Alignment shows the seed region of the top three microRNAs binding to the 3′UTR of DNMT1 in the Targetscan database. (B) The levels of miR-152-3p, miR-148a-3p, and miR-148b-3p expression in the spinal cords of mice with DNP. (C) Typical images of AAV-EGFP viral expression in the spinal cord after intrathecal injection of AAV-miR-152-3p. Scale bar: 200 μm (D) Level of miR-152-3p expression after intrathecal injection of AAV-miR-152-3p. (E) Level of DNMT1 protein expression in the spinal cord after intrathecal injection of AAV-miR-152-3p. (F) Level of DNMT1 protein expression in the spinal cord after intrathecal injection of AAV-miR-152-3p. (G) The MWT of mice after intrathecal injection of AAV-miR-152-3p. (H) The TWL of mice after intrathecal injection of AAV-miR-152-3p. (I) The FBG concentration in mice after intrathecal injection of AAV-miR-152-3p. All data are presented as the mean±SEM. n=6; ^* P<0.05; ^** P<0.01; ns, not significant.

To further validate the role of miR-152-3p in DNP, miR-152-3p was overexpressed by intrathecal injection of an AAV expressing miR-152-3p and EGFP (AAV-miR-152-3p) and control mice were injected with an AAV expressing EGFP alone. Figure 4C shows the expression of EGFP fluorescence in the spinal cord, indicating that AAVs were successfully transfected. The results of RT-qPCR confirmed that intrathecal injection of AAV-miR-152-3p upregulates the level of miR-152-3p in mice with DNP (Fig 4D). Moreover, intrathecal injection of AAV-miR-152-3p significantly reduced the levels of DNMT1 mRNA and protein in mice with DNP compared to mice injected with AAV-EGFP (Fig 4E, F), indicating that DNMT1 is the miR-152-3p target. Furthermore, upregulation of miR-152-3p improved mechanical allodynia and thermal hypersensitivity in mice with DNP (Fig 4G, H) but had no influence on the FBG concentration in mice with DNP (Fig 3I). Taken together, the results suggest that miR-152-3p may be involved in the development of DNP by targeting DNMT1.

2.5. DNMT1 is essential for miR-152-3p regulation of DNP

To confirm whether miR-152-3p targets and regulates DNMT1 to affect the occurrence and development of DNP, miR-152-3p knockdown (AAV-miR-152-3p-TuD) carrying EGFP was intrathecally injected into naive mice to determine the effect of miR-152-3p downregulation on pain behavior and DNMT1 expression. Furthermore, based on the injection of AAV-miR-152-3p-TuD, AAV-DNMT1 shRNA was simultaneously injected to determine if DNMT1 knockdown reversed the effect of miR-152-3p knockdown on DNP. Figure 5A shows that EGFP and mCherry fluorescence is co-expressed in the spinal cord, indicating that AAVs were successfully transfected. In addition, the intrathecal injection of AAV-miR-152-3p-TuD reduced the level of miR-152-3p (Fig 5B; P<0.01), upregulated DNMT1 mRNA and protein expression (Fig 5C–E; P<0.01 and P<0.01, respectively) in the mouse spinal cord, and induced mechanical allodynia and thermal hypersensitivity in naive mice compared to control mice (Fig 5F, G; P<0.01 and P<0.01, respectively). However, intrathecal injection of AAV-DNMT1 shRNA reversed the increased DNMT1 expression resulting from miR-152-3p knockdown (Fig 5C–E; P<0.01 and P<0.01, respectively). The decreased MWT and TWL induced by miR-152-3p knockdown was also reversed by DNMT1 knockdown (Fig 5F, G; P<0.01 and P<0.01, respectively). In addition, the FBG concentration was not influenced by miR-152-3p knockdown or knockdown of miR-152-3p and DNMT1 (Fig 5H). Taken together, the results demonstrated that downregulation of miR-152-3p is involved in the development of DNP by upregulation of DNMT1.

Figure5 |

DNMT1 is essential for miR-152-3p to regulate DNP. (A) Typical images of AAV-EGFP and AAV-mCherry viral expression in the spinal cord of naive mice after intrathecal injection of AAV-miR-152-3p TuD and AAV-DNMT1 shRNA. Scale bar: 200 μm (B) Expression of miR-152-3p in the spinal cords of naive mice after intrathecal injection of AAV-miR-152-3p TuD. (C) The level of DNMT1 mRNA expression in the spinal cords of naive mice after intrathecal injection of AAV-miR-152-3p TuD and AAV-DNMT1 shRNA. (D, E) The level of DNMT1 protein expression in the spinal cords of naive mice after intrathecal injection of AAV-miR-152-3p TuD and AAV-DNMT1 shRNA. (F) The MWT of naive mice after intrathecal injection of AAV-miR-152-3p TuD and AAV-DNMT1 shRNA. (G) The TWL of naive mice after intrathecal injection of AAV-miR-152-3p TuD and AAV-DNMT1 shRNA. (H) The FBG concentration of naive mice after intrathecal injection of AAV-miR-152-3p TuD and AAV-DNMT1 shRNA. All data are presented as the mean±SEM. n=8; ^** P<0.01; ns, not significant.

3. DISCUSSION

DNP is one of the most common complications of DM and significantly reduces the quality of life [17,18]. There remains a lack of effective treatment strategies for DNP. Recently, DNA methylation has been increasingly recognized in neuropathic pain [19]. Moreover, intraperitoneal or intrathecal injection of 5-Aza, a DNMT inhibitor, attenuates neuropathic pain [20,21]. However, there are differences in the expression of DNMTs in various pain models [9,22–24]. Therefore, the present study examined the role of DNMTs in the development of DNP.

In this study a high-fat fed/low-dose STZ-induced diabetic mouse model was constructed. Mice exhibited thermal hyperalgesia and mechanical allodynia on day 21 after STZ injection, indicating that the diabetic mice had developed significant pain hypersensitivity, which is consistent with our previous results in rats [25]. Moreover, the levels of DNMT1 mRNA and protein, but not DNMT3a or DNMT3b, were increased in the spinal cords of mice with DNP, suggesting that DNMT1 may be involved in the development of DNP. Furthermore, DNMT1 was primarily localized in spinal neurons but not in astrocytes or microglia, suggesting that neuronal DNMT1 is an important regulator of DNP. To further validate this result, AAV-hSyn-DNMT1 shRNA was intrathecally injected to specifically knock down DNMT1 in spinal neurons of mice with DNP. DNMT1 knockdown in spinal neurons reversed the mechanical allodynia and thermal hyperalgesia in mice with DNP. In agreement with our findings, it has been reported the neuronal location of DNMT1 in the DRG and the expression of DNMT1 are significantly upregulated in damaged DRG and may induce neuropathic pain by inhibiting the expression of the Kcna2 gene in the DRGs of mice [9]. However, there are still some differences between the results reported by Sun et al. and the present study [9]. Sun et al. validated the role of DRG DNMT1 in the genesis of SNL-induced neuropathic pain [9]. In our previous study we demonstrated that significant mechanical allodynia and thermal hyperalgesia developed on day 21 after STZ injection [25]. Therefore, in the current study, the role of DNMT1 on day 21 after STZ injection was determined and the important role of spinal DNMT1 in the maintenance stage of DNP was identified. However, there was no evidence indicating that spinal DNMT1 is involved in the genesis of DNP in the present study.

Recent studies have shown that miRNAs have critical roles in the regulation of gene expression, with >60% of genes being targeted by miRNAs [26]. Preclinical and clinical studies have also shown that altered miRNA expression profiles are involved in the pathogenesis of neuropathic pain and miRNA expression in the blood can serve as a biomarker of pain and have predictive value for the patient response to analgesic therapy [27–29]. Hence, we further focused on identifying potential miRNAs that regulate DNMT1 expression. Bioinformatic analysis identified the top 3 miRNAs targeting DNMT1 (miR-152-3p, miR-148a-3p, and miR-148b-3p). It was further shown that the level of miR-152-3p decreased significantly in the spinal cords of mice with DNP, while the levels of miR-148a-3p and miR-148b-3p did not change significantly, suggesting that miR-152-3p is involved in the development of DNP. As expected, overexpression of miR-152-3p in the spinal cord markedly relieved pain behaviors and downregulated DNMT1 expression in mice with DNP, while miR-152-3p knockdown in the spinal cord induced pain behaviors and increased DNMT1 levels in naive mice. Moreover, the effect of miR-152-3p knockdown was abolished by DNMT1 knockdown. These results suggested that miR-152-3p may be involved in the development of DNP by targeting DNMT1. Consistent with the results herein, several studies also reported that miR-152-3p is involved in bladder cancer, glioblastomas, and systemic lupus erythematosus by targeting DNMT1 [30–32]. In addition, recent studies have demonstrated that DNMT1 mediates methylation of miR-152-3p and decreases miR-152-3pexpression [33,34], suggesting that there is a feedback loop between DNMT1 and miR-152-3p. However, further research is needed to determine whether DNMT1-mediated methylation of miR-152-3p is involved in the development of DNP.

There were some limitations in this study. First, it has been reported that DNMT1 is upregulated via activation of the transcription factor, CREB, in DRGs after peripheral nerve injury [9]. Whether CREB is involved in DNMT1 upregulation in DNP remains to be investigated. Second, miR-152-3p has been reported to regulate the expression of other genes in the nervous system, such as postsynaptic density protein-93 (PSD-93) and CaMKIIα [35,36], which have also been shown to be associated with neuropathic pain [37,38]. Therefore, we cannot rule out the possibility that other genes are involved in the role of miR-152-3p in DNP. Third, the mechanism by which DNMT1 regulates DNP has not yet been elucidated. DNMT1 has been shown to contribute to the genesis of neuropathic pain in injured DRGs, in part through repression of DRG Kcna2 gene expression [9]. Our previous study indicated that DNMT1 mediates chronic pain-related depression by inhibiting GABAergic neuronal activation in the central amygdala [39]. In the spinal cord, DNMT1 was shown top be primarily localized in neurons. Therefore, further research is needed to determine whether DNMT1 participates in the development of DNP by regulating spinal neuronal activation or by other mechanisms.

In summary, the present study demonstrated that downregulation of miR-152-3p in the spinal cord upregulates the expression of its downstream target gene, DNMT1, thereby participating in the development of DNP (Fig 6). The current study findings enhance our understanding of the pathogenesis of DNP and provides a potential therapeutic target for DNP.

Figure6 |

DNMT1 contributes to diabetic neuropathic pain mediated by miR-152-3p downregulation. Downregulation of miR-152-3p in the spinal cord is involved in the development of DNP by upregulating DNMT1.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

SUPPLEMENTARY DATA

Supplementary data related to this article can be retrieved online: https://npt-journal.org/wp-content/uploads/2024/11/Supplementary.pdf.

REFERENCES

1. Khan MAB, Hashim MJ, King JK, Govender RD, Mustafa H, Al Kaabi J. Epidemiology of type 2 diabetes – global burden of disease and forecasted trends. J Epidemiol Glob Health. 2020. Vol. 10(1):107–111

2. Sun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, et al.. IDF diabetes atlas: global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. Diabetes Res Clin Pract. 2022. Vol. 183:109119

3. Li C, Wang W, Ji Q, Ran X, Kuang H, Yu X, et al.. Prevalence of painful diabetic peripheral neuropathy in type 2 diabetes mellitus and diabetic peripheral neuropathy: a nationwide cross-sectional study in mainland China. Diabetes Res Clin Pract. 2023. Vol. 198:110602

4. Iqbal Z, Azmi S, Yadav R, Ferdousi M, Kumar M, Cuthbertson DJ, et al.. Diabetic peripheral neuropathy: epidemiology, diagnosis, and pharmacotherapy. Clin Ther. 2018. Vol. 40(6):828–849

5. Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology. 2013. Vol. 38(1):23–38

6. Jeltsch A. Molecular enzymology of mammalian DNA methyltransferases. Curr Top Microbiol Immunol. 2006. Vol. 301:203–225

7. Shao C, Gao Y, Jin D, Xu X, Tan S, Yu H, et al.. DNMT3a methylation in neuropathic pain. J Pain Res. 2017. Vol. 10:2253–2262

8. Zhao JY, Liang L, Gu X, Li Z, Wu S, Sun L, et al.. DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons. Nat Commun. 2017. Vol. 8:14712

9. Sun L, Gu X, Pan Z, Guo X, Liu J, Atianjoh FE, et al.. Contribution of DNMT1 to neuropathic pain genesis partially through epigenetically repressing Kcna2 in primary afferent neurons. J Neurosci. 2019. Vol. 39(33):6595–6607

10. Mohr AM, Mott JL. Overview of microRNA biology. Semin Liver Dis. 2015. Vol. 35(1):3–11

11. Pan Z, Shan Q, Gu P, Wang XM, Tai LW, Sun M, et al.. miRNA-23a/CXCR4 regulates neuropathic pain via directly targeting TXNIP/NLRP3 inflammasome axis. J Neuroinflammation. 2018. Vol. 15(1):29

12. Yao C, Ren J, Huang R, Tang C, Cheng Y, Lv Z, et al.. Transcriptome profiling of microRNAs reveals potential mechanisms of manual therapy alleviating neuropathic pain through microRNA-547-3p-mediated Map4k4/NF-kappab signaling pathway. J Neuroinflammation. 2022. Vol. 19(1):211

13. Tavares-Ferreira D, Lawless N, Bird EV, Atkins S, Collier D, Sher E, et al.. Correlation of miRNA expression with intensity of neuropathic pain in man. Mol Pain. 2019. Vol. 15:1744806919860323

14. Francés R, Mata-Garrido J, de la Fuente R, Carcelén M, Lafarga M, Berciano MT, et al.. Identification of epigenetic interactions between MicroRNA-30c-5p and DNA methyltransferases in neuropathic pain. Int J Mol Sci. 2022. Vol. 23(22):13994

15. Fuso A, Raia T, Orticello M, Lucarelli M. The complex interplay between DNA methylation and miRNAs in gene expression regulation. Biochimie. 2020. Vol. 173:12–16

16. Zhang TJ, Zhang LC, Xu ZJ, Zhou JD. Expression and prognosis analysis of DNMT family in acute myeloid leukemia. Aging (Albany NY). 2020. Vol. 12(14):14677–14690

17. Zhou C, Zhang Y, Jiao X, Wang G, Wang R, Wu Y. SIRT3 alleviates neuropathic pain by deacetylating FoxO3a in the spinal dorsal horn of diabetic model rats. Reg Anesth Pain Med. 2021. Vol. 46(1):49–56

18. Zhang Z, Ding X, Zhou Z, Qiu Z, Shi N, Zhou S, et al.. Sirtuin 1 alleviates diabetic neuropathic pain by regulating synaptic plasticity of spinal dorsal horn neurons. Pain. 2019. Vol. 160(5):1082–1092

19. Jiang W, Tan XY, Li JM, Yu P, Dong M. DNA Methylation: a target in neuropathic pain. Front Med (Lausanne). 2022. Vol. 9:879902

20. Wang Y, Liu C, Guo QL, Yan JQ, Zhu XY, Huang CS, et al.. Intrathecal 5-azacytidine inhibits global DNA methylation and methyl-CpG-binding protein 2 expression and alleviates neuropathic pain in rats following chronic constriction injury. Brain Res. 2011. Vol. 1418:64–69

21. Li X, Liu D, Dai Z, You Y, Chen Y, Lei C, et al.. Intraperitoneal 5-azacytidine alleviates nerve injury-induced pain in rats by modulating DNA methylation. Mol Neurobiol. 2023. Vol. 60(4):2186–2199

22. Mao Q, Wu S, Gu X, Du S, Mo K, Sun L, et al.. DNMT3a-triggered downregulation of K_2p 1.1 gene in primary sensory neurons contributes to paclitaxel-induced neuropathic pain. Int J Cancer. 2019. Vol. 145(8):2122–2134

23. Miao XR, Fan LC, Wu S, Mao Q, Li Z, Lutz B, et al.. DNMT3a contributes to the development and maintenance of bone cancer pain by silencing Kv1.2 expression in spinal cord dorsal horn. Mol Pain. 2017. Vol. 13:1744806917740681

24. Pollema-Mays SL, Centeno MV, Apkarian AV, Martina M. Expression of DNA methyltransferases in adult dorsal root ganglia is cell-type specific and up regulated in a rodent model of neuropathic pain. Front Cell Neurosci. 2014. Vol. 8:217

25. Zhou CH, Zhang MX, Zhou SS, Li H, Gao J, Du L, et al.. SIRT1 attenuates neuropathic pain by epigenetic regulation of mGluR1/5 expressions in type 2 diabetic rats. Pain. 2017. Vol. 158(1):130–139

26. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009. Vol. 19(1):92–105

27. Zhao YY, Wu ZJ, Zhu LJ, Niu TX, Liu B, Li J. Emerging roles of miRNAs in neuropathic pain: from new findings to novel mechanisms. Front Mol Neurosci. 2023. Vol. 16:1110975

28. Morchio M, Sher E, Collier DA, Lambert DW, Boissonade FM. The role of miRNAs in neuropathic pain. Biomedicines. 2023. Vol. 11(3):775

29. López-González MJ, Landry M, Favereaux A. MicroRNA and chronic pain: from mechanisms to therapeutic potential. Pharmacol Ther. 2017. Vol. 180:1–15

30. Liu P, Wu L, Chand H, Li C, Hu X, Li Y. Silencing of miR-152 contributes to DNMT1-mediated CpG methylation of the PTEN promoter in bladder cancer. Life Sci. 2020. Vol. 261:118311

31. Tao B, Xiang W, Li X, He C, Chen L, Xia X, et al.. Regulation of toll-like receptor-mediated inflammatory response by microRNA-152-3p-mediated demethylation of MyD88 in systemic lupus erythematosus. Inflamm Res. 2021. Vol. 70(3):285–296

32. Sun J, Tian X, Zhang J, Huang Y, Lin X, Chen L, et al.. Regulation of human glioma cell apoptosis and invasion by miR-152-3p through targeting DNMT1 and regulating NF2: MiR-152-3p regulate glioma cell apoptosis and invasion. J Exp Clin Cancer Res. 2017. Vol. 36(1):100

33. Xu SS, Ding JF, Shi P, Shi KH, Tao H. DNMT1-induced miR-152-3p suppression facilitates cardiac fibroblast activation in cardiac fibrosis. Cardiovasc Toxicol. 2021. Vol. 21(12):984–999

34. Wang C, Ma X, Zhang J, Jia X, Huang M. DNMT1 maintains the methylation of miR-152-3p to regulate TMSB10 expression, thereby affecting the biological characteristics of colorectal cancer cells. IUBMB Life. 2020. Vol. 72(11):2432–2443

35. Sun T, Tan L, Liu M, Zeng L, Zhao K, Cai Z, et al.. Tilianin improves cognition in a vascular dementia rodent model by targeting miR-193b-3p/CaM- and miR-152-3p/CaMKIIalpha-mediated inflammatory and apoptotic pathways. Front Immunol. 2023. Vol. 14:1118808

36. Zhang A, Qian Y, Qian J. MicroRNA-152-3p protects neurons from oxygen-glucose-deprivation/reoxygenation-induced injury through upregulation of Nrf2/ARE antioxidant signaling by targeting PSD-93. Biochem Biophys Res Commun. 2019. Vol. 517(1):69–76

37. Zhang B, Tao F, Liaw WJ, Bredt DS, Johns RA, Tao YX. Effect of knock down of spinal cord PSD-93/chapsin-110 on persistent pain induced by complete Freund’s adjuvant and peripheral nerve injury. Pain. 2003. Vol. 106(1-2):187–196

38. Ogawa A, Dai Y, Yamanaka H, Iwata K, Niwa H, Noguchi K. Ca(2+)/calmodulin-protein kinase IIalpha in the trigeminal subnucleus caudalis contributes to neuropathic pain following inferior alveolar nerve transection. Exp Neurol. 2005. Vol. 192(2):310–319

39. Ding X, Lin Y, Chen C, Yan B, Liu Q, Zheng H, et al.. DNMT1 mediates chronic pain-related depression by inhibiting GABAergic neuronal activation in the central amygdala. Biol Psychiatry. 2023. Vol. 94(8):672–684

Graphical abstract

Highlights

• The expression of DNMT1 is increased in the spinal neurons in diabetic neuropathic pain mice.

• miR-152-3p is involved in the development of diabetic neuropathic pain by targeting DNMT1.

In brief

The downregulation of miR-152-3p in the spinal cord is involved in the development of DNP by upregulating DNMT1.