Whole Genomic DNA Methylation Profiling of CpG Sites in Promoter Regions of Dorsal Root Ganglion in Diabetic Neuropathic Pain Mice

Abstract

DNA methylation and demethylation play an important role in neuropathic pain. In general, DNA methylation of CpG sites in the promoter region impedes gene expression, whereas DNA demethylation contributes to gene expression. Here, we evaluated the methylation status of CpG sites in genomic DNA promoter regions in dorsal root ganglions (DRGs) of diabetic neuropathic pain (DNP) mice. In our research, streptozotocin (STZ) was intraperitoneally injected into mice to construct DNP models. The DNP mice showed higher fasting blood glucose (above 11.1 mmol/L), lower body weight, and mechanical allodynia than control mice. Whole-genome bisulfite sequencing (WGBS) revealed an altered methylation pattern in CpG sites in the DNA promoter regions in DRGs of DNP mice. The results showed 376 promoter regions with hypermethylated CpG sites and 336 promoter regions with hypomethylated CpG sites. In addition, our data indicated that altered DNA methylation occurs primarily on CpG sites in DNA promoter regions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that differentially methylated CpG sites annotated genes were involved in activities of the nervous and sensory systems. Enrichment analysis indicated that genes in these pathways contributed to diabetes or pain. In conclusion, our study enriched the role of DNA methylation in DNP.

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Data Availability

All data and materials described in the study are available from the corresponding author on reasonable request.

Abbreviations

DNP:

Diabetic neuropathic pain

DRG:

Dorsal root ganglion

KEGG:

Kyoto Encyclopedia of Genes and Genomes

P2X3R:

Purinergic P2X ligand-gated ion channel 3 receptor

PWT:

Paw withdrawal threshold

STZ:

Streptozotocin

WGBS:

Whole-genome bisulfite sequencing

References

  1. Chaplan SR, Bach FW, Pogrel JW, Chung JW, Yaksh TL (1994) Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53(1):55–63. https://doi.org/10.1016/0165-0270(94)90144-9

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Chen W, Chi YN, Kang XJ, Liu QY, Zhang HL, Li ZH, Zhao ZF, Yang Y, Su L, Cai J, Liao FF, Yi M, Wan Y, Liu FY (2018) Accumulation of CaV3.2 T-type calcium channels in the uninjured sural nerve contributes to neuropathic pain in rats with spared nerve injury. Front Mol Neurosci 11: 24. https://doi.org/10.3389/fnmol.2018.00024

  3. Cheng C, Kobayashi M, Martinez JA, Ng H, Moser JJ, Wang XL, Singh V, Pharm M, Fritzler MJ, Zochodne DW (2015) Evidence for epigenetic regulation of gene expression and function in chronic experimental diabetic neuropathy. J Neuropathol Exp Neurol 74(8):804–817. https://doi.org/10.1097/NEN.0000000000000219

    CAS  Article  PubMed  Google Scholar 

  4. Chidambaran V, Zhang X, Martin LJ, Ding L, Weirauch MT, Geisler K, Stubbeman BL, Sadhasivam S, Ji H (2017) DNA methylation at the mu-1 opioid receptor gene (OPRM1) promoter predicts preoperative, acute, and chronic postsurgical pain after spine fusion. Pharmgenomics Pers Med 10:157–168. https://doi.org/10.2147/PGPM.S132691

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Ciccacci C, Latini A, Colantuono A, Politi C, D’Amato C, Greco C, Rinaldi ME, Lauro D, Novelli G, Spallone V, Borgiani P (2020) Expression study of candidate miRNAs and evaluation of their potential use as biomarkers of diabetic neuropathy. Epigenomics 12(7):575–585. https://doi.org/10.2217/epi-2019-0242

    CAS  Article  PubMed  Google Scholar 

  6. Ehrlich M (2019) DNA hypermethylation in disease: mechanisms and clinical relevance. Epigenetics 14(12):1141–1163. https://doi.org/10.1080/15592294.2019.1638701

    Article  PubMed  PubMed Central  Google Scholar 

  7. Farooqui AA, Horrocks LA (2001) Plasmalogens: workhorse lipids of membranes in normal and injured neurons and glia. Neuroscientist 7(3):232–245. https://doi.org/10.1177/107385840100700308

    CAS  Article  PubMed  Google Scholar 

  8. Feinmann J (2016) Advice on sugar and starch is urged in type 2 diabetes counselling. BMJ 355:i6543. https://doi.org/10.1136/bmj.i6543

    Article  PubMed  Google Scholar 

  9. Feldman EL, Nave KA, Jensen TS, Bennett DLH (2017) New horizons in diabetic neuropathy: mechanisms, bioenergetics, and pain. Neuron 93(6):1296–1313. https://doi.org/10.1016/j.neuron.2017.02.005

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Frisardi V, Panza F, Seripa D, Farooqui T, Farooqui AA (2011) Glycerophospholipids and glycerophospholipid-derived lipid mediators: a complex meshwork in Alzheimer’s disease pathology. Prog Lipid Res 50(4):313–330. https://doi.org/10.1016/j.plipres.2011.06.001

    CAS  Article  PubMed  Google Scholar 

  11. Garriga J, Laumet G, Chen SR, Zhang Y, Madzo J, Issa JPJ, Pan HL, Jelinek J (2018) Nerve injury-induced chronic pain is associated with persistent DNA methylation reprogramming in dorsal root ganglion. J Neurosci 38(27):6090–6101. https://doi.org/10.1523/JNEUROSCI.2616-17.2018

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Gombert S, Rhein M, Winterpacht A, Münster T, Hillemacher T, Leffler A, Frieling H (2020) Transient receptor potential ankyrin 1 promoter methylation and peripheral pain sensitivity in Crohn’s disease. Clin Epigenetics 12(1):1. https://doi.org/10.1186/s13148-019-0796-9

    CAS  Article  Google Scholar 

  13. Guo K, Elzinga S, Eid S, Figueroa-Romero C, Hinder LM, Pacut C, Feldman EL, Hur J (2019) Genome-wide DNA methylation profiling of human diabetic peripheral neuropathy in subjects with type 2 diabetes mellitus. Epigenetics 14(8):766–779. https://doi.org/10.1080/15592294.2019.1615352

    Article  PubMed  PubMed Central  Google Scholar 

  14. He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, Ding J, Jia Y, Chen Z, Li L, Sun Y, Li X, Dai Q, Song CX, Zhang K, He C, Xu GL (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333(6047):1303–1307. https://doi.org/10.1126/science.1210944

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Iwasa T, Afroz S, Inoue M, Arakaki R, Oshima M, Raju R, Waskitho A, Inoue M, Baba O, Matsuka Y (2019) IL-10 and CXCL2 in trigeminal ganglia in neuropathic pain. Neurosci Lett 703:132–138. https://doi.org/10.1016/j.neulet.2019.03.031

    CAS  Article  PubMed  Google Scholar 

  16. Jolivalt CG, Frizzi KE, Guernsey L, Marquez A, Ochoa J, Rodriguez M, Calcutt NA (2016) Peripheral neuropathy in mouse models of diabetes. Curr Protoc Mouse Biol 6(3):223–255. https://doi.org/10.1002/cpmo.11

    Article  PubMed  PubMed Central  Google Scholar 

  17. Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13(7):484–492. https://doi.org/10.1038/nrg3230

    CAS  Article  PubMed  Google Scholar 

  18. Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28(1):27–30. https://doi.org/10.1093/nar/28.1.27

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Khan J, Ramadan K, Korczeniewska O, Anwer MM, Benoliel R, Eliav E (2015) Interleukin-10 levels in rat models of nerve damage and neuropathic pain. Neurosci Lett 592:99–106. https://doi.org/10.1016/j.neulet.2015.03.001

    CAS  Article  PubMed  Google Scholar 

  20. Kohli RM, Zhang Y (2013) TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502(7472):472–479. https://doi.org/10.1038/nature12750

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Li E, Zhang Y (2014) DNA methylation in mammals. Cold Spring Harb Perspect Biol 6(5):a019133. https://doi.org/10.1101/cshperspect.a019133

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Liu C, Jiao C, Wang K, Yuan N (2018a) DNA methylation and psychiatric disorders. Prog Mol Biol Transl Sci 157:175–232. https://doi.org/10.1016/bs.pmbts.2018.01.006

    CAS  Article  PubMed  Google Scholar 

  23. Liu CL, Li CC, Deng ZY, Du E, Xu CS (2018b) Long non-coding RNA BC168687 is involved in TRPV1-mediated diabetic neuropathic pain in rats. Neuroscience 374:214–222. https://doi.org/10.1016/j.neuroscience.2018.01.049

    CAS  Article  PubMed  Google Scholar 

  24. Luo C, Hajkova P, Ecker JR (2018) Dynamic DNA methylation: in the right place at the right time. Science 361(6409):1336–1340. https://doi.org/10.1126/science.aat6806

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Martins DF, Turnes BL, Cidral-Filho FJ, Bobinski F, Rosas RF, Danielski LG, Petronilho F, Santos ARS (2016) Light-emitting diode therapy reduces persistent inflammatory pain: Role of interleukin 10 and antioxidant enzymes. Neuroscience 324:485–495. https://doi.org/10.1016/j.neuroscience.2016.03.035

    CAS  Article  PubMed  Google Scholar 

  26. Moore LD, Le T, Fan G (2013) DNA methylation and its basic function. Neuropsychopharmacology 38(1):23–38. https://doi.org/10.1038/npp.2012.112

    CAS  Article  PubMed  Google Scholar 

  27. Morgan AE, Davies TJ, Mc Auley MT (2018) The role of DNA methylation in ageing and cancer. Proc Nutr Soc 77(4):412–422. https://doi.org/10.1017/S0029665118000150

    CAS  Article  PubMed  Google Scholar 

  28. Rauscher GH, Kresovich JK, Poulin M, Yan L, Macias V, Mahmoud AM, Al-Alem U, Kajdacsy-Balla A, Wiley EL, Tonetti D, Ehrlich M (2015) Exploring DNA methylation changes in promoter, intragenic, and intergenic regions as early and late events in breast cancer formation. BMC Cancer 15:816. https://doi.org/10.1186/s12885-015-1777-9

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Rideout WM III, Coetzee GA, Olumi AF, Jones PA (1990) 5-methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science 249(4974):1288–1290. https://doi.org/10.1126/science.1697983

    CAS  Article  PubMed  Google Scholar 

  30. Rosenberger DC, Blechschmidt V, Timmerman H, Wolff A, Treede RD (2020) Challenges of neuropathic pain: focus on diabetic neuropathy. J Neural Transm (Vienna) 127(4):589–624. https://doi.org/10.1007/s00702-020-02145-7

    Article  Google Scholar 

  31. Shillo P, Sloan G, Greig M, Hunt L, Selvarajah D, Elliott J, Gandhi R, Wilkinson ID, Tesfaye S (2019) Painful and painless diabetic neuropathies: what is the difference? Curr Diab Rep 19(6):32. https://doi.org/10.1007/s11892-019-1150-5

    Article  PubMed  PubMed Central  Google Scholar 

  32. Sloan G, Shillo P, Selvarajah D, Wu J, Wilkinson ID, Tracey I, Anand P, Tesfaye S (2018) A new look at painful diabetic neuropathy. Diabetes Res Clin Pract 144:177–191. https://doi.org/10.1016/j.diabres.2018.08.020

    Article  PubMed  Google Scholar 

  33. Sun Y, Sahbaie P, Liang DY, Li W, Shi X, Kingery P, Clark JD (2015) DNA methylation modulates nociceptive sensitization after incision. PLoS ONE 10(11):e0142046. https://doi.org/10.1371/journal.pone.0142046

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Zhang L, Ma Z, Wu Z, Jin M, An L, Xue F (2020) Curcumin improves chronic pain induced depression through regulating serum metabolomics in a rat model of trigeminal neuralgia. J Pain Res 13:3479–3492. https://doi.org/10.2147/JPR.S283782

    Article  PubMed  PubMed Central  Google Scholar 

  35. Zhang HH, Hu J, Zhou YL, Qin X, Song ZY, Yang PP, Hu S, Jiang X, Xu GY (2015) Promoted interaction of nuclear factor-kB with demethylated purinergic P2X3 receptor gene contributes to neuropathic pain in rats with diabetes. Diabetes 64(12):4272–4784. https://doi.org/10.2337/db15-0138

    CAS  Article  PubMed  Google Scholar 

  36. Zheng J, Jiang YY, Xu LC, Ma LY, Liu FY, Cui S, Cai J, Liao FF, Wan Y, Yi M (2017) Adult hippocampal neurogenesis along the dorsoventral axis contributes differentially to environmental enrichment combined with voluntary exercise in alleviating chronic inflammatory pain in mice. J Neurosci 37(15):4145–4157. https://doi.org/10.1523/JNEUROSCI.3333-16.2017

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Zheng J, Wang Y, Han S, Luo Y, Sun X, Zhu N, Zhao L, Li J (2018) Identification of protein kinase C isoforms involved in type 1 diabetic encephalopathy in mice. J Diabetes Res 2018:8431249. https://doi.org/10.1155/2018/8431249

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by the National Natural Science Foundation of China (grant no. 81971037 to F. Yang, 81870865 to W. Cui), the Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ201910025026 to F. Yang), Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five–year Plan (CIT&TCD201904092 to Q. Li), and Beijing Postdoctoral Research Foundation (grant no. ZZ 2019–01 to W. Chen).

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All authors contributed to the study conception and design. Wen Chen, Fei Yang, and Weihua Cui prepared the manuscript. Wen Chen, Ting Lan, and Qingyu Sun conducted most of the experiments. Yurui Zhang, Danmin Shen, Tingting Hu, Jing Liu, Yingzi Chong, Peipei Wang, and Qian Li analyzed the data and commented on previous publications. All authors approved the final manuscript.

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Correspondence to Fei Yang.

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All animal procedures were approved by the experimental animal ethics committee of Capital Medical University and were performed in accordance with the National Institute of Health Guidelines for the Treatment Animals. The manuscript does not contain any study on human participants.

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Chen, W., Lan, T., Sun, Q. et al. Whole Genomic DNA Methylation Profiling of CpG Sites in Promoter Regions of Dorsal Root Ganglion in Diabetic Neuropathic Pain Mice. J Mol Neurosci (2021). https://doi.org/10.1007/s12031-021-01847-1

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Keywords

  • Diabetic neuropathic pain
  • DNA methylation
  • Dorsal root ganglion
  • CpG