Epigenetic control in opioid receptor-dependent analgesia: role of miRNA and DNA methylation on the crotalphine antinociceptive effect

• Grant number: 16/22774-6
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Effective date (Start): May 01, 2017
• Effective date (End): July 31, 2019
• Field of knowledge: Biological Sciences - Pharmacology - General Pharmacology
• Principal Investigator: Yara Cury
• Grantee: Flávia Viana Santa Cecília
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated research grant: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling, AP.CEPID

Abstract

It is estimated that between 10-25% of the world population suffers from persistent or chronic pain. Opioids are widely used in the treatment of pain of different origins, however, these drugs are not effective for all types of pain, in addition to having adverse effects, including, tolerance, dependence and delayed hyperalgesia. Thus, the expansion of knowledge about the cellular and molecular mechanisms involved in pain and analgesia, as well as the development of new analgesic drugs with high efficacy and low toxicity, are needed. The crotalphine, a peptide first identified and isolated from the venom of the snake Crotalus durissus terrificus, has potent antinociceptive effect of long duration. This effect is most prominent in the presence of previous sensitization and involves the activation of CB2 cannabinoid receptors and type kappa and delta opioid receptors, with consequent opening of K + channels sensitive to ATP. Recent studies have showed that epigenetic mechanisms are involved in the pain and its control, including inflammatory and neuropathic pain. Among the molecular processes involved in the epigenetic changes, there is the control of gene expression by microRNAs (miRNAs) and by DNA methylation. miRNAs are small sequences of no-coding RNAs endogenously expressed and exert inhibition of protein translation of specific mRNAs. The epigenetic regulation of the opioid system - including opioid receptors, as well as precursors of endogenous opioid peptides - by DNA methylation, chromatin remodeling and miRNA, it has also been demonstrated. It is worth noting that studies on epigenetic regulation of opioids are focused, for the most part, the opioid receptors of the type ¼. The role of DNA methylation in the promoter regions of kappa and delta opioid receptors, is not yet fully elucidated. This project aims to assess the epigenetic control in the antinociceptive effect induced by crotalphine, in a hyperalgesia model induced by prostaglandin E2 (PGE2) in rats using morphine, dynorphin A and selective agonists of opioid receptors, as positive controls. In in vitro studies, there will be used dorsal root ganglia of rats. There will be assessed: 1) miRNAs expression patterns in DRGs, by RT-qPCR using Taqman probes; 2) changes in the expression of a panel of mRNAs known to be associated with inflammatory pain by RT-qPCR arrays, as well as the profile of co-expression of miRNAs and mRNAs, in order to identify miRNAs that interfere with gene expression, and 3) changes in global pattern of DNA methylation in repetitive DNA sequences of the type LINE-1 (Long Interspersed Elements) and on the promoter regions of opioid receptors.The results of this study will: 1) clarify patterns of mRNAs and miRNAs expression during hyperalgesia induced by PGE2, as well as identify possible miRNAs participants of this process, from bioinformatics prediction and subsequent confirmed by functional studies (overexpression and silecing); 2) expanding the understanding of post-transcriptional control of opioid receptors in antinociception entailed by crotalphine; 3) to identify DNA methylation patterns, global and on promoter sequences of opioid receptors, as important new targets for controlling the expression of these receptors. (AU)