Identification of Epigenetic Interactions between MicroRNA-30c-5p and DNA Methyltransferases in Neuropathic Pain

Neuropathic pain is a prevalent and severe chronic syndrome, often refractory to treatment, whose development and maintenance may involve epigenetic mechanisms. We previously demonstrated a causal relationship between miR-30c-5p upregulation in nociception-related neural structures and neuropathic pain in rats subjected to sciatic nerve injury. Furthermore, a short course of an miR-30c-5p inhibitor administered into the cisterna magna exerts long-lasting antiallodynic effects via a TGF-?1-mediated mechanism. Herein, we show that miR-30c-5p inhibition leads to global DNA hyper-methylation of neurons in the lumbar dorsal root ganglia and spinal dorsal horn in rats subjected to sciatic nerve injury. Specifically, the inhibition of miR-30-5p significantly increased the expression of the novo DNA methyltransferases DNMT3a and DNMT3b in those structures. Furthermore, we identified the mechanism and found that miR-30c-5p targets the mRNAs of DNMT3a and DNMT3b. Quantitative methylation analysis revealed that the promoter region of the antiallodynic cytokine TGF-?1 was hypomethylated in the spinal dorsal horn of nerve-injured rats treated with the miR-30c-5p inhibitor, while the promoter of Nfyc, the host gene of miR-30c-5p, was hypermethylated. These results are consistent with long-term protection against neuropathic pain development after nerve injury. Altogether, our results highlight the key role of miR-30c-5p in the epigenetic mechanisms' underlying neuropathic pain and provide the basis for miR-30c-5p as a therapeutic target.Funding: This work was supported by: Grant SAF2016-77732-R funded by MCIN/AEI/10.13039/ 501100011033 and by “ERDF A way of making Europe”; Grant PID2019-104398RB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and Instituto de Investigación Sanitaria Valdecilla (IDIVAL) (NVAL17/23). R.F. was the recipient of a pre-doctoral fellowship of the Foundation Tatiana Pérez de Guzmán el Bueno. Acknowledgments: We acknowledge the excellent technical assistance of Nieves García Iglesias

Mechanisms involved in the analgesic effect of the silencing of miR-30c-5p in an experimental model of neuropathic pain

RESUMEN: El dolor neuropático es una patología prevalente y debilitante, con una carga clínica y económica grave para los pacientes y para el sistema de salud. Profundizar en los mecanismos moleculares responsables del desarrollo y cronificación del dolor neuropático es fundamental para el diseño de nuevos tratamientos eficaces. Estudios previos del laboratorio, sugirieron la contribución relevante de miR-30c-5p al desarrollo del dolor neuropático. Los estudios farmacológicos mostraron que el tratamiento con un inhibidor sintético de miR-30c-5p previene y revierte el desarrollo de dolor neuropático en ratas. En esta tesis se han determinado los mecanismos moleculares responsables del efecto analgésico del inhibidor de miR-30c-5p en el animal de experimentación. Los presentes resultados muestran la implicación de la señalización por TGF-β en el efecto analgésico del inhibidor de miR-30c-5p. Además, hemos mostrado que, la relación bidireccional existente entre miR-30c-5p y TGF-β regula la actividad del sistema opioide endógeno. En un segundo objetivo, hemos profundizado en las fuentes celulares y el tráfico de miR-30c-5p. Nuestros resultados muestran que las secuencias de nucleótidos de miRNAs pueden transitar a través del líquido extracelular neural, el líquido cefalorraquídeo y el torrente sanguíneo. Finalmente, hemos diseñado mecanismos para una terapia dirigida y de precisión.ABSTRACT: Neuropathic pain is a prevalent debilitating disease with a severe clinical and economic burden for patients and the health system. The understanding of the mechanisms responsible for neuropathic pain is essential to design new effective treatments. Previous studies from our laboratory suggested a relevant contribution of miR-30c-5p to neuropathic pain development. The pharmacological studies showed that treatment with a synthetic inhibitor of miR-30c-5p prevents and reverses the development of neuropathic pain in rats. In this thesis, we determined the molecular mechanisms responsible for the analgesic effect of miR-30c-5p inhibitor in the experimental animal. The present results demonstrate that the analgesic effect of the miR-30c-5p inhibitor involves TGF-β-mediated signaling. Moreover, we demonstrate that the cross-talk between miR-30c-5p and TGF-β signaling regulates the activity of the endogenous opioid system. In a second objective, we deepened the cell sources and traffic of miR-30c-5p. Our results demonstrate that miRNA nucleotide sequences can traffic multi-directionally through the neural extracellular fluid, cerebrospinal fluid, and bloodstream. Finally, we designed mechanisms to get targeted and precise therapy.Esta tesis doctoral ha sido financiada con ayudas a la investigación procedentes de: ayuda predoctoral en el área de la Biomedicina, Biotecnología y Ciencias de la Salud del Instituto de Investigación sanitaria Marqués de Valdecilla; proyecto INN-VAL19/08 del Instituto de Investigación sanitaria Marqués de Valdecilla; Ministerio de Economía y Competitividad a través de los proyectos del Plan Estatal: SAF2013-47434-R y SAF02016-77732-R; proyecto BF2-19-20 de la fundación española del dolor