MICRORNA SIGNATURES IN PATIENTS WITH SMALL FIBER NEUROPATHY, WITH OR WITHOUT REDUCED INTRAEPIDERMAL NERVE FIBER DENSITY

A. Gioia¹, M. Marchi², B. Fontana³, A. Strippoli², R. Roncarati⁴, G. Lauria⁵, P. Forget⁶, G. Devigili⁷, M. Ferracin⁸ | ¹Pain Medicine Unit, Ospedale Bellaria, Azienda USL di Bologna, Italy; ²Neuroalgology Unit, Fondazione IRCCS Istituto Neurologico "Carlo Besta", Milan, Italy; ³Department of Medical and Surgical Sciences (DIMEC), University of Bologna, Italy; ⁴Department of Medical and Surgical Sciences (DIMEC), University of Bologna; CNR Institute of Molecular Genetics "Luigi Luca Cavalli-Sforza", Unit of Bologna, Italy; ⁵Neuroalgology Unit, Fondazione IRCCS Istituto Neurologico "Carlo Besta", Milan, Department of Medical Biotechnology and Translational Medicine, University of Milan, Italy; ⁶Aberdeen Centre for Arthritis and Musculoskeletal Health (Epidemiology Group), Institute of Applied Health Sciences, School of Medicine, Medical Sciences and Nutrition, Aberdeen, UK; ⁷Neurophysiological Unit, Parkinson Disease and Movement disorder Unit, Fondazione IRCCS Istituto Neurologico "Carlo Besta", Milan, Italy; ⁸Department of Medical and Surgical Sciences (DIMEC), University of Bologna, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Italy

Introduction. The pathophysiology of neuropathic pain remains only partially understood. There is a growing interest about altered gene and protein expression underlying this pathologic entity. (1) Small fiber neuropathy (SFN) is a neuropathic pain condition characterized by selective impairment of small-diameter A-delta and C-fibers. Diagnosis relies on clinical characteristics and assessment of intraepidermal nerve fiber density (IENFD). (2) MicroRNAs (miRNAs), non-coding regulatory RNAs, have emerged as biological regulators modulating gene expression related to pain sensitization. (3) The aim of this study is to investigate the expression a subset of miRNA as part of the pathomechanism underlying chronic pain in patients with SFN, and to correlate their abundance with the IENFD, in order to identify molecular signatures associated to these characteristics.
Methods. A literature review was conducted to establish scientific rationale for investigation of altered miRNA expression in painful polyneuropathy patients, identifying candidate for expression analysis: miR-146a-5p, miR-155-5p, miR-203a-3p, and miR-21-5p. For the quantitative analysis we selected 16 patients with a diagnosis of SFN: 8 of them showing denervated pattern (“Den”) at skin biopsy and 8, age and sex-matched, with normal (“Norm”) innervation pattern, all recruited at Fondazione IRCCS Istituto Neurologico C. Besta of Milan, Italy. Total RNA was isolated from the epidermis of two 50-um sections per subject, after tissue dissecting under the microscope, using PureLink FFPE Total RNA Isolation Kit (Invitrogen). (4) All RNA samples achieved adequate purity ratios (A260/A280 = 1.4–2.4) and concentration (1.6-6.6 ng/uL). The expression levels of four selected miRNAs were quantified using Digital Droplet PCR (ddPCR) and the values obtained were normalized with ubiquitarian and invariant non-coding RNA sequences SNORD44 and SNORD48 as endogenous controls. (8) Statistical analysis has been conducted with Kolmogorov-Smirnov testing, Mann-Whitney U tests for group comparisons, and Spearman's correlation for reference endogenous control assessment, with significance set at p<0.05. Results are expressed as medians with quartiles for non-parametric data.
Results. “Den” cohort showed significantly higher expression of all four investigated microRNAs compared to “Norm” group (Figure 1): miR-155-5p showed the most significant elevation (p=0.0030), however also miR-146a-5p, miR-203a-3p and miR-21-5p showed significant upregulation (p=0.0092, p=0.0499, p=0.0207 respectively). The expression patterns of these miRNAs were consistent with trends reported in rodent models of nerve injury and experimental neuropathy, with exception for miR-203a-3p.
Conclusions. This study demonstrated that patients diagnosed with SFN and exhibiting signs of denervation presented significantly elevated expression levels of miR-146a-5p, miR-155-5p, miR-203a-3p, and miR-21-5p compared with patients without denervation. These miRNAs are implicated in responses to nerve injury, neuroinflammation, and immune activation. In peripheral neuropathy, they may regulate axonal regeneration, Schwann cell activity, and pathways such as NF-κB, contributing to cytokine production and nociceptor sensitization. The findings provide translational evidence in humans, as most data supporting a role for these miRNAs derive from animal models, and support their potential as biomarkers of ongoing nerve damage and inflammatory activity.