2. miR-382-5p and miR-34a-5p in Migraine

There is mounting evidence that miR-382-5p and miR-34a-5p are dysregulated in migraine (Table 1 and Table 2).

Andersen et al. first detected the aberrant serum expression of 32 miRNAs in migraine patients in 2016 and identified a nine-fold increase in miR-34a-5p expression with a four-fold increase in miR-382-5p expression during the migraine attack when compared to the interictal phase ^[1][46]. Intriguingly, they also detected a significant higher miR-382-5p expression in migraine patients during the interictal period when compared to healthy subjects, and thus proposed this miRNA as a possible actor in migraine pathophysiology and a specific biomarker of the disease ^[1][46]. A main limitation of the study was that the migraine population was not well characterized, thus it was not possible to correlate the results obtained with the migraine phenotype (episodic or chronic) and with headache features.

A previously suggested hypothesis was that miR-382-5p serum expression was explained by increased blood–brain barrier permeability, which was suggested to occur during the migraine attack in a pre-clinical model ^[9][47], but this hypothesis has never been confirmed in humans and needs further confirmation.

Previous studies revealed that both miR-382-5p and miR-34a-5p negatively modulate target genes linked to GABAergic facilitating signaling, a neurotransmitter involved in trigeminal pain analgesia ^[1][46]. This may represent a protective compensatory mechanism in migraine self-limitation; indeed, higher GABA levels have been detected in the cerebrospinal fluid and in the saliva during migraine attacks ^[10][11][48,49].

miR-382-5p is also involved in Interleukin (IL)-10 anti-inflammatory signaling, where it acts via a negative modulation of the interleukin 10 receptor alpha subunit (IL-10RA), an endogenous inhibitor of pro-inflammatory IL-1β signaling ^[1][46].

The seminal work of Gallelli et al. paved the way for the identification of miRNAs as potential peripheral biological markers of drug response. They demonstrated lower miR-34a-5p and miR-375-5p expression in serum and saliva of children and adolescents treated with magnesium and acute nonsteroidal anti-inflammatory drugs (NSAIDs) compared to untreated subjects with migraine without aura ^[7][50]. It is noteworthy that in the first cohort of patients, the expression of these specific miRNAs was comparable to a group of healthy controls ^[7][50].

A recent study specifically analyzed miR-382-5p and miR-34a-5p expression in subjects with EM or CM and MOH (CM–MOH) ^[2][51]. The authors described a significantly higher interictal expression of miR-34a-5p and miR-382-5p in the plasma of CM–MOH subjects ^[2][51]. This analysis detected for the first time the association between specific miRNAs and the migraine phenotype as well as disease severity ^[2][51]. Notably, in this study CGRP plasma levels positively correlated with miR-382-5p and miR-34a-5p expression ^[2][51].

Although analyzed as a secondary endpoint, miR-382-5p and miR-34a-5p expression was reduced two months after detoxification from acute medications (in-hospital detoxification program), regardless of the clinical outcome ^[2][51]. This observation suggests the possibility of a direct role for acute medications in the expression of both miRNAs.

miR-382-5p and miR-34a-5p were also negatively modulated by erenumab, a monoclonal antibody directed against the CGRP receptor, in a population of difficult-to-treat CM patients with and without MOH ^[6][52]. The expression of each miRNA was reduced after three monthly s.c. administrations of erenumab, irrespective of the clinical outcome, which suggests a direct role for CGRP in miRNA regulation ^[6][52]. By contrast, central sensitization improved only in migraine patients who responded to erenumab treatment with an at least 30% reduction in monthly migraine days ^[6][52].

miR-34a expression was reported to be altered in several human diseases besides migraine, including osteoarthritis ^[12][13][53,54], Alzheimer’s disease ^[14][55], cancer ^[15][16][56,57] and leukemia ^[17][58]. The connection between miR-34a and these pathologies resides in its strong contribution to the regulation of cell cycles and apoptosis, with miR-34a being a major transcriptional target of p53 ^[18][19][59,60]. The involvement of miR-34a in several painful and non-painful conditions clearly suggests that, when considered alone, it cannot represent a migraine-specific biomarker. Nonetheless, it may still play a role as part of a multi-biomarker panel signature of migraine ^[20][7].

Regarding pain-related disorders, miR-34a plays a role in the modulation of the inflammatory response ^[21][22][61,62]. Upregulation of mir-34a has been reported in osteoarthritic tissues specimens and in patients with low back pain related to intervertebral disc degeneration ^[23][24][25][63,64,65]. miR-34a expression was enhanced by IL-1β release ^[12][53], although miR-34a itself can up-regulate the IL-1β/cyclooxygenase 2 (COX-2)/Prostaglandin E2 (PEG2) inflammation pathway ^[26][66] by targeting the deacetylase silencing information regulator 1 (SIRT1) ^[26][27][28][66,67,68]. miR-34a also inhibits SIRT1 by increasing the acetylation of the nuclear factor kappa B (NF-κB) P65 subunit, ultimately enhancing the transcription of inflammatory mediators, including IL-1β, IL-6 and tumor necrosis factor-α ^[27][29][67,69]. In an animal model of inflammatory pain, Chen et al. reported an increase in miR-34a expression in the spinal cord and showed that its inhibition by a miR-34a antagonist exerted analgesic effects by increasing spinal SIRT1 expression ^[28][68]. The inhibition of SIRT1 was reported to enhance the release of CGRP in rat trigeminal ganglia in vitro, probably as a consequence of IL-1β/COX-2/PGE2 pathway activation ^[26][66]. Other genes coding for proteins that are involved in chronic pain pathways have been validated as targets of miR-34a, such as sodium voltage-gated channel beta subunit 2 (Scn2b) and vesicle-associated membrane protein 2 (VAMP2) ^[30][70]. This latter belongs to the SNARE (soluble N-ethyl-maleimide-sensitive factor attachment protein receptor) protein complex that regulates presynaptic neurotransmitter release as well as neurotransmitter receptor trafficking and synaptic plasticity ^[31][71], whose variants have been reported to be linked with migraine susceptibility ^[32][72]. It must be noted that miR-34a was found to be down-regulated in rats’ dorsal root ganglions 12 days after the experimental induction of neuropathic pain, with the concomitant up-regulation of VAMP2 ^[30][70]. Since the increase of synaptic proteins is related to higher glutamate release ^[33][73], this may ultimately lead to an enhancement of nociception. Nonetheless, miR-34a was predicted to target the genes coding for specific GABA receptors subunits (GABBR2, Gamma-aminobutyric acid type B receptor subunit 2; GABRA3, Gamma-aminobutyric acid receptor subunit alpha-3) ^[1][46]. In this context, an up-regulation of this microRNA, as reported in serum of migraine patients, could contribute to the reduction of the GABA inhibitory signal ^[1][46]. Although some controversies exist, it is reasonable to assert that the role of miR-34a may vary across different pain disorders according to the time of evaluation and, more importantly, depending on specimen collection. However, together these observations strongly point towards the contribution of miR-34a to the regulation of pain-related neurotransmission.

Like miR-34a, miR-382-5p was predicted to target the GABRA5 (Gamma-aminobutyric acid receptor subunit alpha-5) GABA receptor subunit and also IL-10RA ^[1][46]. Thus, the observed up-regulation in the serum levels of miR-382-5p in migraine patients may be explained by reduced GABAergic and anti-inflammatory signaling ^[1][34][35][46,74,75]. The increased miR-382-5p serum levels in migraine patients may be also related to high protein levels of transforming growth factor β (TGF-β). In agreement with this hypothesis, increased TGF-β levels were found in the plasma and cerebrospinal fluid of patients with EM ^[36][37][76,77]. TGF-β is able to increase the expression of miR-382-5p ^[38][39][40][78,79,80] by inhibiting transcription factors’ repressors ^[41][81]. TGF-β modulated miR-382-5p in vitro (human CD34+ stem cells), resulting in the increased accumulation of reactive oxygen species (ROS). ROS accumulation was explained by miR-382-5p-induced inhibition of superoxide dismutase 2 (SOD2), a direct target of this mRNA ^[39][40][79,80]. This latter issue appears to be relevant in the context of migraine pain, since oxidative stress and mitochondrial dysfunction was suggested as actors in migraine pathophysiology ^[42][43][44][82,83,84]. miR-382-5p regulation is also influenced by the long non-coding RNA Ftx (lncRNA Ftx), which exerts its action as a competing endogenous RNA (ceRNA), competing with miR-382-5p at targets level ^[45][85]. An increase in miR-382-5p expression together with a down-regulation of lncRNA Ftx and neuregulin-1 (Nrg-1) was reported in an animal model of neuropathic pain ^[45][85]. The lack of inhibition exerted by lncRNA Ftx on miR-382, is supposed to increase the inhibition of Nrg-1, thus facilitating pain and inflammation ^[45][85]. The involvement of Nrg-1 in pain was also reported in an animal model of inflammatory pain. Its reduction has been found in the dorsal root ganglia and spinal cord of mice, contributing to hypersensitivity ^[46][86]. Notably, increasing Nrg-1 expression facilitates analgesia and tissue repair by modulating glial cells proliferation, central nervous system injury repair and the reduction of the release of pro-inflammatory cytokine ^{[45][47][48][49]}[85,87,88,89]. Increased miR-382-5p expression in pain conditions may also be related to nuclear factor kappa B (NF-κB) signaling, implicated in migraine pain ^[50][51][52][90,91,92]. NF-κB acts as a transcription factor for many miRNAs ^[3][93], including miR-382 ^[4][94]. Indeed, the inhibition of NF-κB significantly suppressed miR-382 in vitro ^[4][94].

These observations suggest that the activation of the inflammatory pathways may lead to an up-regulation of miR-34a and miR-382 which in turn modulate the release of inflammatory and pain mediators (Figure 1).