miRNA-711 Binds and Activates TRPA1 Extracellularly to Evoke Acute and Chronic Pruritus.

The conventional role of microRNAs (miRNAs) is to regulate gene expression by binding to the 3’ untranslated region of messenger RNA (mRNA) by ~7 base pair seed sequence complementarity. This intracellular role of miRNAs can negatively impact gene expression either by mRNA degradation or translational repression. Though much of the emphasis in identifying circulating miRNAs associated with disease states has been on their utility as biomarkers, investigating the function of these miRNAs can unravel the molecular mechanisms underlying a disorder. In an interesting study by Han et al., Dr. Ru-Rong Ji’s group elucidates a novel role for a circulating miRNA, miR-711, in driving acute and chronic itch by binding extracellularly to TRPA1 ion channels on pruriceptive neurons, thus demonstrating a novel function for small noncoding RNA. They demonstrate that inhibition of miR-711, or blocking peptide-induced disruption of miR-711 and TRPA1 interaction, can alleviate chronic itch.

Patients with cutaneous T cell lymphoma (CTCL) suffer from severe itching, and skin biopsies have shown upregulation of miR-21, miR-155, miR-326, and miR-711. Among the several novel aspects reported in the study is the establishment of a novel translationally relevant rodent model of CTCL by intradermal inoculation of human Myla cells. The mice developed lymphoma and chronic itch. There was also an increase in hsa-miR-711 in the serum that was released by the human cancer cells. Though both the human and mouse miR-711 have identical seed sequences, the differences in adjacent nucleotides enable quantitative real-time RT-PCR (qPCR) to differentiate mouse- and human-specific miRNAs.

The authors first sought to determine the role of four different miRNAs upregulated in CTCL skin biopsies in mediating itch and pain using a cheek model. This model can distinguish pain (wiping) and itch (scratching) (Shimada and LaMotte, 2008). They found that intradermal injection of miR-711 induced scratching but not wiping. The onset of miR-711-induced pruritus was rapid, and the latency was shorter than the classic pruritogens chloroquine and histamine, suggesting that miRNA may trigger pruritus through different mechanisms.

TRP channels regulate acute and chronic pruritus and are expressed by pruriceptive and nociceptive DRG neurons. The authors investigated if itch from miR-711 is mediated by the TRP channels TRPA1 and TRPV1. Using knockout mice, they confirmed that miR-711 evokes itch via TRPA1 but not TRPV1. Most known agonists of TRPA1 covalently bind cysteine residues at the intracellular C-terminal. Typical TRPA1 agonists are pungent natural compounds such as allyl isothiocyanate (AITC), eliciting pain after intradermal injection (Ross, 2011).

Patch-clamp recordings showed miR-711 can activate TRPA1 and is more potent than AITC. Inside-out patch recordings in Trpa1-expressing HEK293 cells showed that bath application of AITC, but not miR-711, on the intracellular surface elicited single-channel opening events. Thus, unlike AITC, miR-711 activates TRPA1 on the extracellular side. Calcium imaging data indicate that miR-711 activates a subset of TRPA1+ sensory neurons in mice, but 10 μM miR-711 did not alter calcium currents in DRG neurons. The translational impact of this study was demonstrated using human-specific hsa-miR-711, which evoked TRPA1-dependent inward currents in human DRG neurons.

Since miR-711 was activating the channel extracellularly, the authors used computational modeling to predict binding of the miR-711 core sequence GGGACCC to TRPA1. A series of mutation studies showed that extracellular residues of TRPA1 can interact with the miRNA core sequence to regulate TRPA1 activation. Using blocking peptides, the authors demonstrate that this interaction was required to elicit itch. Employing several pharmacological and genetic approaches to target miR-711, the authors also demonstrate that an miR-711 inhibitor, TRPA1 antagonists, and an miR-711/TRPA1 interaction-blocking peptide attenuate chronic itch in the mouse model of CTCL.

The only other miRNA reported to have an unconventional role in extracellular signaling is let-7b, which was shown to bind the Toll-like receptor, TLR7, in cortical neurons, leading to neurodegeneration (Lehmann et al., 2012) and neuronal excitation in nociceptive neurons (Park et al., 2014). miR-711-induced pruritus was unaltered in Tlr7-/- mice, indicating a TLR7-independent action of miRNA. Thus, the two extracellular miRNAs induce distinct sensory responses: pruritus by miRNA-711 but nociception by let-7b.

TRPA1 mediates both pain and itch, and the activation of the same receptor triggers two different sensory responses. The authors postulate that rapid and transient activation of TRPA1 from the extracellular side in a subset of TRPA1+ neurons by miR-711 can cause itch, whereas slow and persistent activation of TRPA1 from the intracellular side by AITC results in pain. Differences in TRPA1 activation may underlie distinct sensory behaviors resulting from different downstream signaling.

RNAs can function as ligands for specific receptors. For example, TLR7 and TLR8 recognize single-stranded RNAs as their natural ligand. It is interesting to consider the dual function of miRNAs as a negative regulator of gene expression and as a ligand for the receptor, albeit at much different concentrations, and whether the two functions of the miRNA evolved independent of each other. This study raises the question whether there is a localized increase in miR-711 release near TRPA1 receptors at a concentration high enough to activate the channel, though this is difficult to measure in vivo. It is possible that miRNA release from cancer cells could activate nearby nerve terminals, since lymphoma is innervated by nerve fibers. miR-711 could also be transported at a higher concentration by being packaged into exosomes. It is also not known if the functional sequence of miR-711 is present in itch-inducing agents, including microbes.

In summary, the data presented in the current study demonstrate a previously unknown role for miR-711 as a mediator of itch. The findings have broad clinical implications for treatment of several dermatologic diseases. Targeting extracellular miR-711 using a specific inhibitor with a complementary sequence, or disrupting the miR-711/TRPA1 interaction with a blocking peptide, can be strategies that can be pursued to alleviate chronic itch.