Researchers from Duke University this week reported new data suggesting that an extracellular microRNA can mediate pain by interacting with a member of the Toll-like receptor (TLR) family of immune response proteins.
To Duke investigator and senior author of the study Ru-Rong Ji, the data suggest that this miRNA could potentially be used as a biomarker for pain — a condition that can be difficult to quantify — and even as a therapeutic target, although he cautioned that the findings are preliminary.
In 2010, Ji and colleagues reported on the discovery that TLR7 is expressed in dorsal root ganglion (DRG) sensory neurons and played a role in inducing itch sensations in mice. Other work, meanwhile, has demonstrated that TLR7 is involved in triggering pain and itch, and that its administration to mice causes itching.
Given that endogenous ligands of TLR7 have yet to be identified, and in light of data showing that miRNAs in nociceptive primary sensory neurons of DRG regulate pain, the Duke researchers looked at whether the small, non-coding RNAs act as TLR7 agonists to activate DRG neurons and trigger pain.
They focused on let-7b, which contains the GUUGUGU immune stimulatory motif shown to activate TLR7, testing whether the miRNA would elicit electrophysical responses in DRG neurons, and found exposure of the neurons to the miRNA "induced robust inward currents" of ions into such cells of wild-type mice, they wrote in a paper appearing in Neuron. In the neurons of mice engineered to lack TLR7, however, the currents were abolished.
The scientists then asked whether the TLR7-associated ion channel TRPA1 is involved in the process, and discovered that let-7b-responsive neurons also responded to a TRPA1 agonist. All miRNA-induced inward currents were blocked, however, by a TRPA1 antagonist and absent in TRPA1-null mice, according to the paper.
The investigators then tested whether the miRNA-induced neuronal activation observed with let-7b is sequence dependent. They found that miR-599, which also contains the GUUGUGU motif triggered a TLR7-dependent current. On the other hand, miR-29a and miR-21, which both lack the motif, had no such effect. Importantly, let-7a — a close family member of let-7b with just a single nucleotide change in the GUUGUGU motif — failed to induce any inward ion current.
Taken together, the results indicate that a GU-rich core is "critically required" for inducing miRNA-induced inward currents, they wrote in Neuron. In additional experiments, Ji and his team found that coexpression of TLR7 and TRPA1 is also required for this response, which is abolished in mice lacking either.
To see whether let-7b could activate TLR7 and TRPA1 in a heterologous cell system, the team examined the miRNA's activity in HEK293 human embryonic kidney cells overexpressing the two. Indeed, let-7b evoked "robust inward currents in these cells," which were completely blocked by TRPA1 antagonism, they wrote in their study. The miRNA also had no effect in HEK293 cells overexpressing TRPA1 alone.
Further experimentation showed that let-7b binds to TLR7 and enhances its interaction with TRPA1 in HEK293 cells. Additionally, the miRNA was found to induce TLR7/TRPA1-dependent single-channel activities in DRG neurons and HEK293 cells over-expressing TLR7/TRPA1.
To test the practical implications of their findings, the investigators intradermally injected let-7b into the feet of mice. They found that this triggered a rapid and transient pain response as revealed by licking and lifting behaviors, and that this effect could be mitigated by pretreatment with a TRPA1 antagonist or let-7b inhibitor.
Low doses of let-7b were also found to induce persistent TRPA1-dependent mechanical allodynia, which could also be eliminated with let-7b inhibitor pretreatment.
In light of these results, Ji speculated that let-7b might prove to be a therapeutic target for controlling pain, noting that the miRNA's inhibition can easily be achieved using single-stranded RNA.
Ji and his team also demonstrated in Neuron that let-7b levels are significantly higher in DRG tissue than spinal cord, cortex, and skin tissues, and the endogenous let-7b can be released from DRG neurons in an activity-dependent manner, opening the door to the miRNAs use as a biomarker of pain.
As part of Duke University Medical Center, Ji's lab has access to clinical blood samples, and he said he is now aiming to test whether serum levels of let-7b can be correlated to pain associated with various diseases and conditions in humans.