In this study, to our knowledge, we provide the first evidence that TET1 interacts with miR-30b-5p and regulates Nav1.6 expression in affected DRG neurons in OXA-induced peripheral neuropathy. The decrease in TET1 correlates with a loss of hydroxymethylation at the promoter region of the Mir30b, thus downregulating the miR-30b-5p/Nav1.6 signaling and leading to neuropathic pain. Blocking the TET1 decrease can reverse the reduction of hydroxymethylation in DRG neurons, destabilize the OXA-induced Nav1.6 upregulation, and alleviate chemotherapy‐associated pain hypersensitivities.
Recent reports have strongly linked MicroRNA to nociceptive processing.[39–41] miR-30b-5p is highly conserved across species,[42] and it modulates various disease processes, such as immune response, inflammation, cancer, and nociceptive processing.[43–46] Our previous studies showed that miR-30b-5p was involved in neuropathic pain targeting Nav1.3 and Nav1.7 after peripheral nerve injury in rats.[47, 48] In addition, as our recent report demonstrated, miR-30b-5p is implicated in the mechanism of chemotherapy-induced neuropathic pain via targeting Nav1.6 in rats, supporting a crucial role of miR-30b-5p in pain genesis.[27] In the current study, we replicated the OXA-induced pain model in mice and observed a decrease in miR-30b-5p expression, which was in line with our previous findings that demonstrated the conserved expression of miR-30b-5p in different models and species, further supporting the involvement of miR-30b-5p in pain development. Moreover, we examined the expression of pain-related voltage-gated sodium channel genes, including Scn3a (encoding Nav1.3), Scn8a (encoding Nav1.6), Scn9a (encoding Nav1.7), Scn10a (encoding Nav1.8), and Scn11a (encoding Nav1.9). Among these genes, we observed a significant increase in Scn8a, but not Scn3a, Scn9a, Scn10a, or Scn11a, indicating the conserved expression of Scn8a and different expression patterns of Scn3a, Scn9a, Scn10a, and Scn11a in nerve injury- or chemotherapy-induced neuropathic pain models. Notably, the alteration of Nav1.6 expression was the most significant, which was consistent with the finding that Nav1.6 was closely related to cold allodynia induced by chemotherapy.[27, 48] Furthermore, the identification of the expression patterns of miR-30b-5p/Nav1.6 signaling and other Nav isoforms in different species or neuropathic pain models provides additional evidence for the development of analgesic targets. In addition, we observed the enhancement of Nav1.6-mediated current in DRGs during OXA-induced pain. Compared to our previous study, we provide additional direct evidence that Nav1.6 is involved in pain induction.
In recent years, epigenetic modification has been extensively researched in antitumor-immunity,[49] neurodegeneration, neuroprotection,[50] congenital diseases,[51] as well as in chronic pain.[52] As a demethylase, TET1 was suggested to be implicated in pathological pain, such as neuropathic and inflammatory pain.[17, 18, 53] However, the TET1 expression pattern and function remain controversial. For example, some studies demonstrated that TET1 was upregulated in the ipsilateral spinal cord dorsal horn in spinal nerve ligation-induced neuropathic pain and formalin- or complete Freund’s adjuvant-induced inflammatory pain, while other evidence suggests that DRG TET1 overexpression alleviates pain-like behaviors.[53, 54] In our study, we observed a decrease in TET1 in DRGs after OXA treatment and found that DRG TET1 overexpression effectively attenuated OXA-induced mechanical and cold allodynia. Comparatively, our findings were consistent with the data that DRG TET1 overexpression mitigates spinal nerve ligation-induced neuropathic pain through targeting µ-opioid receptor and Kv1.2,[17] contrary to the role of spinal TET1 after OXA or SNL.[18, 55] These findings revealed the role of endogenous TET1 in DRGs and spinal dorsal horn neurons in pain genesis and suggested that the TET1 expression pattern and function may be region-specific.
Furthermore, we determined the effect of TET1 expression on genome-wide 5hmc level through TET1 overexpression or knockout. TET1 overexpression significantly rescued the impaired 5hmc level induced by OXA, while TET1 conditional knockout led to a marked decrease in 5hmc level in naïve mice. Given that TET1 has the potential to regulate MicroRNAs in diseases (miR-124; miR-34a; and miR-26),[28, 56, 57] we investigated the relationship between TET1 and miR-30b-5p. To probe whether TET1 regulated miR-30b-5p, we selected four possible methylation fragments in the Mir30b promoter region through bioinformatics and conducted a ChIP assay to confirm the potential interactions. Interestingly, our results demonstrated that TET1 could bind to the Mir30b promoter fragment (-1 103bp to -1 079bp), which may be modified by methylation. More importantly, we observed the increased binding activity between them in OXA-treated groups, indicating that TET1 contributed to OXA-induced peripheral neuropathy by triggering the demethylation of the Mir30b promoter. Further analyses revealed that microinjection of TET1-lentiviral activation into the DRGs blocked the decrease in miR-30b-5p and the increase in Nav1.6 induced by OXA. Microinjection of AAV-Cre in the DRGs of Tet1flox/flox mice induced a reduction in miR-30b-5p and an elevation in Nav1.6 expression, leading to pain-like behaviors. Moreover, in OXA-induced pain, the reduced expression of TET1 and the elevated expression of Nav1.6 suggest that TET1 does not regulate Nav1.6 through demethylation. These findings further confirmed the regulation of miR-30b-5p/Nav1.6 by TET1 and suggested that TET1 was involved in OXA-induced neuropathic pain by regulating miR-30b-5p/Nav1.6 signaling, at least through its triggered demethylation of Mir30b promoter in the DRGs.
Although we reported the unique regulatory mechanism of TET1/miR-30b/Nav1.6 in chemotherapy-induced pain, the present study still had some limitations. First, previous studies suggested that DNA demethylase or other methylases also contribute to pain development. For example, DNA Methyltransferase 3 Alpha (DNMT3a) has been reported to contribute to the development and maintenance of bone cancer pain by silencing Kv1.2 expression in the spinal cord dorsal horn.[31] In addition, TET3 was also found to display differential upregulation after injury, suggesting a potential role in neuropathic pain.[58] However, in the present study, we did not detect changes in DNMT3a, TET3, or TET2 expression patterns, which should be explored in subsequent studies. Second, we proposed the regulatory mechanism of TET1 and miR-30b-5p. It is unknown whether TET1 participates in OXA-induced pain by regulating other miRNAs. Third, since spinal TET1 in peripheral injury and chemotherapy-induced pain is inconsistent with that observed in DRGs,[15, 41] the role of spinal TET1 after OXA administration should also be explored to provide more information. Fourth, considering the major role of small- and medium-sized DRG neurons in pain sensation [59–61] as well as the difficulty in performing effective whole-cell recording, we only analyzed the excitability and Nav currents in the small- and medium-sized DRG neurons instead of the large neurons. However, despite the predominant expression of miR30b in small- to medium-sized neurons, TET1 was also expressed in large DRG neurons. The possibility of TET1 regulating the excitability of large neurons and its potential contribution to pain cannot be disregarded. Lastly, due to the absence of a specific blocker or activator of the Nav1.6 currents, the exact change magnitude in the Nav1.6 currents could not be measured accurately. The potential impact of TET1 on other TTX-s Nav channels remains to be further investigated.
In summary, our study presented novel evidence that TET1 modulated miR-30b-5p to upregulate Nav1.6 protein expression, resulting in heightened neuronal excitability during pain. Conversely, TET1 overexpression could mitigate these effects and alleviate pain. These findings could offer valuable insights into the pathophysiology of pain and suggest TET1 as a promising therapeutic target for analgesic drug development.