TET1 participates in oxaliplatin-induced neuropathic pain by regulating microRNA-30b/Nav1.6

Abstract

Chemotherapy-induced neuropathic pain poses significant clinical challenges and severely impacts patient quality of life. Sodium ion channels are crucial in regulating neuronal excitability and pain. Our research indicates that the microRNA-30b (miR-30b) in rat dorsal root ganglia (DRG) contributes to chemotherapy-induced neuropathic pain by regulating the Nav1.6 protein. Additionally, ten-eleven translocation methylcytosine dioxygenase 1 (TET1) plays a crucial role in pain generation by altering gene expression. We established a chemotherapy-induced neuropathy model using intraperitoneal oxaliplatin (OXA) injections and measured TET1 and Nav1.6 protein in the DRG. Using lentivirus and Tet1^flox/flox mice, we modulated TET1 expression and assessed pain behaviors, DRG neuronal excitability, Nav1.6 currents, miR-30b-5p, and demethylation of the Mir30b promoter region. We employed chromatin immunoprecipitation to pinpoint TET1-binding sites on the Mir30b promoter. The impacts of miR-30b agomir or antagomir on Nav1.6 expression and pain responses were assessed postintrathecal injections. The results showed that OXA reduced TET1, increasing neuronal excitability, Nav1.6 currents, and miR-30b-5p in the DRG. TET1 knockdown exacerbated these effects and induced pain behaviors. Conversely, TET1 overexpression reversed these effects. TET1 also targeted and enhanced demethylation at the Mir30b promoter (-1103 bp to -1079 bp). miR-30b agomir reduces Nav1.6, whereas miR-30b antagomir reverses TET1's effects on Nav1.6 and pain. In OXA-induced neuropathy, decreased TET1 reduces miR-30b, elevating Nav1.6 expression and currents and contributing to pain. We hypothesize that TET1 mediates this process by regulating the demethylation of the Mir30b promoter.

Keywords: 5-hydroxymethylcytosine; DRG; Nav1.6; TET1; epigenetics; microRNA; oxaliplatin; pain; sodium channels.

Figure 1

Oxaliplatin caused mechanical allodynia, cold hyperalgesia, and downregulated TET1 expression.A, schematic diagram of drug injection and behavioral tests. B and C, 14 days post-OXA injection, mice in the OXA group showed mechanical allodynia upon 0.07 g von Frey filament stimulation of both hind paws, in contrast to the vehicle group (two-way ANOVA. n = 12). D and E, in the OXA group, the PWF for the left hind paw (D) was increased on day 7 postinjection, and for the right hind paw (E) on day 21, following 0.4 g von Frey filament stimulation, relative to the vehicle group (two-way ANOVA. n = 12). F, mice in the OXA group exhibited cold hyperalgesia relative to the vehicle group (two-way ANOVA. n = 12). G–I, on day 14 after OXA injection, the expression of TET1 protein (G and H) and mRNA (I) in bilateral DRG of mice decreased by about 52.83% and 31.85%, respectively (unpaired t test. n = 7–8). J–N, cells (arrows) were labeled for TET1 (red), NeuN (green), GS (green), NF200 (green), CGRP (green), and IB4 (green) in WT mice (n = 4). Scale bar represents 50 μm. O, percentage of TET1+NeuN- and TET1+GS-positive cells relative to TET1-positive cells (n = 4). P, percentage of TET1+NF200-, TET1+IB4-, and TET1+CGRP-positive cells relative to TET1-positive cells (n = 3 in TET1+CGRP/TET1, n = 6 in TET1+IB4/TET1, and n = 4 in TET1+NF200/TET1). CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglia; GS, glutamine synthetase; IB4, isolectin B4; NeuN, neuron; NF200, neurofilament 200; OXA, oxaliplatin; PWF, paw withdrawal frequency; PWL, paw withdrawal latency; Veh, vehicle; WT, wild type.

Figure 2

Specific knockdown of TET1 mimicked OXA-induced pain hypersensitivity.A, schematic of the TET1 knockdown and behavioral test strategies. B, expression of GFP (green), NeuN (red), and DAPI (blue) in DRG at days 0 and 14 after injection of AAV-eGfp (n = 10). Scale bar represents 50 μm. C, the proportion of GFP-positive neurons in the DRG was quantified, showing that 40.11% of neurons expressed GFP protein (n = 10). D–F, the DRG was evaluated on day 28. Protein (D and E) and mRNA (F) expressions of TET1 in the AAV-Cre group were downregulated after virus microinjection (unpaired t test. Each sample contained at least six DRG from two mice. n = 4 in the AAV-eGfp group and n = 3 in the AAV-Cre group in D–E. n = 7 in the AAV-eGfp group and n = 5 in the AAV-Cre group in F). G–J, in the AAV-Cre group, the PWF in the ipsilateral hind paw increased in response to 0.07 g (G) and 0.4 g (I) von Frey filament stimulation starting 7 days postinjection, compared to the AAV-eGfp group. However, there was no change in the contralateral hind paw (H, J) compared to the AAV-eGfp group (two-way ANOVA. n = 20 in AAV-eGfp group and n = 18 in AAV-Cre group). K, TET1 knockdown induced cold hyperalgesia (two-way ANOVA. n = 20 in AAV-eGfp group and n = 18 in AAV-Cre group). AAV, Adeno-associated virus; DRG, dorsal root ganglia; GFP, green fluorescent protein; PWF, paw withdrawal frequency; PWL, paw withdrawal latency.

Figure 3

TET1 overexpression alleviated OXA-induced mechanical allodynia and cold hyperalgesia.A, the virus was microinjected into DRG 14 days before the experiment. Then, we injected 5% glucose/OXA intraperitoneally and detected DRG 14 days later. B and C, on day 14 after injection of OXA or 5% glucose, the expression of TET1 protein increased in the OXA+TET1-OE group compared to the OXA+TET1-Control group (unpaired t test. Each sample contained at least six DRG from two mice, n = 4 in each group). D, the expression of Tet1 mRNA in the OXA+TET1-OE group was upregulated compared to the OXA+TET1-Control group (unpaired t test. Each sample contained at least six DRG from two mice. n = 3 in OXA+TET1-Control group and n = 6 in OXA+TET1-OE group). E, immunofluorescent labeling of TET1-positive cells in vehicle, OXA, OXA+TET1-Control, and OXA+TET1-OE groups of DRG (n = 3). Scale bar represents 50 μm. F–I, upon stimulation with a 0.07 g/0.4 g von Frey filament, both paws of the OXA+TET1-Control group and the contralateral paw of the OXA+TET1-OE group showed mechanical allodynia compared to the Naive group. However, the ipsilateral paw of the OXA+TET1-OE group demonstrated a higher mechanical pain threshold than the OXA+TET1-Control group (two-way ANOVA. n = 15 in Veh+TET1-OE group, n = 18 in OXA+TET1-OE group, and n = 16 for others). J, relative to the Naive group, mice in both the OXA+TET1-Control and OXA+TET1-OE groups displayed cold hyperalgesia on days 7 and 14 postinjection. However, by day 14 postinjection, cold hyperalgesia was alleviated in the OXA+TET1-OE group compared to the OXA+TET1-Control group. By day 21 postinjection, the PWL in the OXA+TET1-OE group equaled that of the Naive group (two-way ANOVA. n = 15 in Veh+TET1-OE group, n = 18 in OXA+TET1-OE group, and n = 16 for others). DRG, dorsal root ganglia; OXA, oxaliplatin; PWF, paw withdrawal frequency; PWL, paw withdrawal latency; TET1-OE, TET1-Lentiviral Activation Particles; TET1-Control, TET1-Control Lentiviral Activation System; Veh, vehicle.

Figure 4

TET1 inhibits the OXA-induced increase in action potentials in DRG neurons.A, representative action potential traces of DRG neurons. B–E, the RMP (B) of DRG neurons in the OXA group was higher, and the rise time (D) was shorter than the vehicle group. However, rheobase (C) and amplitude (E) were similar to those in the vehicle group (unpaired t test. n = 15–16 in Veh group and n = 20–21 in OXA group). F, the spike number at twice the rheobase in DRG neurons of the OXA group was increased compared to the vehicle group (unpaired Mann Whitney test. n = 16 in Veh group and n = 19–21 in OXA group). G, representative action potential traces of DRG neurons. H, the RMP of DRG neurons in the AAV-Cre group was similar to that in the AAV-eGfp group (unpaired t test. n = 28 in the AAV-eGfp group and n = 26 in the AAV-Cre group). I–K, the rise time (J) of DRG neurons in the AAV-Cre group was shorter than in the AAV-eGfp group, while rheobase (I) and amplitude (K) were similar to those in the AAV-eGfp group (unpaired t test. n = 29 for I, n = 30 for J, and n = 30 for (K). L, the spike number at twice the rheobase in DRG neurons of the AAV-Cre group was increased compared to the AAV-eGfp group (unpaired Mann Whitney test. n = 26–28 in AAV-eGfp group and n = 29 in AAV-Cre group). M, representative action potential traces of DRG neurons. N, the RMP was significantly decreased in the OXA+TET1-OE group compared to OXA+TET1-Control group (unpaired t test. n = 16 in OXA+TET1-Control group and n = 21 in OXA+TET1-OE group). O–Q, the rise time (P) was longer in the OXA+TET1-OE group compared to the OXA+TET1-Control group, while rheobase (O) and amplitude (Q) were similar to those in the OXA+TET1-Control group (unpaired t test. n = 17–22). R, there was no difference in the spike number at twice the rheobase between DRG neurons of the OXA+TET1-OE group and the OXA+TET1-Control group (unpaired Mann Whitney test. n = 15–17 in OXA+TET1-Control group and n = 16–19 in OXA+TET1-OE group). AAV, Adeno-associated virus; DRG, dorsal root ganglia; GFP, green fluorescent protein; OXA, oxaliplatin; RMP, resting membrane potential; TET1-Control, TET1-Control Lentiviral Activation System; TET1-OE, TET1-Lentiviral Activation Particles; Veh, vehicle.

Figure 5

OXA upregulated Nav1.6 in DRG neurons.A, fourteen days post-OXA injection, while Scn3a, Scn9a, Scn10a, and Scn11a mRNA levels remained unchanged, only Scn8a mRNA was significantly upregulated in the OXA group compared to the Vehicle group (unpaired t test. Scn3a n = 6, Scn8a n = 7, Scn9a n = 6, Scn10a n = 7, and Scn11a n = 6). B and C, expression of Nav1.6 protein was increased after OXA injection on day 14 (unpaired t test. n = 5). D, percentage of Nav1.6+NeuN- and Nav1.6+GS-positive cells relative to Nav1.6-positive cells (unpaired t test. n = 6). E, percentage of Nav1.6+NF200-, Nav1.6+IB4-, and Nav1.6+CGRP-positive cells relative to Nav1.6-positive cells (n = 5 in Nav1.6+CGRP/Nav1.6, n = 4 in Nav1.6+IB4/Nav1.6, and n = 5 in Nav1.6+NF200/Nav1.6). F–I, cells (arrows) were labeled for Nav1.6 (red), IB4 (green), NF200 (green), CGRP (green), and NeuN (green) in WT mice (n = 5). Scale bar represents 50 μm. J, immunofluorescent Nav1.6 (red), GS (green), and DAPI (blue) triple-labeling of DRG cells (arrows) in WT mice (n = 5). Scale bar represents 50 μm. CGRP, calcitonin gene-related peptide; GS, glutamine synthetase; IB4, isolectin B4; NeuN, neuron; NF200, neurofilament 200; WT, wild type.

Figure 6

Overexpression of TET1 decreases Nav1.6 protein expression and Nav1.6 current.A–C, expressions of Nav1.6 protein (A and B) and mRNA (C) were increased after TET1 knockdown on day 28 (unpaired t test. Each sample contained at least six DRG from two mice. n = 3 in A–B and n = 4 in (C). D and E, TET1 overexpression blocked the increase in Nav1.6 caused by OXA (one-way ANOVA. Each sample contained at least six DRG from two mice. n = 6 in each group). F, Scn8a mRNA levels increased in the OXA+TET1-Control group relative to the vehicle group but decreased in the OXA+TET1-OE group compared to the OXA+TET1-Control group (one-way ANOVA. Each sample contained at least six DRG from two mice. n = 5 in vehicle group, n = 5 in OXA+TET1-Control group, and n = 4 in OXA+TET1-OE group). G and H, the total Nav currents density (−20 mV) was lower in the OXA+TET1-OE group than the OXA+TET1-Control group (one-way ANOVA. n = 15 in Veh and OXA+TET1-OE groups, and n = 10 in OXA+TET1-Control group). I and J, there was no difference in TTX-sensitive currents density (−20 mV) among the three groups (one-way ANOVA. n = 9 in Veh, n = 6 in OXA+TET1-Control groups, and n = 7 in OXA+TET1-OE group). K and L, the Nav1.6 currents density (−20 mV) was lower in the OXA+TET1-OE group than in the OXA+TET1-Control group (one-way ANOVA. n = 5 in Veh group, and n = 6 in OXA+TET1-OE and OXA+TET1-Control groups). M and N, there was no difference in Nav1.8 currents density (−20 mV) among the three groups (one-way ANOVA. n = 6). DRG, dorsal root ganglia; GFP, green fluorescent protein; OXA, oxaliplatin; TET1-Control, TET1-Control Lentiviral Activation System; TET1-OE, TET1-Lentiviral Activation Particles.

Figure 7

TET1 modulates Nav1.6 and pain via miR-30b.A, miR-30b-5p expression in the DRG of the OXA group was decreased compared to the vehicle group (unpaired t test. n = 8 in Veh group and n = 9 in OXA group). B, miR-30b-5p expression in the DRG of AAV-Cre group mice was lower than the AAV-eGfp group (unpaired t test. Each sample contained at least six DRG from two mice. n = 3). C, fourteen days after DRG microinjection, TET1 upregulation could alleviate the decrease in miR-30b-5p caused by OXA injection (unpaired t test. Each sample contained at least six DRG from two mice. n = 5 in OXA+TET1-Control group and n = 4 in OXA+TET1-OE group). D, genomic DNA hydroxymethylation levels in the DRG decreased after OXA injection (unpaired t test. n = 4). E and F, after TET1 knockdown, DNA demethylation levels (E) in the DRG of AAV-Cre group mice decreased compared to the AAV-eGfp group; however, DNA methylation levels (F) remained consistent between the two groups (unpaired t test. Each sample contained at least six DRG from two mice. n = 5 in E and F). G, the whole genome DNA hydroxymethylation level increased after TET1 overexpression in the OXA+TET1-OE group compared to the OXA+TET1-Control group (one-way ANOVA. Each sample contained at least six DRG from two mice. n = 10). H, detection of possible TET1-binding sites in the Mir30b promoter region by CHIP (each sample contained at least 30 DRG from five mice, n = 2). I, demethylated DNA levels at the Mir30b promoter in the DRG decreased following OXA injection (unpaired t test. n = 6). J, following TET1 overexpression, demethylation levels in the Mir30b promoter region from −1103 to −1079 bp in mouse DRG were enhanced (unpaired t test. Each sample contained six DRG from two mice, n = 5). K, in situ hybridization to label miR-30b-5p (green) positive cells in DRG. Simultaneously, immunofluorescent labeling was employed to mark Nav1.6 (red) and TET1 (white) positive cells in DRG (n = 5). Scale bar represents 20 μm. L and M, intrathecal injection of miR-30b agomir reduces the OXA-induced elevation of Nav1.6 protein levels (unpaired t test. Each sample contained six DRG from two mice, n = 5). N, the PWF of the ipsilateral paw in response to 0.4 g von Frey filament stimulation was lower in the OXA+miR-30b agomir group than the OXA+miR-NC group (two-way ANOVA. n = 8). O and P, overexpressing TET1 in OXA-treated mice reduced Nav1.6 protein levels, while intrathecal injection of miR-30b antagomir reversed this effect (one-way ANOVA. Each sample contained six DRG from two mice, n = 4 OXA/OXA+TET1-OE+miR-NC, n = 3 OXA+TET1-OE+miR-30b antagomir). Q, on days 7 and 14 after OXA injection, the PWF of the ipsilateral paw in the OXA+TET1-OE+miR-NC group in response to 0.4 g von Frey filament stimulation was lower than that in the OXA group. However, on day 14, the PWF in the OXA+TET1-OE+miR-30b antagomir group was higher than that in the OXA+TET1-OE+miR-NC group (two-way ANOVA. n = 8). AAV, Adeno-associated virus; DRG, dorsal root ganglia; GFP, green fluorescent protein; OXA, oxaliplatin; TET1-OE, TET1-Lentiviral Activation Particles; TET1-Control, TET1-Control Lentiviral Activation System; Veh, vehicle.

Figure 8

Proposed mechanism by which TET1 participates in neuropathic pain. In physiological conditions, TET1 binds to the promoters of Mir30b (−1103 bp to −1079) and promotes the expression of miR-30b-5p, which inhibits the expression of Nav1.6. In pathological and chronic pain states, TET1 expression decreases in the nucleus. Lack of TET1 binding to the promoters of Mir30b results in a decrease in Mir30b transcription and miR-30b-5p expression, leading to an increase in the expression of the Nav1.6 protein. The enhanced Nav currents subsequently facilitate the spike firing of nociceptors. In summary, TET1 participates in oxaliplatin-induced neuropathic pain in mice by regulating miR-30b/Nav1.6 signaling.