MiR-31-5p regulates the neuroinflammatory response via TRAF6 in neuropathic pain

Abstract

Background: Neuropathic pain is chronic pain and has few effective control strategies. Studies have demonstrated that microRNAs have functions in neuropathic pain. However, no study has been conducted to demonstrate the role and mechanism of microRNA (miR)-31-5p in neuropathic pain. Accordingly, this study sought to determine the pathological role of miR-31-5p in chronic constriction injury (CCI) -induced neuropathic pain mouse models.

Methods: We used CCI surgery to establish mouse neuropathic pain model. Behavioral tests were performed to evaluate pain sensitivity of mice. Expressions of miR-31-5p and inflammatory cytokines in dorsal root ganglion (DRG) were examined by polymerase chain reaction. Animals or cells were received with/without miR-31-5p mimic or inhibitor to investigate its role in neuropathic pain. The mechanism of miR-31-5p was assayed using western blotting, immunofluorescence staining and dual-luciferase reporter assay.

Results: We found that CCI led to a significant decrease in miR-31-5p levels. Knockout of miR-31-5p and administration of miPEP31 exacerbated pain in C57BL/6 mice. Meanwhile, miR-31-5p overexpression increased the paw withdrawal threshold and latency. TRAF6 is one of the target gene of miR-31-5p, which can trigger a complex inflammatory response. TRAF6 was associated with pain and that reducing the DRG expression of TRAF6 could alleviate pain. In addition, miR-31-5p overexpression inhibited the TRAF6 expression and reduced the neuroinflammatory response.

Conclusions: All the results reveal that miR-31-5p could potentially alleviate pain in CCI mouse models by inhibiting the TRAF6 mediated neuroinflammatory response. MiR-31-5p upregulation is highlighted here as new target for CCI treatment.

Keywords: Neuroinflammatory response; Neuropathic pain; TRAF6; miR-31-5p.

Figure. 1

MiR-31-5p knockout aggravated pain hypersensitivity and activate inflammatory response in mice. a Mechanical withdrawal threshold in WT and miR-31-5p^−/− male mice, ^** P < 0.01, two- tailed unpaired t-test, n = 8 mice. b Heat withdrawal latency in WT and miR-31-5p^−/− male mice, ^** P < 0.01, two- tailed unpaired t-test, n = 8 mice. c Cold withdrawal latency in WT and miR-31-5p^−/− male mice, ^** P < 0.01, two- tailed unpaired t-test, n = 8 mice. d Mechanical withdrawal threshold in WT and miR-31-5p^−/− female mice, ^** P < 0.01, two- tailed unpaired t-test, n = 8 mice. e Heat withdrawal latency in WT and miR-31-5p^−/− female mice, ^** P < 0.01, two- tailed unpaired t-test, n = 8 mice. f Cold withdrawal latency in WT and miR-31-5p^−/− female mice, ^** P < 0.01, two- tailed unpaired t-test, n = 8 mice. RT-qPCR analysis of TNF‐α (g), IL‐6 (h) and IL‐1β (i) expression in the DRG of WT and miR-31-5p^−/− mice. ^**P < 0.01, two- tailed unpaired t-test, n = 6 mice.

Figure. 2

MiPEP31 administration could lead to a decrease in murine pain threshold. a–f Mice received Scr(scPEP) or PEP31(miPEP31) (50ug in 100 µL phosphate-buffered saline) via tail vein injections. a Relative expression of miR-31-5p in the DRG of mice on day 3 after injection. ^*P < 0.05 vs. naive. One-way ANOVA, n = 7 mice. b Mechanical withdrawal threshold in three groups, ^* P < 0.05, ^** P < 0.01 vs. naive. two-way ANOVA, n = 8 mice. c Heat withdrawal latency in three groups, ^*P < 0.05, ^**P < 0.01 vs. naive. Two-way ANOVA, n = 8 mice. RT-qPCR analysis of TNF‐α (d), IL‐6 (e) and IL‐1β (f) expression in the DRG of mice injected with miPEP31after 3 days. ^**P < 0.01, two- tailed unpaired t-test, n = 6 mice.

Figure. 3

MiR-31-5p inhibitor intrathecal injection induced pain hypersensitivities in naive mice. a The description of the animal experimental process. Behavior tests were performed after Scr(scramble) or inhibitor (miR-31-5p inhibitor) administration. b Relative expression of miR-31-5p in the DRG of navie mice treated with intrathecal miR-31-5p inhibitors/scrambles was determined by qRT-PCR. ^**P < 0.01 vs. naive. one-way ANOVA, n = 7 mice. c Effects of downregulated miR-31-5p on mechanical allodynia were assessed by withdrawal threshold on post-induction days 1, 2, and 3. ^* P < 0.05, ^** P < 0.01 vs. naive; Two-way ANOVA, n = 8 mice. d Effects of the downregulated miR-31-5p on thermal hyperalgesia were assessed by heat withdrawal latency at post-induction days 1, 2, and 3. ^*P < 0.05, ^**P < 0.01 vs. naïve. Two-way ANOVA, n = 8 mice. e–f Western blot analysis of P-ERK, ERK and GFAP protein expression in the spinal cord of naive mice at 3 days injected with intrathecal miR-31-5p inhibitor. ^**P < 0.01 vs. naive; two-way ANOVA, n = 3. RT-qPCR analysis of TNF‐α (g), IL‐6 (h) and IL‐1β (i) expression in the DRG of mice injected with Scr or inhibitor after 3 days. ^**P < 0.01, two- tailed unpaired t-test, n = 6 mice.

Figure. 4

Downregulated miR-31-5p levels in the CCI mouse model. a The CCI model was established to induce neuropathic pain in mice. b Mechanical withdrawal threshold in sham and CCI mice on postoperative day 0, 3, 7, 10, and 14, ^*P < 0.05, ^**P < 0.01 vs. sham. Two-way ANOVA, n = 8 mice. c Heat withdrawal latency in sham and CCI mice on postoperative day 0, 3, 7, 10, and 14.^*P < 0.05, ^**P < 0.01 vs. sham. Two-way ANOVA, n = 8 mice. d RT-qPCR analysis of miR-31-5p expression in the DRG of CCI mice at the different time points, ^* P < 0.05, ^**P < 0.01 vs. sham. Two-way ANOVA, n = 6 mice. RT-qPCR analysis of TNF‐α(E), IL‐6(F) and IL‐1β(G) expression in the DRG of sham and CCI mice. ^**P < 0.01, two- tailed unpaired t-test, n = 6 mice. CCI, chronic constriction injury; ANOVA, analysis of variance; qRT-PCR: quantitative reverse transcription‐polymerase chain reaction; DRG, dorsal root ganglion.

Figure. 5

Intrathecal miR-31-5p mimic attenuated neuropathic pain. a The description of the animal experimental process. Behavior tests were performed on postoperative days and after scramble or miR-31-5p mimic administration. b Relative expression of miR-31-5p in the DRG of CCI mice treated with intrathecal miR-31-5p mimics/scrambles was determined by qRT-PCR. ^**P < 0.01 vs. sham + scramble; ^##P < 0.01 vs. CCI + scramble. one-way ANOVA, n = 7 mice. c Effects of the upregulated miR-31-5p on mechanical allodynia were assessed by withdrawal threshold on post-induction days 5, 7, 9, and 14. ^**P < 0.01 vs. sham + scramble; ^##P < 0.01 vs. CCI + scramble. Two-way ANOVA, n = 8 mice. d Effects of upregulated miR-31-5p on thermal hyperalgesia were assessed by heat withdrawal latency on post-induction days 5, 7, 9, and 14. ^** P < 0.01 vs. sham + scramble; ^##P < 0.01 vs. CCI + scramble. Two-way ANOVA, n = 8 mice. RT-qPCR analysis of TNF‐α (e), IL‐6 (f) and IL‐1β (g) expression in the DRG of CCI mice injected with Scr or mimic. ^**P < 0.01, two- tailed unpaired t-test, n = 6 mice.

Figure. 6

TRAF6 could relieve neuropathic pain a The sequence of miR‐31-5p and the position of TRAF6 3′‐UTR. b, c Western blotting analysis of TRAF6 on days 0, 3, 7 and 14 after CCI surgery. ^*P < 0.05 vs. sham. ^**P < 0.01 vs. sham, one -way ANOVA, n = 3. d RT-qPCR analysis of TRAF6 on days 0, 3, 7 and 14 after CCI surgery. ^**P < 0.01 vs. sham, one -way ANOVA, n = 3. e, f Fluorescence images of TRAF6 (red), NeuN(green) and DAPI (blue) were obtained in DRGs of sham and CCI mice, The scale bar is 50μm, ^**P < 0.01 vs. sham, Two-tailed unpaired t-test, n = 3. g, h Western blotting analysis of TRAF6 in the neuron 2a cells with TRAF6 siRNA or scramble transfection. ^** P < 0.01 vs. scramble. Two- tailed unpaired t-test, n = 3. DRG microinjection of sham and CCI mice received Scr or TRAF6 siRNA. i Mechanical withdrawal threshold in four groups, ^**P < 0.01 vs. sham+ Scr. ^##P < 0.01 vs. CCI+ Scr, two-way ANOVA, n = 8 mice. j Heat withdrawal latency in four groups, ^**P < 0.01 vs. sham+ Scr, ^#P < 0.05 vs. CCI+ Scr, ^##P < 0.01 vs. CCI+ Scr, two-way ANOVA, n = 8 mice.

Fig. 7

MiR-31-5p targeted TRAF6 by binding to its 3’-UTR region. a The relative expression of miR-31-5p with mimic transfection in the neuron 2a cells was determined by qRT-PCR. ^** P < 0.01 vs. scramble. Two-tailed unpaired t-test, n = 3. b, c Western blotting analysis of TRAF6 in the neuron 2a cells with miR-31-5p mimic transfection. ^** P < 0.01 vs. scramble. Two- tailed unpaired t-test, n = 3. d Relative expression of miR-31-5p with inhibitor transfection in the neuron 2a cells was determined by qRT-PCR. ^** P < 0.01 vs. scramble. Two-tailed unpaired t-test, n = 3. e, f Western blotting analysis of TRAF6 in the neuron 2a cells with miR-31-5p inhibitor transfection. ^* P < 0.05 vs. scramble. Two- tailed unpaired t-test, n = 3. g, h Western blotting analysis of TRAF6 protein expression in the DRG of naive mice on day 3 after treated with intrathecal miR-31-5p inhibitors. ^**P < 0.01 vs. naive; two-way ANOVA, n = 3. i, j Western blot analysis of TRAF6 protein expression in the DRG of CCI mice at 14 days injected with intrathecal miR-31-5p mimic. ^** P < 0.01 vs. naive; ^## P < 0.01 vs. CCI + NS. two-way ANOVA, n = 3. k TRAF6 promoter luciferase reporter (TRAF6-Luc) was co-transfected with miR-31-5p mimic in 293T cells for 24 h, and then relative luciferase activity was analyzed. ^** P < 0.01 vs. naïve, ^# P < 0.05 vs. previous group, ^{# #}P < 0.01 vs. previous group. one -way ANOVA

Fig. 8

MiR-31-5p regulated the neuroinflammatory response via TRAF6 in CCI mice models. Neuron 2a cells were transfected with inhr (miR-31-5p inhibitor) or Scr(scramble) for 48 h. a, b Western blotting analysis of TRAF6 protein expression in five groups. ^** P < 0.01 vs. naive; ^## P < 0.01 vs. inhr. two-way ANOVA, n = 3. c RT-qPCR analysis of miR-31-5p expression in five groups. ^** P < 0.01 vs. naive; two-way ANOVA, n = 3. RT-qPCR analysis of TNF‐α (d), IL‐6 (e) and IL‐1β (f) expression in five groups.^*P < 0.05 vs. naive ^** P < 0.01 vs. naive; ^## P < 0.01 vs. inhr. two-way ANOVA, n = 6. DRG microinjection of miR-31-5p^−/− mice received Scr or TRAF6 siRNA. RT-qPCR analysis of TNF‐α (g), IL‐6 (h) and IL‐1β (i) expression in the DRG of miR-31^−/− mice injected with Scr or TRAF6 siRNA. ^* P < 0.05, ^** P < 0.01, two- tailed unpaired t-test, n = 6 mice. j Mechanical withdrawal threshold in miR-31-5p^−/− mice with TRAF6 siRNA, ^** P < 0.01. two-way ANOVA, n = 8 mice. k Heat withdrawal latency in miR-31-5p^−/− mice with TRAF6 siRNA, ^* P < 0.05 vs. miR-31^−/− + Scr, ^** P < 0.01 vs. miR-31^−/− + Scr. two-way ANOVA, n = 8 mice

Fig. 9

Schematic diagram showing miR-31-5p inhibited TRAF6 transcription and further suppressed the neuroinflammatory response, leading neuroprotective effects against CCI in mice