Korean J Pain.  2024 Oct;37(4):320-331. 10.3344/kjp.24196.

Down-regulation of microRNA-382-5p reduces neuropathic pain by targeting regulation of dual specificity phosphatase-1

• Xu A
• Shen H
• Mei S
• Wang Z
• Xie Q
• Cui H
• Chu Y
• Feng B

• KMID: 2559694
• DOI: http://doi.org/10.3344/kjp.24196

Abstract

Background
MicroRNA (miRNA) plays a crucial role in neuropathic pain (NP) by targeting mRNAs. This study aims to analyze the regulatory function and mechanism of miR-382-5p/dual specificity phosphatase-1 (DUSP1) axis in NP.
Methods
We utilized rats with chronic constriction injury (CCI) of the sciatic nerve as the NP model. The levels of miR-382-5p and DUSP1 were reduced by intrathecal injection of lentiviral interference vectors targeting miR-382-5p and DUSP1. The mRNA levels of miR-382-5p and DUSP1 in the dorsal root ganglions (DRGs) were measured by RT-qPCR assay. The pain behavior was evaluated by mechanical nociceptive sensitivity and thermal nociceptive sensitivity. The expression levels of interleukin-6 (IL)-6, IL-1β, and tumor necrosis factor-α in the DRGs were analyzed by ELISA assay. The targeting relationship between miR-382-5p and DUSP1 was verified by DLR assay and RIP assay.
Results
Compared to the Sham group, the CCI rats exhibited higher levels of miR-382-5p and lower levels of DUSP1. Overexpression of miR-382-5p significantly decreased DUSP1 levels. Reducing miR-382-5p levels can lower the mechanical nociceptive sensitivity and thermal nociceptive sensitivity of CCI rats and inhibit the over-activation of pro-inflammatory factors. Reduced miR-382-5p levels decreased NP in CCI rats. DUSP1 is the target of miR-382-5p, and down-regulation of DUSP1 reverses the inhibitory effect of reduced miR-382-5p levels on NP.
Conclusions
Down-regulation of miR-382-5p inhibits the over-activation of pro-inflammatory factors by targeting and regulating the expression of DUPS1, thereby alleviating NP.

Keyword

Chronic Pain; Down-Regulation; Dual-Specificity Phosphatases; Hyperalgesia; Inflammation; MicroRNAs; Neuralgia; Nociception; Pain Management; Sciatic Nerve

Figure

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  Fig. 1 Experimental design flow chart. LV-anti-NC: empty lentivirus, LV-anti-miR-382-5p: lentivirus that inhibits miR-382-5p, sh-NC: empty lentivirus, sh-DUSP1: lentivirus that inhibits DUSP1, DUSP1: dual specificity phosphatase-1, CCI: chronic constriction injury, RT-qPCR: real-time quantitative reverse transcription polymerase chain reac-tion, ELISA: enzyme-linked immunosorbent assay.

• 

  Fig. 2 Elevated miR-382-5p levels in DRGs of NP rats. (A, B) Mechanical nociceptive sensitivity and thermal nociceptive sensitivity were measured in CCI rats on days 0, 3, 7, 14 and 21 of the CCI surgery. (C–E) On day 21 of CCI surgery, the levels of proinflammatory factors (IL-6, IL-1β, TNF-α) in DRGs were measured by ELISA assay. (F) The level of miR-382-5p in DRGs was measured by RT-qPCR on days 0, 3, 7, 14 and 21 of the CCI surgery. Values are presented as mean ± standard deviation. DRGs: dorsal root ganglions, NP: neuropathic pain, CCI: chronic constriction injury, IL-6: interleukin-6, IL-1β: interleukin-1β, TNF-α: tumor necrosis factor-α, PWT: paw withdrawal threshold, PWL: paw withdrawal latency. **P < 0.01; ***P < 0.001 vs. Sham. n = 4.

• 

  Fig. 3 Intrathecal injection of lentiviral interference vector targeting miR-382-5p (LV-anti-miR-382-5p) alleviated pain behavior in CCI rats. (A) The mRNA level of miR-382-5p in DRGs was detected by RT-qPCR on the day 21 of CCI surgery. (B, C) Mechanical nociceptive sensitivity and thermal nociceptive sensitivity were measured in CCI rats on days 0, 3, 7, 14 and 21 of the CCI surgery. Values are presented as mean ± standard deviation. CCI: chronic constriction injury, DRGs: dorsal root ganglions, RT-qPCR: real-time quantitative reverse transcription polymerase chain reaction, PWT: paw withdrawal threshold, PWL: paw withdrawal latency. **P < 0.01; ***P < 0.001 vs. Sham. #P < 0.05; ##P < 0.01; ###P < 0.001 vs. CCI + LV-anti-NC. n = 13.

• 

  Fig. 4 Intrathecal injection of lentiviral interference vector targeting miR-382-5p (LV-anti-miR-382-5p) inhibited the over-activation of proinflammatory factors in DRGs of CCI rats. (A–C) On day 21 of CCI surgery, the levels of proinflammatory factors (IL-6, IL-1β, TNF-α) in DRGs were measured by ELISA assay. Values are presented as mean ± standard deviation. DRGs: dorsal root ganglions, CCI: chronic constriction injury, IL-6: interleukin-6, IL-1β: interleukin-1β, TNF-α: tumor necrosis factor-α, ELISA: enzyme-linked immunosorbent assay. ***P < 0.001 vs. Sham. ###P < 0.001 vs. CCI + LV-anti-NC. n = 13.

• 

  Fig. 5 DUSP1 is a target gene of miR-382-5p. (A) There are complementary binding sites between miR-382-5p and DUSP1. (B) DLR assay confirms the targeting relationship between miR-382-5p and DUSP1. (C) RIP assay shows that both miR-382-5p and DUSP1 could be enriched in immunoprecipitation complexes. (D–F) The mRNA and protein levels of DUSP1 in DRGs were measured by RT-qPCR and ELISA on days 0, 3, 7, 14 and 21 of the CCI surgery. (G) On day 21 of CCI surgery, RT-qPCR was used to detect the effect of lentivirus interfering vector (LV-anti-miR-382-5p) on DUSP1 mRNA levels in DRGs. Values are presented as mean ± standard deviation. DUSP1: dual specificity phosphatase-1, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, DLR: dual-luciferase reporter, RIP: RNA immunoprecipitation, DRGs: dorsal root ganglions, RT-qPCR: real-time quantitative reverse transcription polymerase chain reaction, ELISA: enzyme-linked immunosorbent assay, CCI: chronic constriction injury, WT: wild type, MUT: mutant. *P < 0.05; **P < 0.01; ***P < 0.001 vs. Sham group. ###P < 0.001 vs. CCI + LV-anti-NC. n = 13.

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  Fig. 6 Down-regulation of DUSP1 inhibited the alleviating effect of down-regulation of miR-382-5p on neuropathic pain. (A) The mRNA level of DUSP1 in DRGs was detected by RT-qPCR on the day 21 of CCI surgery. (B, C) Mechanical nociceptive sensitivity and thermal nociceptive sensitivity were measured in CCI rats on days 0, 3, 7, 14 and 21 of the CCI surgery. (D–F) On day 21 of CCI surgery, the levels of proinflammatory factors (IL-6, IL-1β, TNF-α) in DRGs were measured by ELISA assay. Values are presented as mean ± standard deviation. DUSP1: dual specificity phosphatase-1, DRGs: dorsal root ganglions, RT-qPCR: real-time quantitative reverse transcription polymerase chain reaction, CCI: chronic constriction injury, IL-6: interleukin-6, IL-1β: interleukin-1β, TNF-α: tumor necrosis factor-α, ELISA: enzyme-linked immunosorbent assay, PWT: paw withdrawal threshold, PWL: paw withdrawal latency. ###P < 0.001 vs. CCI + LV-anti-NC. &&P < 0.01; &&&P < 0.001 vs. CCI + LV-anti-miR-382-5p + sh-NC. n = 13.

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