miRNA-155 silencing reduces sciatic nerve injury in diabetic peripheral neuropathy

in Journal of Molecular Endocrinology
Correspondence should be addressed to F Yang: dryang_fengruiyang@163.com

*(J Chen and C Li contributed equally to this work)

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Neuropathic pain represents one of the most common complications associated with diabetes mellitus (DM) that impacts quality of life. Accumulating studies have highlighted the involvement of miRNAs in DM. Thus, the current study aimed to investigate the roles of miR-155 in diabetic peripheral neuropathy (DPN). In vitro DPN models were established using rat Schwann cells (SCs) by treatment with 5.5 mM glucose. Gain- or loss-of-function studies were conducted to determine the effect of miR-155 on Nrf2, cellular function, reactive oxygen species and inflammation. Rat DNP models were established by streptozotocin injection and damage of sciatic nerve. Next, miR-155 antagomir or agomir was employed to investigate the effects associated with miR-155 on motor and sciatic nerve conduction velocity (MNCV, SNCV), angiogenesis and inflammatory response in vivo. Nrf2 was identified to be a target of miR-155 by dual-luciferase reporter gene assay. Silencing of miR-155 or restoration of Nrf2 promoted cell proliferation, inhibited apoptosis and alleviated inflammation in vitro. miR-155 antagomir-induced inhibition increased MNCV and SNCV, strengthened angiogenesis and alleviated inflammation in DPN rats. Additionally, the effects exerted by miR-155 were reversed when Nrf2 was restored both in vitro and in vivo. Taken together, the key findings of our study provide evidence indicating that miR-155 targeted and suppressed Nrf2 in DPN. miR-155 silencing was found to alleviate sciatic nerve injury in DPN, highlighting its potential as a therapeutic target for DPN.

 

      Society for Endocrinology

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    Interactions between miR-155 and Nrf2 in DPN. (A) Binding sites between miR-155 and Nrf2 are predicted by online software. (B) Luciferase activity of Nrf2-WT and Nrf2-MUT after transfection with miR-155, detected by dual-luciferase reporter gene assay. *P < 0.05 vs cells co-transfected with NC and Nrf2-WT. (C) mRNA expression of miR-155 and (D and E) Protein expression of NRF2 normalized to GAPDH in the normal rats and rats from the STZ-induced DPN model determined by Western blot analysis. *P < 0.05 vs normal group. (F) mRNA expression of miR-155 and Nrf2 determined by RT-qPCR after delivery of miR-155 inhibitor, miR-155 mimic or Nrf2. *P < 0.05 vs blank group. (G and H) protein expression of NRF2 normalized to GAPDH determined by Western blot analysis after delivery of miR-155 inhibitor, miR-155 mimic or NRF2; *P < 0.05 vs blank group. Cell experiments were repeated three times. DPN, diabetic peripheral neuropathy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MUT, mutant; Nrf2, nuclear factor, erythroid 2-like 2; WT, wild type.

  • View in gallery

    Effects of silencing miR-155 or restoring Nrf2 on Schwann cells. (A) Representative micrographs showing RSC96 cell proliferation after EdU assay in different groups (×200). (B) The number of EdU-positive cells. (C) Representative micrographs showing cell apoptosis in TUNEL assay (×200). (D) The number of TUNEL-positive cells. (E) mRNA expression of apoptosis-related genes determined by RT-qPCR. (F and G) Levels of apoptosis-related proteins determined by Western blot. (H) ROS activity in RSC96 cells. (I) VEGF, IL-1β, IL-6 and TNF-α levels in RSC96 cells detected by ELISA. *P < 0.05 vs blank group. Cell experiments were repeated three times. Nrf2, nuclear factor, erythroid 2-like 2; ROS, reactive oxygen species; TUNEL, TdT mediated dUTP nick end labeling; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2 associated protein X; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; VEGF, vascular endothelial growth factor; IL, interleukin; TNF, tumor necrosis factor. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0067.

  • View in gallery

    Effects of silencing miR-155 or restoring Nrf2 on DPN rats. (A) Motor nerve conduction velocity in sciatic nerves. (B) Sensory nerve conduction velocity in sciatic nerves. (C) Representative micrographs illustrating the morphological changes in sciatic nerve observed under a transmission electron microscope (10,000×). (D) Number of blood vessels in sciatic nerve from each group. (E) Number of nerve fibers in each group. (F) Axon diameter in myelinated nerve fibers in each group. (G) Myelin sheath thickness of sciatic nerves in each group. (H) Number of tube formation in each group. (I) mRNA expression of Vegfr2 in rat EPCs from each group. (J) Activities of NQO1, GST and GPX in each group determined by ELISA. (K) Representative micrographs showing cell apoptosis in TUNEL assay (×400). (L) Number of TUNEL positive cells in each group. (M) Representative micrographs showing immunofluorescence staining of NeuN and CD31 in sciatic nerve from different groups (×400). (N) γH2AX expression in sciatic nerve from each group detected by immunofluorescence (×400). (O) VEGF, IL-1β, IL-6 and TNF-α levels in each group determined by ELISA. *P < 0.05 vs blank group. n = 6/group. Nrf2, nuclear factor, erythroid 2-like 2; DPN, diabetic peripheral neuropathy; EPC, endothelial progenitor cell; Vegfr2, vascular endothelial growth factor receptor 2; NQO1, NAD (P) H quinone oxidoreductase 1; GST, glutathione S-transferase; GPX, glutathione peroxidase; TUNEL, TdT mediated dUTP nick end labeling; VEGF, vascular endothelial growth factor; IL, interleukin; TNF, tumor necrosis factor. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0067.

  • View in gallery

    Schematic diagram showing potential mechanism of miR-155 and Nrf2 on the development of DPN. DPN, diabetic peripheral neuropathy; Nrf2, nuclear factor, erythroid 2-like 2; RISC, miRNA-induced silencing complex. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0067.

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