LncRNA 4933431K23Rik modulate microglial phenotype via inhibiting miR-10a-5p in spinal cord injury induced neuropathic pain

Abstract

Abstract Neuropathic pain (NP) is caused by primary damage and dysfunction of nervous system, in which spinal cord injury (SCI) is a common cause of NP. Evidence shows that neuroinflammation and oxidative stress are related to the pathophysiology of NP, in which the activation of microglia and astrocytes in spinal is significant. Therefore, understanding the molecular mechanism of NP after SCI is of great significance. The rat model of SCI was established and BV2 cell was treated with LPS. The exosomes derived from astrocytes were extracted by centrifugation. The morphology of the exosomes was observed by electron microscope and the surface markers were detected by Western blot. LncRNA in astrocytes and astrocyte-derived exosomes were detected by qRT-PCR. The expression of microglia activation markers CD68 and Iba-1 was detected by immunohistochemistry. The von Frey test was applied to assess mechanical hypersensitivity. The heat plate analgesia instrument was used to evaluate Paw withdrawal latency (PWL). QRT-PCR used to detect expression of LncRNA49rik and miR-10a-5p. Western blot was used to detect MAPK/PI3K/AKT / mTOR signal pathway and COX2, iNOS. The content of MDA and the activity of SOD were detected by oxidative stress kit. The concentrations of IL-6, IL-1β, IL-18 and IFN-αwere detected by ELISA. The targeting relationship between LncRNA49rik and miR-10a-5p was analyzed by bioinformatics and double luciferase activity, Rip and FISH experiments. LncRNA49rik was highly expressed in astrocytes and its derived exosomes. SCI stimulated astrocytes to release exosome containing LncRNA49rik and promote microglia activation to increase inflammatory response. At the same time, overexpression of LncRNA49rik increased the incidence of NP and aggravated the level of inflammation and oxidative stress in rats with SCI. MiR-10a-5p is the target of LncRNA4933431K23Rik. Overexpression of LncRNA49rik significantly inhibited the up-regulation of miR-10a-5p. Overexpression of miR-10a-5p inhibited hyperalgesia and inflammation in SCI rats. In addition, transfection of miR-10a-5p mimics significantly inhibited the expression of MAPK/PI3K/AKT and up-regulated the expression of mTOR. Mechanism studies have shown that overexpression of miR-10a-5p weakens the phenotypic induction of microglia induced by LncRNA4933431K23Rik. LncRNA4933431K23Rik regulates microglial phenotype through inhibiting miR-10a-5p, which is responsible for NP induced by SCI.

Keywords

• LncRNA4933431K23Rik
• miR-10a-5p
• Astrocytes
• Spinal cord injury
• Neuropathic pain