Early over-expressing of microRNA-145 effectively precludes the development of neuropathic mechanical hyperalgesia via suppressing Nav1.8 in diabetic rats

Abstract

Background: Painful diabetic neuropathy (PDN) is a common complication secondary to diabetes mellitus. Nav1.8 is an isoform of voltage-gated sodium channels and its expression regulation is closely related with PDN. MicroRNA-145 (miR-145) is involved in the occurrence and development of neuropathic pain. TargetScan software has revealed that Nav1.8 (SCN10A) is the major target of miR-145. However, its function between miR-145 and Nav1.8 in PDN is unknown. Objectives: We aim to explore the regulatory effect of miR-145 on the expression and function of Nav1.8, which plays a pivotal role in precluding the advancement of neuropathic mechanical hyperalgesia in diabetic pain. Study Design: An experimental, animal study. Setting: An animal research facility at Nanjing Maternal and Child Health Institute, China. Methods: The paw mechanical withdrawal threshold (PMWT) of rats was assessed with the von Frey test. The adverse regulation of Nav1.8 by miR-145 was confirmed by a dual luciferase detection system in HEK293T cells. The mRNA level and expression of Nav1.8 in dorsal root ganglion (DRG) neurons were assessed with real-time polymerase chain reaction (real-time PCR), western blotting and immunofluorescence assays following intrathecal injection of agomiR-145 in vitro and in vivo. Whole-cell patch-clamping was applied to assess alterations in the tetrodotoxin-resistant (TTX-R) sodium current (Nav1.8) in DRGs. Results: The PMWT was significantly decreased in rats following streptozotocin (STZ) injection on Day 7 and was maintained at a lower level on Day 28; this change was accompanied by changes in the expression of Nav1.8 in DRG neurons, which was increased 3 days after STZ injection and reached a maximal level on Day 14. The early knockdown of Nav1.8 with siRNA or agomiR-145 treatment on Day 8 effectively precluded the deterioration of pain behaviors in STZ-treated rats. The luciferase intensity was significantly decreased in HEK293T cells expressing wild-type SCN10A infected with miR-145 mimic. In addition, Nav1.8 overexpression was significantly repressed via overexpression of miR-145 in cultured DRG neurons, and neuronal hyperexcitability was concomitantly decreased. Furthermore, the intrathecal administration of agomiR-145 elicited a significant decrease in Nav1.8 expression in DRG neurons from STZ-treated rats on Day 14. Limitations: The causes of PDN are likely to be multifactorial and inflammatory markers, such as IL-6, IL-2, and TNF-α, are elevated in hyperglycemia and might be the precipitating factors that contribute to miR-145 dysregulation. The curative effect of miR-145 upregulation in reversal of pain behaviors at the stage of well-established PDN wasn’t investigated in this study. Conclusion: Early infection with a lentiviral vector overexpressing miR-145 adversely regulated the expression and function of TTX-resistant Nav1.8 and abrogated the development of PDN. Therefore, miR-145 might be a potential therapeutic target for preventing PDN in the near future.